Brock Biology of Microorganisms, Global Edition, 14E Michael T Madigan Solution Manual with Test Ban

Type:

Solution Manual and Test Bank

Resource:

Brock Biology of Microorganisms,

Edition:

14th Edition

Author(s):

Michael Madigan Kelly Bender Daniel Buckley W. Sattley David Stahl

INSTRUCTOR’S MANUAL AND TEST BANK W. Matthew Sattley INDIANA WESLEYAN UNIVERSITY

Christopher A. Gulvik GEORGIA INSTITUTE OF TECHNOLOGY

Sherry L. Seston ALVERNO COLLEGE

Heather Williamson Jordan UNIVERSITY OF TENNESSEE

GLOBAL EDITION CONTRIBUTIONS BY

Sumitra Datta, Shanmugha Arts, Science, Technology and Research Academy

Brock Biology of Microorganisms GLOBAL EDITION

FOURTEENTH EDITION

Madigan/Martinko/Bender/Buckley/Stahl

Table of Contents

INSTRUCTOR’S MANUAL CHAPTER 1

Microorganisms and Microbiology

1

CHAPTER 2

Microbial Cell Structure and Function

11

CHAPTER 3

Microbial Metabolism

29

CHAPTER 4

Molecular Microbiology

39

CHAPTER 5

Microbial Growth and Growth Control

52

CHAPTER 6

Microbial Genomics

64

CHAPTER 7

Metabolic Regulation

76

CHAPTER 8

Viruses and Virology

87

CHAPTER 9

Viral Genomes and Diversity

95

CHAPTER 10 Genetics of Bacteria and Archaea

107

CHAPTER 11 Genetic Engineering and Biotechnology

117

CHAPTER 12 Microbial Evolution and Systematics

131

CHAPTER 13 Metabolic Diversity of Microorganisms

142

CHAPTER 14 Functional Diversity of Bacteria

157

CHAPTER 15 Diversity of Bacteria

170

CHAPTER 16 Diversity of Archaea

181

CHAPTER 17 Diversity of Eukaryotic Microorganisms

188

CHAPTER 18 Methods in Microbial Ecology

196

CHAPTER 19 Microbial Ecosystems

205

CHAPTER 20 Nutrient Cycles

214

CHAPTER 21 Microbiology of the Built Environment

220

CHAPTER 22 Microbial Symbioses

228

CHAPTER 23 Microbial Interactions with Humans

237

TABLE OF CONTENTS

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CHAPTER 24 Immunity and Host Defense

245

CHAPTER 25 Immune Mechanisms

253

CHAPTER 26 Molecular Immunology

260

CHAPTER 27 Diagnostic Microbiology

267

CHAPTER 28 Epidemiology

278

CHAPTER 29 Person-to-Person Bacterial and Viral Diseases

287

CHAPTER 30 Vectorborne and Soilborne Bacterial and Viral Diseases

300

CHAPTER 31 Water and Food as Vehicles of Bacterial Diseases

307

CHAPTER 32 Eukaryotic Pathogens: Fungal and Parasitic Diseases

318

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CHAPTER

1

Microorganisms and Microbiology

Summary Chapter 1 introduces the study of microbiology for students, most of whom will have had little or no exposure to the subject. Consequently, you have a good opportunity to provide an overview of microbiology that demonstrates the important roles microorganisms play in human activities and in the ecology of the entire biosphere. The ability of microorganisms to exist independently in nature as free-living cells (Figure 1.1) confers enormous adaptive advantages over cells of macroorganisms, which are incapable of an independent existence.

1.1 | What Is Microbiology About and Why Is It Important?

Describe to students ways in which microbiology serves as both a basic and applied biological science. From a basic perspective, the study of microorganisms has been the primary means by which the fundamental genetic and biochemical properties of living cells have been revealed. From an applied perspective, microorganisms directly affect the quality of human life in both detrimental and beneficial ways. Although microorganisms are the causative agents of some of the most important human, animal, and plant diseases, they are also used for the industrial production of antibiotics, pharmaceuticals, and foods. Microorganisms are also increasingly being used for beneficial purposes as diverse as bioremediation of polluted sites, gene therapies for genetic diseases, and the production of biofuels. Microbiology is therefore a science of far-reaching scope, with applications that affect the quality of human life in a variety of ways.

You should also emphasize to students the importance of microorganisms in the emergence and maintenance of higher forms of life. From the production of molecular oxygen (by cyanobacteria) to the biogeochemical cycling of key elements, such as carbon, nitrogen, and sulfur, microorganisms play a major role in sustaining all life on the planet. Point out in your course introduction that for all the reasons summarized in this section, microbiology is the foundation of all biological sciences.

1.2 | Structure and Activities of Microbial Cells Because microorganisms generally exist as free-living cells, it is important to discuss the characteristics of cells in general. Emphasize that all cells exhibit a nonrandom organization with a semipermeable membrane boundary that encompasses an internal system that is not in equilibrium with its environment. Point out that prokaryotic cells (i.e., all Bacteria and .

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Archaea) do not contain membrane-bound, internal organelles as traditionally described for eukaryotic cells (the Eukarya; Figure 1.2). In particular, the organization of prokaryotic DNA as a nucleoid, an aggregated mass of genetic material within the cytoplasm, is in stark contrast to the compartmentalized, multichromosomal configuration typically found in eukaryotes. However, despite the structural and morphological similarities of Bacteria and Archaea, make sure the students are aware early on that these groups of microorganisms have quite distinct evolutionary lineages and are, therefore, not closely related on a genetic level. This concept is discussed in more detail in Section 1.3.

The ability of cells to maintain a thermodynamic energy flow far from equilibrium defines what we refer to as a living system. All living systems display some form of metabolism in which both energy-yielding (catabolic) and energy-consuming (anabolic) biochemical reactions are catalyzed simultaneously. These chemical transformations allow for biosynthesis of new cell structures and, ultimately, cell division (microbial growth). Figure 1.3 shows the characteristics that define cellular life, some of which are universal (e.g., metabolism and evolution) and some of which occur only in some cells (e.g., differentiation and motility).

1.3 | Evolution and Diversity of Microbial Cells Although Earth is believed to be about 4.6 billion years old, several lines of evidence indicate that the appearance of multicellular life did not occur until about 600 million years ago (Figure 1.4). By contrast, microbial cells (single-celled prokaryotes) likely appeared between 3.8 and 3.9 billion years ago and therefore were the only inhabitants of the planet for about 80 percent of its history! The appearance of multicellular life was necessarily preceded by the accumulation of significant amounts of molecular oxygen (O2) in the atmosphere, a phenomenon that resulted from the activity of cyanobacteria and the evolution of a chlorophyll molecule that could use H2O as electron donor (see Chapter 13). Develop this concept for students in some detail at an early stage in the course to provide proper historical perspective of important events in cellular evolution. You may wish to start with the evidence supporting the hypothesis that the first self-replicating “entities” may have been RNA molecules, and the incorporation of these entities within lipid membranes may have represented the first cell type(s). This topic will be more thoroughly discussed in Chapter 12.

• Discuss with your students some of the events that led to “higher” forms of life. The evolution of anaerobic phototrophic metabolisms likely began within 500 million years after the appearance of the first cells, followed nearly a billion years later by the evolution of cyanobacteria from these early phototrophs (Figure 1.5), thus starting the slow process of oxygenating the atmosphere and the evolutionary path to multicellularity.

• Also discuss the methods now routinely used in the development and refinement of the phylogenetic tree of life (Figure 1.6a). Introduce to your students the enormous impact ribosomal RNA gene sequencing has had on our understanding of microbial diversity and on the classification of living organisms into three distinct domains (Figure 1.6b).

1.4 | Microorganisms and Their Environments .

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The traditional approach to teaching students about microorganisms is from a pure culture perspective (e.g., a bacterial species and its effect on a host). It is more appropriate to introduce microorganisms as populations of cells occupying a particular habitat in which communities of different populations interact in complex ways. These interactions affect both the habitat and its inhabitants, and they have a profound impact on the larger, multicellular inhabitants of an ecosystem. In fact, the very existence of multicellular life depends upon the nutrient cycling activities of microorganisms. Point out to students that pure cultures of microorganisms almost never occur in nature. Many students may have more interest in ecology than in what appears to them a more abstract area, such as microbiology. Therefore, stress the fact that the biochemical conversions and molecular interactions of microorganisms significantly control the balance and community structure of all living organisms in an ecosystem.

Students will likely be unaware of the abundance and ubiquitous nature of microbial life.

Microorganisms can be found in even the most inhospitable environments by human standards, and students are often amazed at the extremes in which microorganisms can thrive (Table 1.1). Point out the fact that the total amount of carbon, nitrogen, and phosphorus in microbial cells exceeds that in all plant and animal biomass. Microbial cells represent the major reservoirs of life’s essential nutrients.

In this connection, prokaryotes comprise the major portion of Earth’s total biomass.

Most of this biomass consists of marine and terrestrial subsurface microbial populations (Table 1.2). These communities contain a reservoir of genetic diversity that is largely unexplored and that could benefit humankind in as yet unknown ways. The effect of microorganisms on animals, plants, and global ecosystems is a topic interwoven throughout a number of chapters in this text. These concepts will come as a surprise for most students—who generally consider microorganisms “simple,” insignificant creatures that only cause disease—and will set the stage for more detailed study in future chapters.

1.5 | The Impact of Microorganisms on Humans Students probably understand the importance of microorganisms as agents of infectious disease, and indeed, this has been a driving force in the development of microbiology as a distinct scientific discipline. The effects of microorganisms upon human affairs have been remarkable, particularly when one compares the foremost causes of death in the United States at the beginning of the twentieth century (before the widespread use of antibiotics and vaccines) with the major causes of death in this century (Figure 1.8). While the deleterious effects of pathogenic bacteria to human wellness should be discussed here, it is important to also stress the key beneficial roles microorganisms play in agriculture (Figure 1.9), in human digestive health (Figure 1.10), in the food and beverage industries (Figure 1.11), and in the growing fields of biotechnology and alternative and renewable energy (Figure 1.12). With this discussion, students should become aware of the tremendous influence of microorganisms in human society.

The genomes of microorganisms hold incredible potential for the production of commercial products. The traditional methods of employing microorganisms for human use (e.g., fermentation) have evolved into what students now recognize as “biotech”—the biotechnology industry. The industry arose from the development by microbiologists of tools for genet.

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ically manipulating the genome of a bacterial cell to produce a desired product. An excellent example is the production of human insulin by Escherichia coli, an innovation that made the treatment of diabetes affordable for people from all economic backgrounds.

In addition to producing a product directly, bacterial cells can be used as a vehicle to introduce specific genes into the genomes of other organisms for commercial production of a desired product. For example, genetic engineering of Agrobacterium tumefaciens, a plant pathogen that naturally transfers DNA directly into plant cells upon infection, has led to the use of plants for the production of human antibodies with potential anticancer or antiviral properties.

1.6 | The Discovery of Microorganisms It is important to emphasize the historical roots of microbiology, particularly considering its rather recent beginnings as a systematic field of science in the nineteenth century and the fact that several new fields have arisen from this discipline (e.g., molecular biology and biotechnology; see Table 1.3). Lead the students on a chronological journey, beginning with Robert Hooke’s first description of a microorganism (a fungal mold) in 1665 (Figure 1.13) and Antoni van Leeuwenhoek’s microscope and publication of the first observation of bacteria in 1684 (Figure 1.14). Perhaps the most important early figure in general microbiology is Ferdinand Cohn (a contemporary of Louis Pasteur and Robert Koch) who is widely regarded as the founder of bacteriology. Several important events followed the early observations of van Leeuwenhoek, a few of which are listed below: • Nineteenth-century scientists tested the issues of spontaneous generation and the nature of infectious disease, leading to the genesis of the field of medical microbiology.

• Improvements in microscopy allowed for Cohn’s observations of bacteria, which included a preliminary characterization of the sulfur-oxidizing species Beggiatoa mirabilis (Figure 1.15) and his discovery of heat-resistant endospores in the genus Bacillus. Cohn discovered that boiling killed the vegetative form of Bacillus but not the endospore stage.

• Cohn laid the foundation for classification of bacteria and designed the first methods for preventing contamination of culture media; these methods were later adopted and improved upon by German physician Robert Koch.

1.7 | Pasteur and Spontaneous Generation As a young researcher, Louis Pasteur was formally trained as a chemist with expertise in the field of crystallography. His knowledge of the optical properties of crystals provided the basis for his early investigations into the biological sciences. Begin the discussion of his celebrated scientific achievements by pointing out that it was his observation that living organisms discriminate between optical isomers of metabolites during growth (Figure 1.16) that led him to begin a series of experiments that would result in the defeat of the centuries old theory of spontaneous generation. Pasteur’s experimental design that finally disproved this theory introduced the now famous swan-necked flask (Figure 1.17). His work is a classic example of the effectiveness of using the scientific method and controlled experimentation to resolve questions rather than personal observation and interpretation, which often lead to unsubstan.

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tiated (and often erroneous) conclusions. Point out to students that disproving spontaneous generation resulted in the development of effective procedures for controlling microbial growth, including the familiar process of pasteurization. Outline the major career accomplishments of Pasteur, pointing out the fact that although his professional training pertained to chemistry rather than biology, his laboratory experimentation led him to the study of microorganisms.

• As a chemist, Pasteur’s work with optical isomers of amyl alcohol, a byproduct of alcohol fermentation by beets, led him to confirm that fermentation was not a strictly chemical phenomenon but that living yeast cells were required for the process. The outcome of his fermentation studies led him to the classic experiments that defeated spontaneous generation (Figure 1.17).

• Discuss the many other accomplishments of Pasteur, such as the development of vaccines for anthrax, fowl cholera, and rabies. The dramatic results of his rabies vaccine, which saved a young boy, startled the scientific world and led the French government to build the Pasteur Institute in Paris in 1888 (Figure 1.18). Together with Robert Koch, whose scientific achievements are discussed next, Pasteur played a major role in establishing the field of medical microbiology.

1.8 | Koch, Infectious Disease, and Pure Cultures Explain to your students that for centuries many thought that some unknown agent from a diseased person could be transmitted to healthy people, but no real proof of this existed until the work of Robert Koch (Figure 1.19). Although Koch was 21 years younger than Pasteur, several of their greatest achievements were carried out around the same time during the late 1800s. The following list serves as an introduction to the works of Koch for your students.

• Koch’s work on the germ theory of disease (1876) was perhaps the most important stimulus for the development of medical microbiology. Given the popular focus on bioterrorism in recent years, students may find it interesting that Koch’s work leading to this important theory involved the study of the endospore-forming bacterium Bacillus anthracis, the causative agent of anthrax. Carefully discuss Koch’s postulates, the use of which has since led to discoveries for the prevention and cure of many infectious diseases (Figure 1.20). However, be sure to note that while Koch’s postulates are the “gold standard” for determining the causative agent of a disease, it is not always possible to carry them out in full, especially considering that many pathogenic bacteria resist laboratory culture. It is in these cases that genomics is proving useful in linking a causative agent to a particular disease in the absence of cultivable microorganisms (see feature box Explore the Microbial World: The Black Death Decoded).

• Koch’s postulates require that the suspected organism be obtained in pure culture. Koch developed clever ways of obtaining pure cultures, including the use of solidifying agents such as gelatin and agar to obtain single colonies. Point out to students that a colony is a population of cells arising from a single cell, and thus each colony contains a single type of microorganism, an observation that Koch himself was quick to realize.

• Ferdinand Cohn had also been active in his research during this time, and he and Koch regarded each other’s work with mutual respect. Several of Cohn’s innovations for prevent.

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ing contamination of pure cultures were adopted by Koch and his associate Walter Hesse (Figure 1.21), thereby aiding many of Koch’s studies of pathogenic bacteria.

• Koch was the first to isolate Mycobacterium tuberculosis and demonstrate that it was the causative agent of tuberculosis (Figure 1.22). Considering the huge number of individuals that succumbed to this disease during Koch’s time (about 15% of all reported deaths), the significance of this achievement could hardly be exaggerated. In addition to this, Koch also discovered and characterized Vibrio cholera, the causative agent of the severe diarrheal disease cholera

1.9 | The Rise of Microbial Diversity Continue your discussion of the historical roots of microbiology by introducing the work of Martinus Beijerinck and Sergei Winogradsky, both of whom are known for their study of microbial diversity. In the same way that Pasteur and Koch propelled the field of medical microbiology to the scientific mainstream, Beijerinck and Winogradsky became the chief figures of environmental microbiology. Both of these researchers were interested in the biogeochemical cycling of nitrogen and sulfur in the environment, including the reduction of atmospheric nitrogen to ammonia by nitrogen-fixing bacteria (Figure. 1.23). In addition, Beijerinck was the first to describe the existence of subbacterial infectious agents of disease (viruses) through his study of tobacco mosaic disease.

• Martinus Beijerinck, a Dutchman, developed the concept of enrichment culture technique for the selective culture of specific types of microorganisms from natural samples. The use of selective culture media and incubation conditions was a major innovation for the isolation of novel strains of microorganisms, and the technique is still used extensively today.

For example, enrichment cultures are routinely used to isolate novel bacteria for industrial and medical use, such as antibiotic-producing microorganisms from soil (see Chapter 15).

It is important to describe the principle of this important technique to students, since this concept will be especially relevant for certain laboratory exercises that usually accompany courses in general microbiology.

• Sergei Winogradsky of Russia used enrichment culture to study bacteria in soil, particularly those that play a role in the cycling of nitrogen and sulfur (Figure 1.22). He isolated the first N2-fixing bacterium, Clostridium pasteurianum, and developed the concept of bacterial fixation—an important ecological discovery. Winogradsky was also interested in bacteria that oxidize inorganic compounds for energy conservation, including the nitrifying and sulfur-oxidizing bacteria, and he showed that these organisms are autotrophic, using CO2 as their sole source of carbon. Winogradsky was perhaps the first scientist to recognize the metabolic significance of biogeochemical processes. Briefly discuss Winogradsky’s major discoveries: chemolithotrophy and nitrogen fixation.

1.10 | Modern Microbiology and Genomics Conclude your discussion of the history of microbiology by summarizing the major subdisciplines that have arisen from the basic and applied directions that the discipline took in the twentieth century (Table 1.3). It is important to note that the distinction between basic and .

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applied is not subdiscipline-specific in a strict sense, as might be implied in the table; it is defined by the questions being asked by the researcher.

• Basic: microbial systematics, microbial physiology, microbial biochemistry, virology, microbial genetics, microbial ecology, molecular biology, and genomics. With the use of molecular tools, microbial ecology has now become an important discipline for identifying novel molecules from microorganisms for biotechnological applications. These tools are allowing scientists to probe the immense and untapped genetic diversity of the most efficient bioengineers—microorganisms. The development of microbial genetics has given rise to the era of molecular biology, genomics, and biotechnology. This is an important point to emphasize to students, many of whom will work in the biotechnology industry and/or use the very products that have developed through discoveries that have come out of this field.

• Applied: immunology, medical microbiology, agricultural microbiology, industrial microbiology, aquatic microbiology, and biotechnology. The subdisciplines of applied microbiology arose directly from the works discussed in previous sections (Pasteur and Koch—medical microbiology and immunology; Beijerinck and Winogradsky— agricultural microbiology, aquatic microbiology, etc.).

• Molecular microbiology: Lastly, discuss the era of molecular microbiology that rapidly emerged during the 1970s, leading to the subdisciplines of biotechnology, genomics, transcriptomics, proteomics, and metabolomics. These topics will be discussed further in Chapter 6.

Answers to Review Questions 1. Microbiology is both a basic biological science (a science of discovery) and an applied biological science (a science of solving problems). Knowledge of the world around us is gained through basic research, and this knowledge can often be applied to resolve challenges that face our society or threaten human welfare.

2. The Bacteria and Archaea consist of prokaryotic, generally free-living cells. The Eukarya are eukaryotic cells (or multicellular organisms consisting of eukaryotic cells) that are generally significantly larger than prokaryotes and have a greater degree of complexity with a defined nucleus and membrane-bound organelles. Cells function as machines in a chemical sense, carrying out metabolic reactions essential for their integrity and survival.

However, information from coding devices is also required so that metabolic events may be initiated or deferred at the appropriate time. Translation of the genetic information encoded in DNA requires ribosomes to coordinate the interaction of messenger RNA with the other components required to assemble the protein product, transfer RNA, and amino acids. Though catalytic and coding functions of cells differ (one specifies what can occur, the other specifies when), neither can function without the other within the confines of the cell boundary. Thus, order is maintained against a thermodynamically unfavorable gradient. A living cell must possess both.

3. After the first 2 billion years of Earth’s existence, during which its atmosphere contained only nitrogen (N2), carbon dioxide (CO2), and a few other gases, Cyanobacteria evolved from anoxygenic phototrophs and began the slow process of oxygenating Earth’s atmosphere. Genes that encode ribosomal RNAs serve as barometers of microbial diversity. .

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Ribosomal RNAs are components of ribosomes. As they are found in all forms of life, a phylogenetic tree that reveals the position of any organism relative to its neighbors can be constructed through its ribosomal RNA genes.

4. An ecosystem consists of the biotic factors (living organisms) together with the abiotic factors (physical and chemical nonliving constituents) of a habitat in the environment. In nature, microorganisms almost never occur in pure culture; they occur as mixed populations composing a larger microbial community. The microorganisms in an ecosystem use nutrients from the environment and excrete waste products of their metabolisms into the environment. The life processes of various microorganisms significantly contribute to primary production, decomposition, and nutrient cycling in an ecosystem.

5. Microorganisms carry out many important functions, with only a small percentage of bacteria (<1%) capable of causing disease. In fact, the human body has far more bacterial cells on and in it than human cells, and while a small fraction of these microorganisms are potentially pathogenic, the vast majority are commensal species essential for maintaining proper human health. In addition, some bacteria fix nitrogen (N2) and are thus important in agriculture. Others are important in food and beverage production. Most microorganisms play crucial ecological roles in the environment.

6. Robert Hooke was the first to observe and describe microorganisms while studying the fruiting structures of molds (fungi). Antoni van Leeuwenhoek was the first person to observe bacteria using microscopes of his own design and build. Both researchers were active and accomplished their most influential work in the late 1600s.

7. Pasteur’s experiments using swan-necked flasks silenced the objection of vitalists who claimed that sealing the mouth of a flask blocked the availability of open air, which they viewed as being essential for spontaneous generation (they were entirely unaware of the existence of anaerobes). Pasteur definitively showed that, even in open air, putrefaction of the heated broth did not occur unless the flask was tipped such that the broth came in contact with dust particles trapped in the neck of the flask, thus proving that the microorganisms that contaminated the broth were carried in the air.

8. A pure culture is a culture that consists of only one type of microorganism. The most common method of obtaining pure cultures is by the isolation of single colonies on solid growth media. Pure cultures may be used to determine the specific effect an organism has on its environment. For example, Koch and others used pure cultures to determine the causative agents for many diseases.

9. Koch’s postulates are a set of criteria for proving that a specific type of microorganism causes a specific disease. The main points are as follows: (1) The suspected microorganism must be present in all cases of disease and not present in healthy individuals; (2) the organism must be cultivated from diseased individuals and isolated in pure culture; (3) inoculation of a healthy individual with organisms from the pure culture must initiate disease in susceptible animals; and (4) the organism should be re-isolated from these experimental animals in pure culture and be identified as the same infecting organism as that found in the original diseased animal. Investigators still use these postulates as a guide to determine the causative agents of diseases and for developing successful treatments for the prevention and cure of many infectious diseases.

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10. While Robert Koch was an eminent early medical microbiologist, Beijerinck and Winogradsky were responsible for developing the field of microbial ecology as it relates to the environment rather than the human body. Their primary contributions to microbiology were in the design and implementation of the enrichment culture technique and the isolation and characterization of bacteria responsible for cycling nitrogen and sulfur, including those having chemolithoautotrophic metabolisms. Before Winogradsky’s work, the use of CO2 as the sole source of carbon by nonphotosynthetic organisms was unknown. Winogradsky isolated and described the first nitrogen-fixing bacterium, the anaerobe Clostridium pasteurianum. Later, Beijerinck described the first aerobic nitrogen-fixing bacterium, Azotobacter chroococcum, from a soil sample.

11. Subdisciplines with an emphasis in basic science are those that primarily pertain to the advancement of knowledge in the area of the fundamental processes of life. Areas such as biochemistry, physiology, genetics, virology, and microbial ecology are all related to the molecular biology of cells and therefore fall largely under this category. Subdisciplines with an emphasis in applied science are those directly linked to important practical problems that affect human welfare, and these include areas of microbiology related to medicine, agriculture, industry, technology, and sanitation.

Answers to Application Questions 1. Before Pasteur’s experiments, and despite some evidence to the contrary, it was thought that microorganisms could arise spontaneously in or on the surface of a nutrientcontaining medium and, by extension, in any environment. Pasteur showed that microorganisms must be carried into the environment from the outside. A medium that was initially sterile remained so until a living cell from outside was introduced. Pasteur’s work provided some of the basic concepts for the development of aseptic technique, as well as methods of food preservation, such as canning and pasteurization. Moreover, with this development, it became possible to think of microorganisms as the causative agents of disease.

2. Koch used the following methodology, which became known as Koch’s postulates, to determine the causative agent of a disease: a. The organism must be isolated from the diseased animal and grown in pure culture. b. The organism must not be present in healthy animals. c. When the microorganism is injected into a healthy animal, the disease must occur. d. The pathogenic agent must be re-isolated from the diseased animal.

Koch succeeded in isolating the organism we now know as Mycobacterium tuberculosis from every animal that died from the disease (postulates a, b). He injected cells of a pure culture of M. tuberculosis into healthy guinea pigs, and these guinea pigs developed systemic tuberculosis (postulate c). These microorganisms were then re-isolated from the newly diseased animals and were shown to be M. tuberculosis (postulate d). Thus, all four criteria were met.

3. a. The Dead Sea is a hypersaline habitat. Any seawater medium with salinity maintained by sodium chloride can be used. b. For isolation of microorganisms from Mammoth Hot Springs, maintenance of a high temperature is a prerequisite. Subsequently, based on the .

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alkaline, acidic or haline nature of the water sample , a modified nutrient media supplemented with required amount of calcium carbonate or sodium chloride could be used.

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CHAPTER

2

Microbial Cell Structure and Function

Summary Chapter 2 is an excellent introductory overview of microscopic techniques and the structure and function of both prokaryotic and eukaryotic cells. For courses designed for nonscience majors, this chapter provides general details on each topic that, if supplemented with material from related chapters later in the text, may be sufficient background for most students. However, it is recommended that Chapter 2 be used to set the stage for more detailed coverage later in the course.

2.1–2.4 | Microscopy The variety of microscopic methods available for observing microorganisms must be introduced early, as much of the presentation of structure–function relationships depends upon the excellent micrographs that appear throughout the book. Although details of microscopy are more easily introduced in the laboratory portion of the course, the material included here is pertinent to effective lecture presentation.

• Discuss the basic principles and components of the compound light microscope, including the relationships between resolution and magnification, and numerical aperture (Figure 2.1). Note that although bright-field microscopy is fine for visualizing pigmented cells (Figure 2.2), it is not an efficient tool for viewing unstained cells with no natural pigmentation, such as nonphototrophic bacteria.

• This deficiency will lead to a discussion of various methods employed to increase contrast.

Discuss the various simple dyes used to stain cells, most of which are positively charged, basic dyes capable of binding to negatively charged cell surfaces (e.g., methylene blue and crystal violet; Figure 2.3). Continue the discussion of differential stains, the most widely used of which is the Gram stain (Figure 2.4).

• Students should understand that while staining procedures increase the contrast of cells against the background to make them more visible, they also kill cells and often distort their appearance. Discuss phase-contrast microscopy and dark-field microscopy (Figure 2.5), two tools that allow one to look at living cells without the need for staining.

• Fluorescence microscopy is widely used in clinical diagnostic microbiology and environmental microbiology (Figure 2.6). Most students who enter the biotechnology industry or .

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medical profession will work with fluorescent molecules (such as those used for fluorescence antibody staining methods). The variety and sensitivity of these molecules has increased dramatically over the past decade. This has allowed the development of a wide variety of nonradioactive alternatives to biological assays that are now routinely used in research.

• Students should be interested in the micrographs from three-dimensional imaging of cells.

Depending upon the level of the course, you may choose to discuss the principles of differential interference contrast microscopy (Figure 2.7) and confocal scanning laser microscopy (Figure 2.8). Lastly, show and discuss the micrographs obtained from electron microscopy (Figures 2.9 and 2.10). Note the differences between scanning electron microscopy (SEM), which provides an image of the external features of a specimen, and transmission electron microscopy (TEM), in which thin sections of the specimen show its detailed internal structure.

2.5 | Cell Morphology Using Figure 2.11, point out the three major morphologies of prokaryotic cells (coccus, rod, and spirillum). Inform your students that, in some species, the cells remain attached following cell division, giving rise to different arrangements that are often genus-specific. For example, coccus cells may exist as short chains (Streptococcus) or grapelike clusters (Staphylococcus).

Less common cell morphologies also exist, such as spirochetes, appendaged (budding) bacteria, and filamentous bacteria (Figure 2.11). Stress to students that these morphologies are only representative of those found in nature. Other unusual shapes have also been described in rare cases (for example, square and star-shaped cells!).

Before the molecular era, morphological and physiological properties were used to classify bacterial species. However, we now know that these criteria are poor predictors of evolutionary relationships. For example, certain species of Archaea may appear identical in size and shape to species of Bacteria under the microscope, but these organisms are of different phylogenetic domains and thus are not closely related to one another on an evolutionary basis.

The cell morphology of a particular species is primarily a result of selective pressures in a given habitat that favored a particular cell shape for enhanced reproductive success.

2.6 | Cell Size and the Significance of Being Small The presentation in the text on the significance of being small is an important concept for students to internalize as they progress in their study of microbiology. Table 2.1 shows the wide size range variability of prokaryotic cells, which range from a diameter of about 0.2 µm to over 700 µm. Use the two examples of unusually large prokaryotes discussed in this section to illustrate the current upper limit of prokaryotic cell size: (1) the surgeonfish gut symbiont Epulopiscium fishelsonsi (>600 µm in length; Figure 2.12a), and (2) the sulfur chemolithotroph Thiomargarita namibiensis (750 µm; Figure 2.12b). The evolutionary “rationale” for the existence of unusually large-celled prokaryotes is a mystery when one considers that the .

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metabolic rate of a cell varies inversely with the square of its size. Ask your students for ideas and/or hypotheses that might explain the selective advantage of large cell size in these two prokaryotes.

The fact that bacteria can live independently as single cells (unlike an individual cell of a multicellular organism) suggests that they must possess some capabilities that provide a selective advantage over their multicellular counterparts that ensure their survival on the planet. Small cells have more surface area to volume (i.e., a higher surface-to-volume ratio), and this alone confers many of the evolutionary advantages of being small, including the following: • Rapid nutrient and waste transport into and out of the cell allows for faster metabolic rates and growth rates.

• Rapid growth rates result in the rapid production of large populations of cells. These populations, in turn, can greatly affect the physiochemical conditions of an ecosystem within a short time period.

• Transport rates are a function of the surface area of the cell membrane relative to cell volume. Use Figure 2.13 to mathematically demonstrate to students that the surface area of a sphere is a function of the square of the radius, whereas the volume of a sphere is a function of the cube of the radius. This means that the surface-to-volume ratio of a spherical cell can be expressed as 3/r, where r equals the radius of the cell. Therefore, a coccus cell having a smaller radius has more surface area per volume, and thus more efficient transport capabilities, than a coccus cell having a larger radius.

• Rates of evolutionary change are higher in smaller, faster growing haploid cells than in larger, slower growing diploid cells. This allows for greater adaptive potential through rapid selection for advantageous mutations and counterselection against deleterious mutations.

The theoretical lower limit of size for a living cell is likely near 0.2 m in diameter. This limit is dictated by the amount of volume required to contain cellular components that are crucial for maintaining life, such as (1) the presence of essential genes on the chromosome; (2) having a sufficient number of ribosomes; and (3) containing a minimal number of metabolic, structural, and transport proteins within the cell. Challenge students to list these and other molecular components a cell would have to contain to maintain life. Remind students that some cells are parasitic in nature. Inform them that, much like viruses, such microorganisms often have streamlined genomes that lack important genes and may make them dependent upon their hosts for growth. Can such organisms truly be considered living? This might make a good outside project for group debate, requiring students to view the cell as a threedimensional physical structure constrained in space and to research a problem that is currently being debated.

2.7 | Membrane Structure

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The structure of the cytoplasmic membrane, a phospholipid bilayer, should be discussed in considerable detail because it plays a critical role in establishing and maintaining the cell’s internal environment. Students must understand that the cytoplasmic membrane is the selectively permeable boundary between the cytoplasm of the cell and the cell’s immediate environment. If the integrity of the membrane becomes compromised, then essential cellular components can leak out of the cytoplasm and into the environment, thereby destroying the cell. Convey to students that the cytoplasmic membrane generally does not confer a specific shape and provide rigid support to the cell (these are roles of the cell wall, to be discussed later), but rather the membrane has a fluid nature that allows for a degree of lateral movement of phospholipids and proteins (Figures 2.14 and 2.15). Proteins embedded in the membrane consist of both hydrophobic regions that are situated within the lipid portion of the phospholipid bilayer and hydrophilic regions that are oriented toward either the external environment or the aqueous cytoplasm of the cell.

In contrast to eukaryotic cells, which contain rigid sterol molecules to strengthen and stabilize membranes (especially those of animal cells, which lack cell walls), most prokaryotic membranes instead contain planar molecules called hopanoids that serve a similar function.

Exceptions to this generalization include methanotrophic bacteria, which contain large amounts of sterols in internal membranes, and the mycoplasmas, a group of parasitic bacteria that lack cell walls.

While members of the Bacteria and Eukarya contain ester linkages that bond the fatty acids to glycerol in their membranes (Figure 2.16a and b), Archaea contain ether linkages between the glycerol and lipid portions of their membranes. In addition, archaeal membrane lipids are not composed of fatty acids but instead consist of repeating five-carbon isoprene units that combine to form 20-carbon phytanyl side chains (Figures 2.16c and 2.17a and b).

Together, the glycerol and phytanyl form a glycerol diether. In some Archaea, glycerol diethers are joined at their hydrophobic ends to create a lipid monolayer of diglycerol tetraethers (Figure 2.17b and e). This structural conformation provides superior thermostability of the membrane, and indeed lipid monolayers are most commonly found in hyperthermophilic archaeal species. Finally, members of the Crenarchaeota often contain crenarchaeol, a unique monolayer membrane lipid having four cyclopentyl rings and one cyclohexyl ring (Figure 2.17c). Despite the molecular differences between archaeal membranes and bacterial/eukaryotic membranes, their basic structural properties are the same in that each possesses hydrophobic interior hydrocarbon chains attached to polar (hydrophilic) glycerophosphate molecules.

Although molecular adaptations of membranes to high and low temperatures are discussed in some detail in Chapter 5, this may be a good opportunity to introduce the topic of saturated versus unsaturated hydrocarbon chains and discuss how they relate to membrane fluidity under high and low temperature extremes (e.g., why vegetable shortening is a solid at room temperature, and vegetable oil is a liquid under the same conditions).

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2.8 | Membrane Function The major functions of the cytoplasmic membrane are summarized in Figure 2.18 and include its role as (1) a permeability barrier, (2) a protein anchor, and (3) a means of energy conservation. With respect to acting as a permeability barrier, impress upon students that even extremely small ions do not freely pass through the hydrophobic interior of the membrane due to their charges (Table 2.2). While water molecules do diffuse through membranes (due to their small size and only weak polarity) in a process called osmosis, the movement of water across membranes is greatly accelerated by water transport proteins called aquaporins. These transport proteins have been identified in the membranes of organisms from all domains of life but are perhaps best studied in the bacterium Escherichia coli.

Introduce students to the concept that a membrane can function much like a battery in that it can store potential energy. By separating protons to the outside of the membrane from hydroxyl ions on the inside, the membrane becomes “energized” (i.e., polarized), and this energized state is referred to as the proton motive force (PMF). The dissipation of this force results in the conversion of potential energy to kinetic energy. When protons stored outside of the membrane return to the inside of the cell through an ATPase enzyme complex, ADP and Pi are converted to ATP, the cell’s energy currency. This concept will be discussed in detail in Chapter 3.

Discuss with your students the necessity for membrane-bound transport proteins by comparing the rate of simple diffusion of a solute across a membrane to the greatly accelerated rate of carrier-mediated transport of a solute across a membrane (Figure 2.19). Transport proteins allow for the accumulation inside a cell of a solute that may be in very low concentration in the environment. Point out that each carrier-mediated transport protein shows high specificity for a given solute.

2.9 | Nutrient Transport Some students may find the variety of nutrient transport mechanisms difficult to comprehend initially, so discuss these mechanisms in detail using Figures 2.20–2.23 to illustrate the concepts and provide examples of each type of transport event. When describing the three classes of membrane transport systems—simple transport, group translocation, and the ABC (ATP-binding cassette) system—highlight the following points to your students: • Some transport mechanisms require only a membrane-spanning component (e.g., the simple transporters shown in Figure 2.21).

• Some require a series of proteins that cooperate in a phosphorylation/dephosphorylation cascade to carry out the transport event (e.g., the group translocation phosphotransferase system; Figure 2.22).

• Some require a membrane-spanning transporter, a substrate-binding protein, and an ATP-hydrolyzing protein (e.g., the monosaccharide ABC transporters; Figure 2.23).

In addition to small molecule transport, larger molecules, such as proteins, need to be inserted into membranes or transported outside the cell (e.g., toxins, amylases, and cellulases). This movement of materials is accomplished by translocases, the most well.

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characterized being the SecYEG system that is found in many prokaryotes and the Type III Secretion System employed for the export of toxins by several pathogenic bacteria.

2.10 | Peptidoglycan The bacterial cell wall warrants extensive coverage in the classroom because research on its structure and function can be traced back to the early history of microbiology. It began with Ferdinand Cohn’s early observation of the differential reaction of various bacterial cells to the Gram stain. This stain distinguished two types of bacteria based on the composition of the cell wall: gram-positive and gram-negative. Research proceeded with the discovery that both lysozyme and penicillin induced cell lysis and with the realization that some of the bacterial cell wall constituents (diaminopimelic acid and N-acetylmuramic acid) were unique. These discoveries were exciting. They increased our understanding of prokaryotic cells and helped to obtain better chemotherapy with which to combat bacterial diseases. The mechanisms of peptidoglycan biosynthesis, cell division (covered in Chapter 5), osmotic lysis, and the activity of penicillin are important topics of discussion because they provide striking examples of the interrelationship of basic knowledge and practical applications of great significance.

Figure 2.24 provides an excellent summary of the differences in structure and appearance of gram-positive versus gram-negative cell walls. Point out the fundamental repeating structure of peptidoglycan (the glycan tetrapeptide; Figure 2.25), which consists of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues. Indicate that the latter of these two sugars is connected to a short peptide chain consisting of four amino acids, including in many bacteria the unique lysine analog diaminopimelic acid (DAP). The peptide chains provide structural rigidity to peptidoglycan by cross-linking the polysaccharide layers such that tensile strength is conferred on the cell wall in both the X and Y directions (Figure 2.26). Although there is some variation in the amino acid composition of these peptide cross-linkages, there is great unity within the bacteria regarding the presence of N-acetylmuramic acid, DAP (which may be replaced by lysine), and D-amino acids (D-alanine and D-glutamic acid) rather than the usual L stereoisomers found in proteins.

However, in contrast to gram-negative bacteria, some NAM residues in the peptidoglycan of gram-positive bacteria contain covalently bound teichoic acids, polyalcohols joined by phosphate esters (Figure 2.27). Teichoic acids contribute to the overall negative charge of the gram-positive cell surface and help to sequester cationic micronutrients (e.g., Ca2+ and Mg2+) from the environment.

At this time you may want to foreshadow the structure of the gram-positive bacterial cell wall in the context of antibiotic therapy and design. Emphasize that examples of unique cell chemistry often provide targets for successful chemotherapy without the problems of host toxicity (Figures 2.25–2.29). The mechanisms of action of both lysozyme and penicillin are good examples of how a chemical agent can destroy peptidoglycan, resulting in bacterial cell lysis.

Finally, note that there are also prokaryotic cells that lack a cell wall, including the mycoplasmas, a group of pathogenic bacteria (see Chapter 15), and species of the archaeal genera Thermoplasma and Ferroplasma (see Chapter 16). As previously noted, the mycoplasmas are unusual among bacteria in that they contain sterols in their membranes. The .

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structural rigidity provided by these molecules presumably helps to maintain cell integrity during mild osmotic stress.

2.11 | LPS: The Outer Membrane The outer membrane of gram-negative bacteria is obvious in TEM sections, where it is seen as a wavy lipid bilayer outside of a thin layer of peptidoglycan. In the outer membrane, lipopolysaccharide (LPS) (Figure 2.28) replaces most of the phospholipids in the outer leaflet, whereas lipoproteins in the inner leaflet function to anchor the outer membrane to peptidoglycan (Figure 2.29). Depending upon the chemistry background of your students, discuss the chemical components of the LPS: (1) Lipid A, the phosphoglycolipid portion of the LPS; (2) the core polysaccharide, consisting of ketodeoxyoctonate (KDO), heptoses (7-carbon sugars), hexoses, and N-acetylglucosamine; and (3) the O-polysaccharide, consisting of repeating sequences of hexoses that form long chains and may be branched (Figure 2.28).

Although the purpose of the outer membrane is structural, it is toxic to animals due to the lipid A component of the LPS. Toxins that are part of the cell wall of gram-negative bacteria are called endotoxins. Provide examples for your students of endotoxic human pathogens (e.g., Shigella, Salmonella, and Escherichia) that elicit ill effects in the host, most of which include gastrointestinal distress.

In contrast to the cytoplasmic membrane, the outer membrane is permeable to small molecules due to membrane channels called porins (Figure 2.29), which vary in specificity from nonspecific to highly specific. Students should be made aware that the periplasm of gram-negative bacteria contains binding proteins that are not present in gram-positive bacteria. The periplasm contains a number of different classes of enzymes, some of which facilitate transport (Section 2.9) or chemotaxis (Section 2.19).

2.12 | Archaeal Cell Walls Cell walls of Archaea do not contain peptidoglycan, but they do possess diverse chemistries that include proteins, polysaccharides, and glycoproteins. Some methanogens (Chapter 16) produce a polysaccharide similar to peptidoglycan called pseudomurein (Figure 2.30). Point out that the -1,3 glycosidic linkage in pseudomurein is different from the -1,4 linkage in peptidoglycan, thus making the former insensitive to the action of lysozyme. There are no known human pathogens from the Archaea, and thus the evolution of lysozyme most probably arose from the interactions of Bacteria with animal hosts over time.

Although not all Archaea contain pseudomurein, nearly all contain a cell wall of some type (exceptions were noted in Section 2.10). Extremely halophilic Archaea have sulfate (SO42–) incorporated into their cell walls to bind excessive Na+ and help shield the cell from its extremely salty environment. Other Archaea (and some Bacteria) have a paracrystalline surface layer, the S-layer, composed of protein or glycoprotein (Figure 2.31). Discuss the potential functions of S-layers, which are varied and may include protecting the cell from osmotic lysis; preventing the access of larger particles, such as viruses, to the cell membrane; and retaining secreted proteins near the cell surface. .

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2.13 | Cell Surface Structures Cell surface structures produced by bacteria that are not an integral part of the cell wall are generally not considered essential to cell survival. However, the presence of capsules and slime layers (Figure 2.32), fimbriae (Figure 2.33), and pili (Figure 2.34) on many prokaryotes suggests that such structures play important ecological roles for these organisms, including the establishment of pathogenic associations with host cells. The attachment of one cell to another is a specific molecular interaction between host and pathogen, and this contact often initiates changes in the host cell resulting in internalization of the pathogen and continuation of its life cycle. Although details of host–pathogen interaction are not part of the material presented here, you could pique student interest by showing a specific example of the role played by these cell surface structures in a specific pathogenesis (e.g., Streptococcus pneumoniae, Yersinia pestis, and Listeria monocytogenes). Clearly define the structural and functional differences of fimbriae, pili, and flagella to students, who may equate these structures based on their similar microscopic appearance.

2.14 | Cell Inclusions Many bacterial cells contain inclusions, such as the storage granules polyhydroxybutyrate (PHB; Figure 2.35) and glycogen, both of which serve as carbon and energy reserves. Additional nutrient inclusions include polyphosphate and elemental sulfur granules (Figure 2.36).

The latter of these inclusions serves as an important secondary energy source for a variety of phototrophic and chemolithotrophic bacteria that oxidize sulfide (H2S) as an electron donor (see Chapter 13).

Other storage inclusions are not necessarily for nutritional purposes. Many prokaryotes catalyze biomineralization, the process of mineral formation by microorganisms. Figure 2.37 shows a beautiful example of benstonite granule accumulation inside a cell of the cyanobacterium Gleomargarita; the function of these structures is unknown but it may be to provide ballast for the cell in its aqueous environment. Your discussion of magnetosomes and magnetotactic bacteria should be of interest to students, who will likely find the idea of “magnetic bacteria” intriguing. Although the function of magnetosomes is also unknown, they are most certainly important to species that form them (Figure 2.38). One hypothesis is that the magnetite in these inclusions acts like a compass, pulling the microaerophilic aquatic bacteria that contain them downward toward the Earth’s magnetic poles and into the sediments where dissolved O2 concentrations are lower. Unlike polyphosphate and elemental sulfur granules, both magnetosomes and PHA inclusions have a “nonunit” (single layer) phospholipid membrane.

2.15 | Gas Vesicles Gas vesicles are rigid, hollow structures in the cytoplasm of some cells that allow vertical migration in a water column. They are therefore considered a mechanism of motility (Figures 2.39–2.41). The proteinaceous shell is permeable to gases, but not to water and solutes. At the molecular level, the shell contains two proteins: GvpA, a rigid -sheet that makes up 97% of .

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the shell; and GvpC, a cross-linking protein made up of -helices (Figure 2.41). These structures are found mostly in aquatic phototrophs, allowing them to regulate their position in the water column where the light intensity required for photosynthesis is optimal. Some nonphototrophs, including some species of Archaea, also contain gas vesicles.

2.16 | Endospores Introduce your discussion of endospores by reminding students of Ferdinand Cohn’s discovery of the endospore-forming genus Bacillus and his research demonstrating the incredible heat resistance of these structures (Chapter 1). Because of Cohn’s efforts, important new methods of sterilization were developed that are still used by the food and medical industries.

To spark student interest, note that many endospore-forming bacteria are also pathogenic and cause some of the most serious diseases known. For example, pathogenic members of the genera Bacillus and Clostridium often produce potent toxins that cause fatal diseases if not treated within a short time. Examples include botulism (C. botulinum), tetanus (C. tetani), gas gangrene (C. perfringens), and anthrax (B. anthracis).

Depending on the level of your course, discuss the structure of endospores and the endosporulation and germination processes in some detail (Figures 2.42–2.47). Some key points to stress include the following: • Describe the unique nature of the core, stressing the functions of the dipicolinic acid (DPA) and Ca2+ complexes, the low water content and low pH, and the role of small acid-soluble spore proteins (SASPs) in protecting the DNA and in serving as a carbon and energy source for the cell during germination.

• Discuss endospore formation as an example of cellular differentiation in prokaryotes, using Bacillus subtilis as a model (Figure 2.47). To impress upon students the remarkable complexity of the differentiation process, mention that more than 200 genes are involved in sporulation, and many details of the process are still being investigated in laboratories around the world.

Finally, students should show interest in a discussion concerning how long endospores can remain viable. The debate on the longevity of these structures has now pushed their life span to millions of years. If experimental evidence from independent research laboratories repeatedly supports these claims, this would indeed be an extraordinary testament to the life-preserving design of these structures.

2.17 | Flagella and Swimming Motility The ability of prokaryotes to move via flagella is intimately connected to their ability to sense and respond to environmental signals through complex signal transduction pathways. Bacteria arrange flagella on their surfaces in a variety of ways (Figures 2.48–2.50), and even many archaea are flagellated (Figure 2.52). The flagellar structure is complex, and its synthesis and assembly involve more than 40 genes in E. coli (Figures 2.51 and 2.53). Rotation of bacterial flagella requires significant energy directly from a proton motive force (PMF; Section 2.8 and Chapter 3). In fact, a single rotation requires the translocation of about 1000 protons across .

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the membrane through the Mot complex (Figure 2.51b). Although bacterial flagella do not rotate at a constant speed, up to 300 revolutions per second are possible, resulting in extremely fast movement of about 60 cell lengths per second. When measured as the number of body lengths moved per second, a bacterium swimming at full speed would be moving nearly 2.5 times faster than a cheetah can run!

Flagella from the different domains of life exhibit significant structural and operational differences. Prokaryotic flagella rotate instead of moving in a whip-like motion (Figure 2.54), as is the case in eukaryotes. Several differences also exist between the flagella of different prokaryotes. Bacterial flagella are about twice as thick as archaeal flagella. In addition, the filament portion of all bacterial flagella is composed of a single type of flagellin protein, whereas the protein composition of archaeal flagellar filaments varies depending on the species. Perhaps the most significant difference between bacterial and archaeal flagella comes from recent evidence indicating that, like eukaryotes, archaeal flagella are powered directly by the hydrolysis of ATP rather than by a PMF, as in bacteria. As is the case for many of their cellular properties, it is interesting to note that Archaea exhibit flagellar characteristics similar to those of both Bacteria and Eukarya without being identical to either one. The fundamental differences between the flagella of the three domains of life suggest that these mechanisms have arisen independently as a result of convergent evolution rather than from a common origin.

2.18 | Gliding Motility Gliding motility (motility without flagella) in bacteria is a relatively underrepresented phenomenon in microbiology texts, and this is probably because of the lack of knowledge of the molecular mechanisms involved in the process. Consequently, it is a good puzzle to present to students following your discussion of flagellar locomotion, about which much is known.

Gliding motility has never been observed in Archaea, but several species of gliding Bacteria are known, including species of cyanobacteria and the genera Myxococcus, Cytophaga, and Flavobacterium (Figure 2.55). Movement by gliding requires a solid surface and is considerably slower than flagellar motility. Several mechanisms of gliding motility have been described. Some bacteria secrete a polysaccharide slime that adheres to a surface and pulls the cell forward. Others exhibit a “twitching motility” in which cell movements are carried out by the repeated extension and retraction of type IV pili. Cells of Flavobacterium johnsoniae appear to glide via the coordinated ratcheting of cytoplasmic membrane proteins with outer membrane proteins, where the latter move along a surface in the opposite direction of the cell itself, much like the movement of a tank on its tracks (Figure 2.56).

2.19 | Chemotaxis and Other Taxes The ability of bacteria to exhibit taxes (i.e., directed movement) confers a selective advantage depending upon environmental conditions. Students should understand that prokaryotes (unlike larger organisms) sense gradients in a temporal (an effect lasting for only a short time) rather than a spatial (a lingering effect) manner. In other words, they must continually compare their current external conditions with those of a few moments before. The studies of .

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chemotaxis in E. coli provided the first genetic model of the process in swimming bacteria.

Chemotaxis will be discussed in Chapter 7 in the context of two-component signal transduction systems, but use Figures 2.57 and 2.58 to show the run and tumble “directed” response and capillary assay system used to evaluate and identify signal molecules that act as attractants or repellants.

Many of the protein components that function in chemotactic pathways are also activated during phototaxis, and flagellar rotation is controlled accordingly. The response of Rhodospirillum centenum to light is a fascinating example of phototaxis. Mention to students that scotophobotaxis (movement away from dark) is not the same as true phototaxis, which involves movement up a light gradient (Figure 2.59). R. centenum is also unusual in that an entire colony of cells on solid media will move toward an infrared light source (the wavelengths absorbed by their photosynthetic pigments) and away from fluorescent light. If one observes the cells within the colony as the colony moves in one direction, the individual cells appear to be moving more or less randomly, suggesting there must be some intercellular communication occurring to generate a net directional movement.

Other taxes have been observed in microorganisms, including directed movement toward or away from oxygen (aerotaxis), toward a specific osmotic condition (osmotaxis), or toward water (hydrotaxis).

2.20–2.22 | Eukaryotic Microbial Cells Students should be familiar with the organelles and general structure of the eukaryotic cell from general biology courses, but you should still present a brief overview of the topic, focusing first on the nucleus and chromosome organization (Figures 2.60 and 2.61). Remind students that eukaryotic DNA is packaged in the nucleus by being wound around positively charged histone proteins. Note that transport of proteins and nucleic acids through nuclear pores requires the energy of GTP. The nucleolus is the site of ribosomal RNA synthesis and assembly of the large and small subunits of the ribosome. Also review mitosis (Figure 2.62) and meiosis with your students, reminding them that these processes, which occur only in eukaryotes, are mechanisms by which a cell divides to create two diploid daughter cells or four haploid gametes (or spores), respectively.

Review mitochondrial structure (Figure 2.63) and function with your students, and allow this to lead into a discussion of the hydrogenosome (Figure 2.64). The latter organelle is found in certain eukaryotes that lack mitochondria, the present-day representatives of which arose early after the divergence of Eukarya from Archaea (see Chapter 12). Unlike mitochondria, hydrogenosomes usually lack cristae and do not contain citric acid cycle enzymes.

Therefore, organisms possessing hydrogenosomes are strictly fermentative. Several are parasites, such as the sexually transmitted pathogen Trichomonas vaginalis, and all are either aerotolerant or obligate anaerobes. Figure 2.64 shows the pyruvate oxidation scheme carried out by the hydrogenosome for ATP synthesis.

Chloroplasts are the structures found in plant cells and many microbial eukaryotes (e.g., dinoflagellates, euglenids, diatoms, and various algae) that carry out photosynthesis and allow for photoautotrophic growth (Figure 2.65). The relationship of chloroplasts, mitochondria, and hydrogenosomes to Bacteria is well-documented and is discussed briefly here as the .

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endosymbiotic hypothesis. Although this topic will be addressed in more detail in future chapters (see Sections 12.3 and 17.1), you may wish to point out key features supporting endosymbiosis to your students at this time: • All three structures contain their own DNA in covalently closed circles. This DNA encodes rRNAs, tRNAs, and some respiratory enzymes.

• These organelles contain their own ribosomes that are bacterial in structure and are sensitive to antibiotics that affect bacterial ribosomes.

• The nuclear DNA of eukaryotic cells contains bacterially derived genes, lending additional evidence to support the endosymbiotic hypothesis (Section 17.1).

• 16S rRNA gene sequence analyses show the evolutionary relatedness of these structures to Bacteria.

Finally, review the function of other eukaryotic cell structures (e.g., the endoplasmic reticulum, Golgi complex, ribosomes, and cytoskeletal elements) mentioned in Section 2.22, which should already be familiar to most students (Figures 2.66–2.68). Point out that eukaryotic flagella are powered by ATP hydrolysis and propel the cell via a whiplike motion rather than by rotation, as seen in prokaryotic cells.

Answers to Review Questions 1. Magnification is the optical enlargement of an object, whereas resolution is the ability to distinguish two objects as distinct and separate, especially in reference to microscopic observation. Although magnification can be increased virtually without limit, increases in resolution are limited by the physical properties of light.

2.

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Bright-field microscopy

Dark-field microscopy

With the bright-field microscope, specimens are visualized because of the slight differences in contrast that exist between them and their surroundings, differences that arise because cells absorb or scatter light to varying degrees. The light source is focused on the specimen by the condenser.

Dyes can be used to stain cells and increase their contrast so that they can be more easily seen in the bright-field microscope.

Although staining is widely used in light microscopy, staining kills cells and can distort their features, unlike that in dark field microscopy where live cells can be viewed. Bright field microscopy is widely used for viewing

Light reaches the specimen from the sides only.

The only light that reaches the lens is that scattered by the specimen, and thus the specimen appears light on a dark background.

Dark-field microscopy is a good way to observe microbial motility, as bundles

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shape of bacterial cells.

Differential interference contrast microscopy employs a polarizer in the condenser to produce polarized light, which passes through a prism that generates two distinct beams.

These beams pass through the specimen and enter the objective lens where they are recombined into one. Because the two beams pass through substances that differ in refractive index, the combined beams are not totally in phase but instead interfere with each other, and this effect enhances subtle differences in cell structure. A confocal scanning laser microscope is a computer-controlled microscope that couples a laser to a fluorescent microscope. The laser generates a bright three-dimensional image and allows the viewer to access several planes of focus in the specimen. To do this, the laser beam is precisely adjusted such that only a particular layer within a specimen is in perfect focus at one time. By precisely illuminating only this single plane, the CSLM eliminates stray light from other focal planes.

3. The major advantage the electron microscope has over the light microscope is greatly increased resolution due to the use of an electron beam for imaging rather than a beam of light. The increased resolution allows for clear imaging at very high magnifications.

A scanning electron microscope would be used to view the three-dimensional features of a cell.

4. The morphology of a cell could be due to several selective forces. Some examples of these might include optimization for nutrient uptake (small cells and others with high surface-to-volume ratios, such as appendaged cells), swimming motility in viscous environments or near surfaces (helical- or spiral-shaped cells), gliding motility (filamentous bacteria), and so on.

5. Prokaryotic cell sizes typically range from about 0.4 µm in length to several hundred micrometers, with most cells averaging 1–3 µm. The maximum size of a prokaryotic cell appears to be in excess of 750 µm, and the smallest is likely to be about 0.2 µm. We are better able to know the lower limit of size of a bacterial cell than the upper limit because we know the minimum amount of volume required for genetic material, ribosomes, proteins, and so on necessary to carry out metabolism. An E. coli cell is 1–2 µm in length and 0.5–0.6 m wide.

6. Hopanoids are sterol-like molecules that strengthen the cytoplasmic membranes of some Bacteria. In a phospholipid membrane, the fatty acids point inward toward each other to form a hydrophobic environment, and the hydrophilic portions remain exposed to the external environment or the cytoplasm.

7. The lipids of Archaea contain ether bonds between glycerol and their hydrophobic side chains. Archaeal lipids thus lack fatty acids, although the hydrophobic side chains play the same functional role as fatty acids. Archaeal lipids are formed from multiple units of the 5-carbon hydrocarbon isoprene. The cytoplasmic membrane of Archaea is formed from either glycerol diethers, which have 20-carbon side chains (the 20-C unit is called a phytanyl group composed of 5 isoprene units), or diglycerol tetraethers, which have 40carbon side chains. In the tetraether lipid, the ends of the phytanyl side chains that point inward from each glycerol molecule are covalently linked. This forms a lipid monolayer .

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instead of a lipid bilayer membrane. In contrast to lipid bilayers, lipid monolayer membranes are extremely resistant to heat and are therefore widely distributed among hyperthermophilic Archaea, organisms that grow best at temperatures above 80°C. Many archaeal lipids contain rings within the hydrocarbon side chains. For example, crenarchaeol, a lipid widespread among species of Thaumarchaeota, contains four 5-carbon (cyclopentyl) rings and one 6-carbon (cyclohexyl) ring. Rings in the hydrocarbon side chains affect the chemical properties of the lipids and thus overall membrane function.

Sugars can also be present in archaeal lipids. For example, the predominant membrane lipids of many Euryarchaeota are glycerol diether glycolipids.

8. Ionized molecules, which have a net charge, are hydrophilic and are therefore repelled by the hydrophobic interior of the membrane. Such molecules are brought into the cell through membrane-spanning transport proteins.

9. The LacY permease system is a symporter system that takes up one molecule of lactose along with one proton, thereby using the energy of the proton motive force to drive uptake of lactose. The phosphotransferase system is a group translocation system in which a series of proteins are alternately phosphorylated and dephosphorylated in a cascading fashion. In the end, the sugar itself (in this case, glucose) is phosphorylated upon being transported into the cell through a membrane-spanning transport protein. The energy to drive the uptake of glucose originates from the high-energy anhydride bond in phosphoenolpyruvate. The ABC transport system uses three components to transport a substance (in this case, maltose) across a membrane: a periplasmic binding protein, a membranespanning transporter, and an ATP-hydrolyzing protein, the latter of which uses the energy released from the hydrolysis of ATP to drive the transport event.

10. Because the rigid layer is a polymer composed of sugars linked by -1,4 glycosidic bonds and polypeptides that cross-link N-acetylmuramic acid residues, the term peptidoglycan (“protein” + “sugar”) is appropriate. Together, the covalent glycosidic and peptide bonds of peptidoglycan provide tensile strength in both the X and Y directions, respectively.

11. The polysaccharide portion of LPS consists of two components, the core polysaccharide and the O-specific polysaccharide. The lipid portion of the LPS, called lipid A, is not a typical glycerol lipid, but instead the fatty acids are connected through the amine groups from a disaccharide composed of glucosamine phosphate. The disaccharide is attached to the core polysaccharide through KDO. LPS replaces much of the phospholipid in the outer half of the outer membrane and functions as an anchor tying the outer membrane to peptidoglycan. Thus, although the outer membrane is technically a lipid bilayer, its structure is distinct from that of the cytoplasmic membrane.

The structural differences between the cell walls of gram-positive and gramnegative Bacteria are responsible for differences in the Gram stain reaction. Recall that in the Gram stain, an insoluble crystal violet–iodine complex forms inside the cell. This complex is extracted by alcohol from gram-negative but not from gram-positive bacteria.

As we have seen, gram-positive bacteria have very thick cell walls consisting primarily of peptidoglycan. During Gram staining, the gram-positive cell wall is dehydrated by the alcohol, causing the pores in the walls to close and preventing the insoluble crystal violet–iodine complex from escaping. By contrast, in gram-negative bacteria, alcohol readily .

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penetrates the lipid-rich outer membrane and extracts the crystal violet–iodine complex from the cell.

12. Peptidoglycan is a polysaccharide present in Bacteria, but absent in Archaea. S-layers consist of protein or glycoprotein in a paracrystalline array. They are found in both Bacteria and Archaea and may serve as an outermost selective sieve around the cell to prevent access of large particles, such as viruses, to the cytoplasmic membrane. Methanogenic Archaea have cell walls composed of pseudomurein, which is similar in structure to peptidoglycan but contains lysozyme-insensitive -1,3 glycosidic bonds.

Extremely halophilic Archaea have polysaccharide cell walls containing large amounts of sulfate, which buffers the cell from its very salty environment by binding Na+.

13. Polysaccharide layers produced by bacteria are referred to as capsules and/or slime layers, and they have several potential functions. They play a major role in the attachment of cells to a surface (a property that allows for biofilm formation), they prevent phagocytosis by immune system macrophages, and they may function to prevent desiccation.

14. Cytoplasmic inclusions include carbon polymers (e.g., poly--hydroxybutyrate and glycogen), inorganic phosphate (polyphosphate), elemental sulfur, and magnetite. Poly-hydroxybutyrate granules serve as a cell energy reserve and consist of a polymeric fatty acid, whereas magnetosomes consist of magnetite (Fe3O4) and serve as small magnetic compasses. Aquatic cells may use magnetosomes as a means to align themselves within zones of favorable oxygen concentration.

15. Because of their buoyancy, gas vesicles provide a means of motility for aquatic cells.

These structures are water impermeable because their membranes consist of highly hydrophobic, rigid, cross-linked proteins. Gas-filled vesicles decrease the density of the cell. Therefore, the number of inflated vesicles in a cell determines its buoyancy and position in the water column.

16. The endospore is a different type of cell than the vegetative cell for several reasons. The cytoplasm of endospores is considerably dehydrated compared to vegetative cells, and endospores have a unique cell surface consisting of multiple layers of proteins. In addition, endospores are “shut down” in the sense that they are essentially metabolically dormant. All of these factors permit significant resistance to heat, radiation, desiccation, and extremes of pH, and they allow for remarkable longevity of these structures.

17. Mature endospores are a form of metabolically inactive, differentiated cells produced by some species of Bacteria in which a marked degree of dehydration has occurred, and the surface has changed to a tough, multilayered protein composition. While endospores represent a dormant stage of an endospore-forming bacterium, vegetative cells are metabolically active cells capable of growth, reproduction, and other activities characteristic of living systems. Germination is the process by which a free, mature endospore is converted back into a vegetative cell upon encountering favorable environmental conditions.

18. The bacterial flagellum is a long, rigid, protein filament of polymerized flagellin that extends from the cytoplasmic membrane and cell wall. It generates cellular motility by rapidly rotating, much like a miniature propeller. A wider hook at the base of the flagellum serves to connect the flagellum to the motor portion (basal body), which is anchored to the cell wall and cytoplasmic membrane. In Bacteria, the proton motive .

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force provides the energy for rotation via the flow of H+ across the membrane. Archaeal flagella are thinner by comparison and may be composed of a diversity of flagellin-type proteins, depending on the species.

19. The gliding motility exhibited by species of Flavobacterium differs from the flagellar motility of E. coli in several respects. Gliding motility, which requires a surface for the cells to move across, is considerably slower than the swimming motility of flagellated cells. The mechanism of gliding in Flavobacterium appears to be by a ratcheting movement between proteins anchored in the cytoplasmic membrane and the outer membrane.

However, gliding motility in species of Flavobacterium and swimming motility in E. coli are both powered by energy from the proton motive force.

20. Chemotaxis is the directed movement of an organism toward (positive chemotaxis) or away from (negative chemotaxis) a chemical gradient (e.g., E. coli). Phototaxis refers to the movement of an organism toward light (e.g., Rhodospirillum centenum).

21. In the experiment shown in Figure 2.58, the control is a capillary that contains neither an attractant nor a repellant. The control is essential because it provides an unmanipulated point of comparison and, therefore, serves to validate the results obtained when a variable (i.e., the attractant or repellant) is introduced to the capillary.

22. Three features that clearly differentiate eukaryotic from prokaryotic cells include the following: (1) Almost all eukaryotes possess a membrane-bound nucleus containing their genomic DNA (although, interestingly, there are a few nucleated prokaryotes among the Planctomycetes, described in Chapter 15); (2) unlike nearly all prokaryotes, eukaryotic cells contain various membrane-bound organelles (e.g., mitochondria and the Golgi complex); and (3) because prokaryotes have no nucleus, they carry out coupled transcription and translation; these processes are separate in eukaryotes because of the presence of a nuclear membrane. Histones are positively charged protein complexes that bind and compact DNA in the nucleus of eukaryotic cells.

23. The hydrogenosome and mitochondrion are about the same size, and they both function to catabolize pyruvate. In mitochondria, pyruvate is oxidized completely using enzymes of the citric acid cycle. CO2 and H2O are the products of this aerobic respiration, and ATP is synthesized by oxidative phosphorylation using a proton motive force established by the mitochondrial electron transport chain. The hydrogenosome is found only in certain anaerobic eukaryotes. Because it lacks citric acid cycle enzymes and cristae containing electron transport chain components, pyruvate is fermented to acetate, CO2, and H2, and ATP is synthesized via substrate-level phosphorylation.

24. Anaerobic eukaryotes that lack mitochondria have hydrogenosomes and carry out a strictly fermentative metabolism. Although similar in size to mitochondria, hydrogenosomes lack citric acid cycle enzymes and also lack cristae. The major biochemical reaction in the hydrogenosome is the oxidation of the compound pyruvate to H2, CO2, and acetate.

On the basis of their relative autonomy, size, and morphological resemblance to bacteria, it was hypothesized that mitochondria and chloroplasts were descendants of respiratory and photosynthetic bacterial cells, respectively. By associating with nonphototrophic eukaryal hosts, the latter gained a new form of energy metabolism while the .

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symbiotic bacterial cells received a stable and supportive growth environment inside the host. Gradually, over time these originally free-living symbionts became an intimate part of the eukaryotic cell. This idea of symbiotic bacteria as the ancestors of the mitochondrion, hydrogenosome, and chloroplast is called the endosymbiotic hypothesis of eukaryotic cell origin.

25. Certain eukaryotic organelles, specifically mitochondria, chloroplasts, and hydrogenosomes, contain circular DNA that is more similar to bacterial DNA than eukaryotic DNA in sequence composition. In addition, these organelles contain their own ribosomes that resemble those of bacteria and, therefore, are sensitive to many of the same antibiotics that target bacterial ribosomes.

26. The eukaryotic cell’s cytoskeletal structure consists of microtubules, microfilaments, and intermediate filaments. Microtubules are tubes about 25 nm in diameter containing a hollow core and are composed of the proteins α-tubulin and β-tubulin. Microfilaments are about 7 nm in diameter, and are polymers of two intertwined strands of the protein actin.

Intermediate filaments are fibrous keratin proteins that form into fibers 8–12 nm in diameter.

Answers to Application Questions 1. The diameter of the smallest object resolvable by any lens in a light microscope is equal to 0.5/numerical aperture (NA). Using a 600-nm wavelength and an NA of 1.32, this would be 227 nm, or ~0.23 µm. The resolution could be improved using this same lens by using a shorter wavelength in the blue light range.

2. The surface/volume ratio for a cell having a diameter of 15 µm is 0.4. The surface/volume ratio for a cell with a diameter of 2 µm is 1.5. As the diameter of the cell decreases, there is more surface area per unit volume. Thus, with a higher surface-tovolume ratio, there is an increased capacity for the cell to interact with its environment per unit of cytoplasm. This generally leads to higher levels of metabolic activity in the cell and, therefore, a faster growth rate.

3. Greigite is a magnetic mineral that imparts a magnetic dipole on a cell, allowing it to orient itself in a magnetic field. It has thus been hypothesized that one function of magnetosomes may be to guide these primarily aquatic cells downward (the direction of Earth’s magnetic field) toward the sediments where O2 levels are lower, especially in anoxic zones, unlike that of E.coli.

Hence, one can easily purify the magnetosomes by using a simple magnet placed below the undisturbed cultures for some period of time. Subsequently, the magnetotactic greigite forming bacteria will exhibit magnetotaxis and settle by the magnet, while the E. coli cells would remain suspended in culture solution.

4. Sixty cell lengths per second for an E. coli cell that is 2 m long equals a speed of 120 µm/sec. A 3-cm capillary tube = 30,000 µm, so at this speed it would take 250 seconds, or nearly 4.2 minutes, for the cell to travel the length of the tube.

5. Unknown cultures can be differentiated in terms of Gram reaction using the following methods: .

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(a) Light microscope: Observe the Gram reaction of the cells following Gram staining of the cultures. (b) Electron microscope: Observe the appearance of the cell wall in thin-section micrographs. Gram-positive cells will have a thick layer of peptidoglycan with no outer membrane, whereas gram-negative cells will have a thin layer of peptidoglycan within a periplasm and a wavy outer membrane containing the LPS layer. (c) Chemical analyses of cell walls: Unlike gram-negative cells, gram-positive cells contain teichoic acids in their cell walls but lack an outer membrane containing the LPS. The presence of the endotoxin lipid A (a component of the LPS) would confirm a gram-negative culture. (d) Phylogenetic analyses: The Gram reaction of an unknown culture can be inferred based on the relationship of the organism to its closest phylogenetic relatives (based on 16S rRNA gene sequence analysis). For example, an organism that is closely related to species of Clostridium would almost certainly be gram-positive.

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CHAPTER

3

Microbial Metabolism

Summary Chapter 3 lays the foundation for introducing students to major concepts of culturing microorganisms and microbial metabolism. The chapter focuses initially on chemoorganotrophs, organisms that use organic compounds as sources of both carbon and energy. The eventual outcomes of the enzymatically controlled manipulation of molecules via either catabolic (energy-yielding) or anabolic (energy-consuming) chemical reactions are growth and reproduction.

3.1 | Cell Chemistry and Nutrition Discuss microbial nutrition in detail. Stress the fact that although just a handful of chemical elements predominate in living systems (Figure 3.1), micronutrients (trace elements) and vitamins are also essential for life (Table 3.1). Microorganisms differ in what nutrients are required (and in what chemical form) to carry out their metabolism for growth and reproduction. Obviously, all cells require carbon, which makes up about 50% of a cell’s dry weight (Figure 3.1). Explain that the next most abundant elements are oxygen and nitrogen (17% and 13% of dry weight, respectively), and then ask your students why these elements are important to cells. The answer, of course, lies in the fact that they are key components of macromolecules, especially polysaccharides, proteins, and nucleic acids. Discuss the roles of other macronutrients (P, S, K, Mg, Ca, Na) in the cell.

Referring to Table 3.1, discuss the critical roles that micronutrients play in cells, usually as components of enzymes and vitamin cofactors. Spend some time discussing the importance of iron in bacterial metabolism (e.g., its incorporation into cytochromes and iron-sulfur proteins for use in electron transport chains). The iron available to most bacteria under oxic conditions is in the form of insoluble ferric iron (Fe3+) minerals, and thus bacteria produce complex iron-binding molecules called siderophores that have a high affinity to bind iron for uptake into the cell. Humans also require iron and produce iron-binding complexes called transferrins and lactoferrins, as will be discussed later. However, the siderophores of many bacterial pathogens have much higher iron-binding affinities than the human counterpart and, thus, can “steal” iron from the host during infection.

• Note that some bacteria do not require iron and substitute Mn2+ as a cofactor in some of their enzymes (e.g., Lactobacillus plantarum and Borrelia burgdorferi, the agent of Lyme disease).

• The iron-poor marine environment also requires certain bacteria to express siderophores for survival.

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In addition to trace metals, growth factors are another important group of micronutrients required by cells in small amounts. These are small organic molecules that include vitamins, which are the most commonly required growth factors, amino acids, purines, and pyrimidines.

3.2 | Media and Laboratory Culture Laboratory culture of microorganisms is the first step toward understanding physiological similarities and differences between various isolated strains and, ultimately, their ecological activities in nature. Moreover, students are able to understand how defined and complex media differ and how to grow microorganisms. Use Table 3.2 to contrast complex and defined culture media. As posed in the text, ask the students, “Which organism, Escherichia coli or Leuconostoc mesenteroides, has a greater biosynthetic capacity?” Many students will probably choose L. mesenteroides based upon the number of different chemicals contained in its growth medium. This initial, and somewhat convoluted, perception mainly results from their lack of exposure to biochemistry. You may want to show them a poster of metabolic pathways and discuss the fact that E. coli can synthesize all of its organic macromolecules by using just glucose as the sole carbon and energy source, along with a few inorganic salts and trace elements. By comparison, L. mesenteroides lacks many of the enzymes (and therefore genes) required for the biosynthesis of diverse organic molecules solely from monosaccharides. Its growth also depends on the availability of amino acids, nucleotides, and other growth factors in its environment. Therefore, E. coli, having the greater metabolic potential, is the more nutritionally versatile organism.

Because most introductory microbiology courses include a laboratory, Section 3.2 will serve to introduce methods that will be discussed and used in the laboratory. You may wish to reserve the details of culture media, pure culture techniques (Figure 3.2), and aseptic technique (Figures 3.3 and 3.4) for the laboratory, where a full appreciation for the fundamental methods of microbiology may be obtained.

3.3 | Energy Classes of Microorganisms This section introduces microbial diversity within the context of the two main metabolic options for energy conservation available to cells: chemotrophy and phototrophy (covered in detail in Chapter 13). Chemotrophy can be further broken down to chemoorganotrophy and chemolithotrophy, depending upon whether an organism uses organic or inorganic substrates as energy sources (electron donors), respectively (Figure 3.5). Students without a strong background in chemistry may have difficulty with the concept of chemical oxidation and reduction, which is key to understanding metabolism and metabolic diversity. Simply put, oxidation involves the removal of an electron (or electrons) from a substance, and reduction involves the addition of an electron (or electrons) to a substance. These processes are coupled during oxidation-reduction reactions. The transfer of electrons eventually results in the production of the high-energy compound ATP, either directly via substrate-level phosphorylation or indirectly via oxidative phosphorylation, as will be discussed in detail later in this chapter. Classifications of microorganisms based upon how they obtain energy are outlined below:

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Copyright © 2015 Pearson Education, Inc.

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• Chemoorganotrophs obtain energy from oxidizing (removing electrons from) organic compounds to synthesize ATP. To impress upon students the tremendous diversity of microorganisms, mention that for almost any organic compound present on Earth, there is a microorganism that can catabolize it for energy. These oxidations can occur either aerobically (in the presence of O2) or anaerobically (in the absence of O2). These organisms represent the majority of microorganisms isolated to date.

• Chemolithotrophs obtain energy from oxidizing inorganic compounds. Inform students that chemolithotrophic metabolisms have only been observed in the prokaryotic domains of life (Bacteria and Archaea). Because these organisms often use the inorganic waste products produced by chemoorganotrophs (e.g., H2 and H2S), they do not compete for metabolic resources within a given environment.

• Phototrophs obtain energy by using light to generate ATP. Students will likely associate photosynthesis with plants rather than bacteria, and in doing so they may recall that this process releases O2 as a by-product, a process referred to as oxygenic photosynthesis.

However, it is estimated that anoxygenic photosynthesis, which does not produce O2, predates the appearance of oxygenic photosynthesis by nearly one billion years (Figure 1.4).

The onset of oxygenic photosynthesis was apparently initiated by the convergence of two distinct photosystems within a single cell (as first occurred in cyanobacteria), with each photosystem derived from a different group of anoxygenic phototrophs. See Chapter 13 for further information distinguishing oxygenic and anoxygenic photosynthesis.

• Organisms are also classified according to their source of cell carbon for biosynthesis: Heterotrophs require various organic compounds as carbon sources, while autotrophs are capable of using CO2 as their sole source of carbon for cellular activities.

3.4 | Bioenergetics Use Table 3.3 to discuss how the free energies of formation (Gf0) of the reactants and products in a given reaction are used to calculate the free energy yield of the reaction. In discussing free-energy changes (∆G) it is important to note that values calculated or given are for standard conditions (∆G0′ = 1 molar, pH 7, 25°C, 1 atm), where substance concentrations are markedly different from those found in a cell. Although the difference between ∆G for standard and practical conditions in nature is rather small, the rapid removal of a product of a reaction may have a profound effect on its free energy. For example, a reaction that is expected to be endergonic may actually become exergonic following immediate removal of one of the end products. Such is the case during butyrate fermentation by Syntrophomonas (see Chapter 13). This organism oxidizes butyrate to acetate plus H2, which is normally highly endergonic (∆G0′ = +48.2 kJ). However, in the presence of a second organism that scavenges H2 (for use as an electron donor) as quickly as it is formed, the original reaction becomes favorable, and growth of Syntrophomonas by butyrate fermentation becomes possible.

Though abstract concepts, such as free energy, may be difficult for students to grasp, a thorough understanding of the material presented in Appendix 1 is essential for those who desire to know rather than memorize. However, it is important for them to understand the variables used to calculate free energies of reactions and that a given reaction can be either endergonic or exergonic, depending upon environmental conditions. The metabolic activities of microorganisms change the concentrations of substrates and products of a reaction, thus .

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incrementally changing the free-energy value of identical subsequent reactions (see Appendix 1, Definition 9).

3.5 | Catalysis and Enzymes The discussion of enzymes and catalysis will be much more meaningful for students if you have already spent time describing protein structure. The three-dimensional structure of a given protein determines the conformation of its active site and its specificity for formation of a transient enzyme–substrate complex. A combination of weak molecular interactions (hydrogen bonds, van der Waals forces, and hydrophobic interactions) solidifies the binding of one or more substrates to the enzyme. Explain to students that enzymes catalyze reactions by lowering the amount of activation energy required to initiate a chemical reaction (Figure 3.6). In addition, students should understand that many enzymes are associated with small nonprotein molecules required for their proper function. These constituents are classified as either prosthetic groups, which are usually covalently attached (e.g, heme groups), or coenzymes, which are loosely attached (e.g., NAD+/NADH).

Students should visualize enzymatic interactions as dynamic—not static—events, as depicted in Figure 3.7. They should understand that the binding of substrate is a reversible phenomenon. Rather than merely “sitting” in the active site until they are converted to product(s), substrates should be visualized as occupying the site for brief moments. Reactions normally proceed in a unidirectional manner when a sufficient number of substrate molecules are present, in which case the active site would be occupied most of the time, and the reaction would proceed as described in the text. This section provides an elementary introduction to enzyme kinetics that can be expanded if desired, depending on the biochemical background of the students.

3.6 | Electron Donors and Acceptors The concept of oxidation–reduction was introduced in Section 3.3 and should be covered in detail here. This material is essential for understanding metabolic pathways and energy generation strategies and for appreciating physiological diversity (Chapter 13), microbial ecology (Chapter 14), and microbial diversity (Chapters 15–17). Emphasize the following key points: • For every oxidation (removal of an electron) reaction, there must be a subsequent reduction (addition of an electron) reaction. Thus, every redox reaction has an electron donor and an electron acceptor. Reduction potentials are expressed for half reactions written as reductions (Figure 3.8).

• Redox couples (e.g., 1/2 O2/H2O and CO2/C6H12O6) are arranged in the redox tower (Figure 3.9) from the strongest reductants (most negative E0′) at the top to the strongest oxidants (most positive E0′) at the bottom. The farther the “drop” of an electron between a donor and an acceptor on the tower, the more energy is released.

• You may choose to spend a few minutes letting the students work in groups to calculate the ∆G for these reactions: (1) oxidation of succinate to fumarate using NO3– as electron acceptor; and (2) oxidation of glucose to CO2 using O2 as electron acceptor (something they are experiencing at this moment!). This activity may serve to reduce the anxiety that .

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some students might feel regarding this important topic. The second of these example reactions is covered in detail later in this chapter (Sections 3.8–3.12), so students will begin to comprehend oxidation–reduction from a biological perspective as opposed to the examples presented in an introductory inorganic chemistry course.

Although it is not typically necessary for students to memorize chemical structures in general microbiology courses, discuss the structures of the reduced and oxidized forms of the electron carrier nicotinamide-adenine dinucleotide (NAD+; Figure 3.10). Focus student attention on the site of oxidation and reduction (the nicotinamide ring moiety), noting that NAD+ reduction requires one proton and two electrons. The redox potential of the NAD+/NADH couple is –0.32 V, placing it high on the electron tower (Figure 4.9) and making NADH a good electron donor. NAD+ can accept electrons from a wide array of electron donors, and thus the cycling of NAD+/NADH (Figure 3.11) increases the diversity of redox reactions in a cell. Cells exploit the storage and transfer of electrons by NAD+ and other electron carriers to energize the cell membrane for production of ATP, as will be seen in Sections 3.7–3.12.

3.7 | Energy-Rich Compounds Cells use a variety of compounds to conserve the energy released from catabolic reactions, including ATP and phosphoenolpyruvate (Figure 3.12). These compounds contain highenergy phosphoanhydride bonds that can be hydrolyzed to release sufficient free energy to drive endergonic chemical reactions. Other key high-energy compounds include coenzyme A derivatives, which contain high-energy thioester bonds that can be hydrolyzed to release sufficient energy to drive the synthesis of ATP from ADP and Pi. ATP (–32 kJ/mol) is the most important energy-storing compound in cells in terms of short-term storage and dynamic turnover. However, as discussed in Chapter 2, cells also synthesize several long-term storage molecules, including glycogen, elemental sulfur, and poly-β-hydroxyalkanoates (PHAs; see Section 2.14).

3.8 | Glycolysis Approach energy conservation options available to chemotrophs (i.e., fermentation and respiration) from a redox reaction perspective. Describe the differences in terminal electron acceptors employed in each process and their respective mechanisms of ATP synthesis (Figure 3.13): • Fermentations occur in the absence of usable terminal electron acceptors, and ATP is generated via substrate-level phosphorylation.

• Respirations use either molecular oxygen (O2) or an alternative terminal electron acceptor (e.g., nitrate, sulfate, or ferric iron), and ATP synthesis occurs via oxidative phosphorylation (requiring the proton motive force).

Many students enrolled in their first course in microbiology will state that they already understand glycolysis and the citric acid cycle. However, the reality will probably be that most of them will have merely memorized the names of steps in the pathway without a conceptual understanding of the chemical strategy involved. Glycolysis (i.e., the Embden-MeyerhofParnas pathway) is the most common pathway employed for the fermentation of glucose .

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(Section 3.9 describes other common fermentations). The reactions of glycolysis occur in three clearly defined stages: (1) preparatory phosphorylation reactions, where 2 molecules of ATP are consumed; (2) initial redox reactions, where 4 molecules of ATP and 2 molecules each of NADH and pyruvate are produced; and (3) final redox reactions, where 2 molecules of NADH are consumed (oxidized back to NAD+), and pyruvate is reduced to one or more fermentation products (Figure 3.14).

Ask students why glycolysis requires that pyruvate be reduced to some fermentation product. The answer relates to the economics of supply and demand and the need for the cell to maintain its redox balance.

• Point out to students that NAD+ is reduced to NADH during the first step of stage II of glycolysis. The cell maintains only relatively small amounts of this molecule, which exists in either the oxidized (NAD+) or reduced (NADH) state. The limited supply of this important electron carrier requires the cell to “regenerate” NAD+ by reducing pyruvate to some other product using NADH as the electron donor. Without this step, glycolysis would cease due to the lack of NAD+ required for oxidation of glyceraldehyde-3-P to 1,3 bisphosphoglycerate (Figure 3.14).

• The reduction of pyruvate results in a variety of fermentation products, depending upon the organism (see next section and Chapter 13). Regardless of the type of fermentation carried out by a particular species, the purpose of making fermentation products is to regenerate the NAD+ required for the continuation of glycolysis.

• Note the energy balance for the fermentation of glucose: The cell has a net gain of 2 ATP molecules.

3.9 | Fermentative Diversity and the Respiratory Option The number of different fermentation strategies used by microorganisms is impressive, but remind students that fermentations have two things in common: (1) With rare exceptions, ATP synthesis occurs via substrate-level phosphorylation and (2) maintenance of redox balance by the production of one or more fermentation products that are generated from the original substrate (Table 3.4). Point out the number of energy-rich compounds available for substrate-level phosphorylation. Use Table 3.4 to show students common fermentations, pointing out that all organisms included in the table use glycolysis for the catabolism of glucose, with the only difference being the mechanism of pyruvate reduction employed.

Fermentations that employ glycolysis are generally classified based upon the products they form (e.g., acetogenic, mixed acid and butyric acid). Some fermentations are essential for the production of certain foods, such as the lactic acid and propionic acid fermentations important in the dairy industry and the alcoholic fermentation carried out by the yeast Saccharomyces cerevisiae (Figure 3.15). Note that baker’s yeast is metabolically versatile, being capable of energy conservation through either respiration (if oxygen is available) or fermentation (if oxygen is absent).

As discussed in Section 3.10, the fermentation of glucose does not yield as much energy as glucose respiration. However, fermentations that produce one or more fatty acids (see Table 3.4) yield more ATP than fermentations that do not produce fatty acids. This is because the fatty acid products are derived from energy-rich coenzyme-A precursors, such as acetyl-CoA (Figure 3.12). Decide how much detail to include with this topic depending upon the level or focus of the course (or the biochemistry background of the students). .

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3.10 | Respiration: Electron Carriers Continue to stress the idea that living systems require mechanisms for maintaining a redox balance that allows cells to conserve energy required for incorporating carbon and other nutrients into cellular biomass. The conservation of energy by respiratory mechanisms requires the participation of cytoplasmic electron carriers, such as NADH, and membraneassociated electron carriers, such as those that comprise electron transport chains (Figures 3.16–3.19). The organization of membrane-associated electron carriers into complexes that progress in an order of increasingly more positive E 0′ allows for controlled and efficient movement of electrons through successive carriers to the terminal electron acceptor (O2 during aerobic respiration; Figure 3.20). The movement of electrons through electron transport systems is coupled to the extrusion of protons outside the cytoplasmic membrane as electrons are donated to electron-only carriers. Summarize the functions of membrane-associated electron carriers in electron transport chains: • NADH dehydrogenases accept hydrogen atoms (electrons plus protons) from NADH and pass them to flavoproteins.

• Flavoproteins accept hydrogen atoms and donate electrons. The two H+ are extruded to the exterior face of the cytoplasmic membrane and contribute to the PMF. • Nonheme iron-sulfur proteins accept electrons only and can have a variety of reduction potentials based on the number and arrangement of Fe and S atoms present in the protein.

• Quinones are small, nonprotein, hydrophobic molecules that are electron-plus-proton carriers. Like flavoproteins, they transfer electrons only to the next carrier in the chain, whereas the protons are deposited to the outside of the cytoplasmic membrane.

• Cytochromes are electron-only carriers. Several types are known, and each donates electrons only to an acceptor with a more positive reduction potential.

3.11 | Respiration: The Proton Motive Force Figure 3.20 is a good representation of an electron transport system from an aerobic-respiring bacterium showing the stepwise movement of electrons through carriers with increasingly more positive reduction potentials. Remind students that this “fall” of electrons to increasingly electropositive carriers occurs via a series of oxidation–reduction reactions catalyzed by protein complexes arranged in the membrane. Most electron transport complexes have access to the inside and the outside of the membrane. Point out to students that the electron transport system in Figure 3.20 is just one example of a transport carrier arrangement found in microorganisms. Some bacterial species may express different membrane carrier complexes, depending upon the nutrient and environmental status of the cell (e.g., anoxic versus oxic conditions). However, students should be aware that all electron transport chains have the following points in common: • Membrane carrier complexes are arranged in an order of increasingly positive E0′.

• Complexes alternate between those that carry electrons only and those that carry electrons plus protons.

• Each system generates a PMF, which is characterized by a pH gradient and an electrochemical potential across the membrane, during electron transport. The PMF will be used to synthesize ATP via oxidative phosphorylation. .

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Now that the cell becomes “charged” like a battery during electron transfers (as in Figure 3.13), and the potential energy created can be converted into kinetic energy to do work. Stress these thermodynamic principles to students; they are key to understanding why a reduced carbon source, such as glucose, is considered a high-energy molecule. Students often do not make the connection that a molecule becomes highly reduced via energy input, and thus the chemical bonds within the molecule are high in potential energy. One could get a sense of the potential energy contained in glucose by igniting it and feeling the tremendous amount of heat released as it is converted to mostly CO2 and H2O. This, of course, converts all the potential energy to heat, and no useful work is done. The cell, however, generates a “controlled combustion” of glucose, gradually extracting electrons for electron transport and generation of a PMF for ATP synthesis.

In reference to ATP synthesis, discuss with students the structure and mechanism of action of the F1/Fo ATP synthase complex (Figure 3.21). Reinforce for the students that this protein complex is actually a tiny molecular motor that converts the potential energy of the PMF to chemical energy stored in ATP. This process is carried out by the movement of protons into the cytoplasm through Fo, ultimately causing a conformational change and the binding of ADP + Pi to the β-subunits of F1. The restoration of the original conformation of the β-subunits catalyzes the synthesis of ATP. Point out that the ATP synthase complex is reversible and can be used to create a PMF at the expense of ATP, in which case the enzyme is referred to as ATPase. This feature is important because other cellular functions depend on the energy of the PMF, such as flagellar rotation and some forms of nutrient transport.

3.12 | Respiration: Citric Acid and Glyoxylate Cycles Continuing from the initial stages in the oxidation of glucose discussed earlier (glycolysis), explain the function and general steps of the citric acid cycle (also called the Krebs cycle and tricarboxylic acid cycle), focusing on the storage of electrons as NADH or FADH2 (Figure 3.22). Remind students that complexes in the electron transport chain oxidize both of these compounds to establish the PMF (Figure 3.20). The final products from the oxidation of glucose (CO2 and H2O) are very low in potential energy (positive reduction potential = poor electron donors). Contrast the overall energy yield for aerobic respiration in bacteria (38 ATP/glucose) with that of alcoholic or lactic acid fermentation (2 ATP/glucose). Point out that the complete oxidation of glucose to six molecules of CO2 instead of to reduced fermentation products, which still contain significant potential energy, accounts for this drastic difference.

You may choose to limit your discussion of the citric acid cycle to its role in energy conservation in introductory microbiology courses, particularly those designed for students of allied health professions. However, for general microbiology courses designed for science majors, a discussion of the glyoxylate cycle is warranted because it requires of the students an understanding of the second role of the citric acid cycle—biosynthesis. The citric acid cycle intermediates α-ketoglutarate, succinyl-CoA, and oxaloacetate are regularly siphoned off for biosynthesis of molecules necessary for cell growth, such as amino acids. When larger C4 or C6 compounds are used for growth, this poses no problem, but when a two-carbon compound, such as acetate, is used as a growth substrate, pools of oxaloacetate would eventually dwindle and stall the cycle because of a net carbon deficit. Discuss the major role played by the glyoxylate cycle in regenerating oxaloacetate when a two-carbon compound, such as acetate, is used as carbon source. Be sure to explain the roles of the two key enzymes of the cycle, .

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isocitrate lyase and malate synthase, in generating glyoxylate and malate, respectively (Figure 3.23).

The catabolism of glucose for ATP production is a highly regulated process in the cell. This is a good opportunity to ask students how the cell “knows” when it needs to speed up or slow down cellular respiration. What are the signals that the cell senses? Some students will see that the cell must be able to “sense” the overall redox state of pools of key electron carrier molecules, such as NAD+ and FAD. Regulation will be discussed in future chapters, and here is a good opportunity to introduce the concept.

3.13 | Catabolic Diversity This section provides an overview of metabolic diversity by showing the variety of strategies employed by microorganisms to make energy. Use Figure 3.24 to compare chemoorganotrophic, chemolithotrophic, and phototrophic metabolisms, noting the diversity of physiological options within each of these categories. Point out that in addition to aerobic respiration and fermentation, anaerobic respiration provides a variety of alternative ways in which chemotrophs can generate ATP and conserve energy. Despite their extensive differences, chemoorganotrophic, chemolithotrophic, and phototrophic metabolisms are all designed to generate a PMF coupled to ATP production to support growth.

3.14–3.16 | Biosynthesis (Sugars and Polysaccharides; Amino Acids and Nucleotides; and Fatty Acids and Lipids) During cellular respiration, only some of the glucose molecules are oxidized completely to CO2 for ATP synthesis. As discussed in Section 3.12, carbon from other glucose molecules is used for biosynthesis of essential molecules to support cell growth. Discuss with your stu- dents the fact that cells use intermediate molecules from glycolysis and the citric acid cycle as starting points for biosynthesis of polysaccharides, nucleotides, amino acids, fatty acids, and other important molecules. Figures 3.25–3.30 provide an excellent overview of the biosyn- thesis of these monomers.

The depth of this overview should be adjusted to the background of your students, as a thorough understanding of the material may require previous exposure to biochemistry. For example, depending on the scope of your course, you may choose not to cover gluconeogenesis (Figure 3.25b) and the pentose phosphate pathway (Figure 3.26) in detail. However, it is useful for students to at least understand the key role these processes play in cell wall and nucleotide biosynthesis, respectively. Similarly, the incorporation of ammonia into cells for amino acid biosynthesis (Figure 3.28) and the mechanism by which fatty acids are produced (Figure 3.30) may be topics best reserved for courses designed for science majors. However, all microbiology students should understand the importance of these mechanisms for the ultimate production of essential macromolecules in the cell.

3.17 | Nitrogen Fixation The biological significance of nitrogen (N2) fixation should be emphasized to all microbiology students, especially given the fact that it is a biosphere-sustaining process that is carried .

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out only by select groups of free-living and symbiotic prokaryotes (Table 3.5). Nitrogen fixation is an intense area of research in many laboratories around the world, and both basic and applied approaches are employed for unraveling the molecular and biochemical events of this process.

Discuss the details of nitrogen fixation by starting with the structure of the nitrogenase complex, consisting of dinitrogenase reductase and dinitrogenase. Focus especially on the iron-molybdenum cofactor of dinitrogenase (FeMo-co)—the actual site of N2 reduction (Figure 3.31). Point out to students that alternative nitrogenases have been identified that contain either vanadium (and iron) or iron only as the metal ligand, and that the expression of these alternative systems is tightly regulated by the cell in response to the availability of molybdenum. Points to stress to your students include the following: • Although many nitrogen-fixing bacteria are obligate aerobes, dinitrogen reductase is oxygen-sensitive. Figure 3.32 nicely illustrates strategies to protect dinitrogenase reductase from O2 inactivation in two aerobes, Azotobacter vinlandii (Figure 3.32a and b) and the filamentous cyanobacterium Anabaena (Figure 3.32c). In A. vinlandii, the diffusion of O2 into the nitrogen-fixing cells is slowed to a rate in which it is quickly respired upon entry.

This is accomplished by the production of a slime layer of variable thickness; the greater the O2 concentration is in the surrounding atmosphere, the thicker the slime layer. By con- trast, Anabaena protects dinitrogenase reductase by coordinating the differentiation of a few evenly spaced cells in the strand into specialized nitrogen-fixing forms called heterocysts. Unlike the rest of the cells in the filament, which are actively carrying out oxygenic photosynthesis, heterocysts maintain anoxic internal conditions to facilitate high levels of nitrogen fixation. Heterocysts provide usable nitrogen (as NH3) for all cells in the strand, whereas vegetative (photosynthesizing) cells supply heterocysts with the energy (ATP and reducing equivalents) required for nitrogen fixation.

• The electron flow diagram in Figure 3.33a is an excellent example of oxidation/reduction cascades (discussed in Section 3.6), although they are employed here for a different purpose. Nitrogen fixation requires a lot of energy (2 ATP for each electron transferred; Figure 3.33b), and it requires dinitrogenase reductase (which carries only a single electron at a time) to associate and disassociate from dinitrogenase eight times to reduce N2 to ammonia! Dinitrogenase accumulates these eight electrons before funneling them to the active site where one mole of N2 is reduced to two moles of NH3 and one mole of H2. Students should understand that the H2 produced is only a consequence of the nonspecific reduction of protons by dinitrogenase and not a desired or important product of the reaction.

• Because nitrogenase enzymes can catalyze the reduction of certain triple-bonded molecules other than N2, the acetylene reduction assay (Figure 3.34) provides a convenient method for indirectly measuring nitrogen fixation. However, the method does not unequivocally demonstrate the process because it shows only the two-electron reduction of the triple-bonded acetylene to ethylene. Definitive evidence of nitrogen fixation comes from providing cells with 15N (a stable isotope) and measuring the production of 15 N-labeled NH3.

Answers to Review Questions 1. All cells require carbon, and most prokaryotes require organic (carbon-containing) compounds as their source of carbon. About 50% of the dry weight of a bacterial cell is carbon. Carbon is obtained from amino acids, fatty acids, organic acids, sugars, nitrogen bases, aromatic compounds, and countless other organic compounds. Some microorganisms are autotrophic .

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and these organisms build their cellular structures from carbon dioxide (CO2). Growth factors are organic micronutrients such as the vitamins, amino acids, purines, and pyrimidines.

2. A selective medium contains compounds that inhibit the growth of some microorganisms but not others. A differential medium is one in which an indicator, typically a dye, is added, which reveals by a color change whether a particular metabolic reaction has occurred during growth 3. Aseptic technique is a series of manipulations used to prevent contamination of sterile objects or pure cultures of microorganisms during handling.

4. Escherichia coli is a chemoorganotroph, whereas Thiobacillus thioparus is a chemolith- otroph.

Table 3.2 indicates this since the organic compound glucose is included in the de- fined culture medium for E. coli, but no organic carbon source is included in the defined culture medium for T. thioparus, which uses CO2 as its sole carbon source.

5. To calculate ∆G0′ one would subtract the sum of the free energies of formation (Gf0) of the reactants (C6H12O6 and 6 O2) from that of the products (6 CO2 and 6 H2O). An exergonic reaction has a negative ∆G0′.

6. ∆G0′ is the free energy of a reaction under standard conditions (pH 7, 25°C, 1 atm, and all reactants and products initially at 1 M concentrations). ∆G means free-energy change under the conditions specified. Gf0, on the other hand, refers to the energy required for formation of the molecule from the elements.

7. The function of enzymes in the cell is to lower the activation energy required for a reac- tion to take place. Enzymes, then, are catalysts that allow chemical reactions to transpire many times more quickly than they would otherwise occur.

8. The greater the potential difference between the redox pair, the greater the potential for energy yield, hence the following series: H2/O2, H2/Fe3+, H2S/O2, methanol/NO3–, H2S/NO3–, NO2–/Fe3+, Fe2+/O2.

9. The reduction potential of the NAD+/NADH couple is –0.32 V (see Figure 3.9).

10. Acetyl phosphate is considered an energy-rich compound because the energy released via hydrolysis of the phosphoanhydride bond in this compound (–44.8 kJ/mol) is far greater than that contained in the phosphoester bond in glucose 6-phosphate (–13.8 kJ/mol).

11. In fermentation, ATP is usually made by substrate-level phosphorylations. In respiration, most of the ATP is synthesized via electron transport coupled to oxidative phosphorylation.

12. In glycolysis, NADH is produced when glyceraldehyde 3-phosphate is oxidized to 1,3-bisphosphoglycerate. It is consumed at the end of the fermentation step (stage III), when pyruvate is reduced via NADH to a fermentation product.

13. Common fermentation products from glucose other than ethanol and lactate include butanol, acetone, 2,3-butanediol, formate, propionate, butyrate, caproate, acetate, CO2, and H2.

14. Important electron carriers found in electron transport chains include NADH dehydrogenase, flavoproteins, iron-sulfur proteins, quinones, succinate dehydrogenase, and cytochromes.

15. The proton motive force is an “energized” state of a membrane created by the expulsion of H+ to the exterior face of the membrane, whereas OH– accumulate on the cytoplasmic face of the membrane. This creates a “charge separation,” much like in a battery (“+” outside; “–” inside) that creates both an electrochemical potential and a pH gradient across the membrane. The dissipation of this charge gradient through a membrane-bound ATP synthase complex provides the energy for ATP synthesis.

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16. The movement of protons (from the PMF) into the cell cytoplasm through Fo drives rotation of the c proteins, generating torque. This causes alternating conformational changes in the β-subunits of F1, thereby catalyzing the production of ATP from ADP and Pi. 17. Organisms can obtain nearly 20 times more ATP when respiring glucose aerobically than when fermenting it.

18. In organisms carrying out respiration, the citric acid cycle functions to extract all of the electrons remaining from the oxidation of glucose to two molecules of pyruvate via a series of reactions that produce an abundance of reduced electron carriers (NADH and FADH2) for ATP synthesis. In its second role, the citric acid cycle produces many of the precursor metabolites required for biosynthesis, especially amino acids.

19. In aerobic respiration, O2 is used as the terminal electron acceptor at the end of an electron transport chain. In anaerobic respiration, a molecule other than O2 serves as the terminal electron acceptor. Aerobic respiration always yields more energy than anaerobic respiration because the O2/H2O redox couple has a more positive E0′ at +0.82 V than any alternative electron acceptor on the redox tower (see Figure 3.9), and ΔG0′ is proportional to ΔE0′.

20. The Embden-Meyerhof-Parnas pathway (glycolysis) supplies the carbon skeletons for sugar biosynthesis, and amino acid precursors come from both glycolysis and citric acid cycle intermediates.

21. The biosynthesis of fatty acids occurs two carbon atoms at a time with the help of a small protein, the acyl carrier protein (ACP). The two carbons are donated from a three-carbon malonate compound, with the concomitant release of a molecule of CO2. These reactions continue until the final chain length is reached (e.g., C16 palmitate).

22. The nitrogenase enzyme complex catalyzes the following reaction: N2 + 8 e– + 8 H+ + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi The ability to fix nitrogen provides a huge advantage in that organisms capable of this process are not obligated to compete for activated nitrogen compounds; nor is it tied to an environment in which such compounds are readily available. If air, and therefore N2, is present in the environment, then a nitrogen-fixing cell has an essentially inexhaustible supply of usable nitrogen for cell maintenance and growth.

Answers to Application Questions 1. Example of enrichment medium for alginate lyase producing marine bacteria: Zobell marine broth + carbon source (0.1% alginate).

A defined medium having KH2PO4, 0.5 g; NH4Cl, 0.5 g; MgSO4, 0.1 g; CaCl2, 0.05 g; KCl, 0.5 g; Na2S2O3, 2 g. Trace elements (Fe, Co, Mn, Zn, Cu, Ni, Mo) 2–10 μg each dissolved in 1000 ml of distilled water with final pH 7 could be used supplemented with 0.1% alginate as a carbon and energy source.

2. ΔG0′ = Gf 0 (H2CO3) − {Gf 0 (H2O) + Gf 0 (CO2)} = 8.52 kJ (endergonic reaction) 3. Azotobacter is aerobic and a chemoorganotroph. It fixes nitrogen, receives energy from redox reactions, and uses organic compounds as electron donors. The laboratory culture requires carbon sources (carbohydrates, alcohols, salts of organic acids), nitrogen sources (nitrates, ammonium ions or amino acids), molybdenum ions for nitrogen, optimal pH for the growth, and nitrogen fixation of 7.0–7.5. Azotobacter can also grow mixotrophically in a nitrogen-free medium containing mannose. Its growth can be validated by an acetylene .

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reduction assay.

4. When Escherichia coli is growing via fermentation, there is no external electron receptor and the energy yield is relatively low. When NO3– is added to the culture, it becomes a suitable electron acceptor, and anaerobic respiration ensues. Under these conditions, the energy yield is greater than under fermentative conditions, but less than under conditions where O2 is the terminal electron receptor (i.e., growth under aerobic conditions).

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CHAPTER

4

Molecular Microbiology

Summary Chapter 4 focuses on the molecular biology of microorganisms and takes the student on a journey from DNA structure and replication to protein folding and secretion. Students should recall that cells are both biochemical catalysts and genetic entities. The chapter provides an excellent overview of the structure, storage, and replication of genetic material; the expression of genes through the process of transcription; and the translation of messenger RNA transcripts into protein products. Our understanding of these processes has been greatly enhanced through intensive study of the model organism Escherichia coli. Indeed, we know more about the structure and function of this bacterium than any other living organism, including humans.

4.1 | Macromolecules and Genes Chapter 4 is organized from DNA structure and replication events to RNA synthesis and processing, and it concludes with protein synthesis, folding, and secretion. This first section describes the structure of nucleotides, the building blocks of nucleic acids (Figures 4.1 and 4.2), and the flow of genetic information from DNA to RNA to proteins (Figure 4.3). Using the figures, show that nucleotides consist of a nitrogenous base, a pentose sugar, and a phosphate group. Distinguish between pyrimidine bases and purine bases, pointing out the nucleic acid molecules (DNA or RNA) in which each of the five bases occurs. Explain that in nucleic acids, individual nucleotides are covalently linked with a phosphate group between the 3-carbon of one sugar and the 5-carbon of the next sugar, forming a phosphodiester bond.

Introduce the terms gene (the functional unit of genetic information) and genetic elements (large nucleic acid molecules containing genes). Note that information flows from DNA to RNA to protein. These cellular components are referred to as informational macromolecules.

By contrast, lipids and polysaccharides are macromolecules that are not considered informational since they have no coding functions.

Discuss the three stages involved in genetic information flow depicted in Figure 4.3: (1) replication, (2) transcription, and (3) translation. All cells employ this central dogma (DNA → RNA → protein) when carrying out essential life functions. However, there are differences in how these steps occur between eukaryotes and prokaryotes, and additional differences exist between the prokaryotic domains Bacteria and Archaea. Point out that in contrast to eukaryotes, prokaryotes often transcribe multiple genes into a single primary mRNA transcript. Moreover, because prokaryotes do not have a compartmentalized nucleus, transcription and translation are coupled processes that occur simultaneously. This is not the .

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case for eukaryotes, in which transcription is confined to DNA in the nucleus, and translation is catalyzed by ribosomes in the cytoplasm. Later, students will learn that some viruses do not adhere to the central dogma of molecular biology in that they have genomes composed of RNA rather than DNA (Chapter 9).

4.2 | The Double Helix Use Figure 4.4 to review DNA structure, focusing on the sugar-phosphate backbone containing phosphodiester bonds and the complementary base-pairing of nitrogenous bases between the two strands of the double helix. Point out that the two strands are held together by hydrogen bonding between the bases⎯two hydrogen bonds between adenine and thymine (or uracil in RNA) and three hydrogen bonds between guanine and cytosine. Students should understand the 5 → 3 orientation of nucleic acids and why the chain is synthesized in this direction. Note also that the DNA strands of the double helix must be antiparallel, which has important implications for how DNA is replicated (Sections 4.4–4.6). The major groove (and to some extent, the minor groove) shown in Figure 4.5 is an important contact point for DNA-binding proteins that play a role in controlling gene expression (Chapter 7).

The size of double-stranded DNA molecules is expressed as the number of nucleotide base pairs per molecule. A molecule of DNA containing 1500 base pairs is expressed as 1500 bp, which is equal to 1.5 kilobase pairs (1.5 kbp). Explain that the length of a single base pair in the helix is 0.34 nm; thus one can calculate the length of a DNA molecule when given the total number of base pairs. You can reinforce this concept by having students calculate the length of the haploid human genome that is 3 billion base pairs long, and convert from metric units to inches or feet, as shown below.

(3  109 bp)  0.34 nm/bp = 1.02  109 nm long = 102 cm (1 cm = 0.0328 ft.) = 3.35 feet (40.2 inches) Section 4.2 also covers DNA supercoiling in the context of DNA structure and packaging. A good illustration of DNA supercoiling for your students is the ability of a bacterial cell to pack over 1 mm of DNA into less than 2 m of cell length. The problem is even more complex in multicellular organisms and requires a much more elaborate packaging scheme employing histone proteins and nucleosome structures (see Figure 2.61). DNA can be supercoiled in either the positive (overwound) or negative (underwound) conformation. Enzymes called topoisomerases catalyze the formation or relaxation of supercoils in DNA by introducing either single-stranded breaks (class I topoisomerases) or double-stranded breaks (class II topoisomerases) in the DNA molecule (Figures 4.6 and 4.7). The breaks are resealed following the introduction of a supercoil. DNA gyrase, a class II topoisomerase, is found in Bacteria and Archaea and introduces negative supercoiling in DNA (Figure 4.7). Quinolones, fluoroquinolones, and novobiocin are antibiotics that inhibit DNA gyrase. Topoisomerases relax supercoils by making transient breaks that allow one strand to untangle. The relaxation of the DNA molecule is necessary due to the changes in the topology of DNA as it is being replicated. As the doubled-stranded helix is unwinding in one direction, the unwound region ahead of the replication fork experiences torsional stress that must be relieved for replication to continue. Supercoiling also plays a role in gene expression in both prokaryotes and eukaryotes. .

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4.3 | Genetic Elements: Chromosomes and Plasmids Section 4.3 introduces students to the concept of chromosomal and extrachromosomal genetic elements. In prokaryotes, the latter group includes virus genomes, plasmids, and transposable elements (Table 4.1). Transposons and insertion sequences are covered in more detail in Chapter 10.

The genomes of most prokaryotes consist of a single, circular chromosome ranging in size from 0.5 Mbp to 10 Mbp in most cases. The genome of E. coli is of approximately average size for a bacterium and depending on the strain, ranges from 4.5 Mbp to 5.5 Mbp. Using Figure 4.8, explain to students how genetic maps are used to illustrate the major properties of chromosomes. The size of the genome, the location of genes or operons, and the direction of transcription of those genes are all features that can be shown with genetic maps. Point out that the genes for some biochemical pathways are clustered into operons (e.g., the lac, gal, trp and his clusters on the right side of the chromosome figure), whereas others are not (e.g., the malT and malS genes are not included in the malKBM operon). Compared to eukaryotic genomes, which usually contain a large percentage of noncoding DNA, the genomes of prokaryotes are quite streamlined in that most of their DNA encodes a product necessary for cell function. About 88% of the genome of E. coli strain K-12 consists of protein-encoding genes, while another 1% encodes ribosomal and transfer RNA molecules. Students may find it interesting that sequenced genomes often contain up to 40% “hypothetical” protein coding sequences for which a function cannot be definitively assigned. Furthermore, it is estimated that a significant portion of genetic content in many genomes was acquired through horizontal gene transfer, a process by which genetic elements are mobilized and transferred from one cell to another (see Chapter 10).

Plasmids are also introduced in this section and are defined as small (typically less than 5% the size of the chromosome), usually circular segments of extrachromosomal doublestranded DNA having their own origin of replication (Figure 4.9). Some bacteria possess multiple plasmids. For example, Borrelia burgdorferi, the causative agent of Lyme disease, has 17 plasmids, some of which are even linear. Plasmid-encoded genes are not essential to cell survival but often confer useful properties on cells, including resistance to antibiotics (via R plasmids; Figure 4.10) and the ability to produce toxins or other virulence factors (Table 4.2). Mention to your students that the increased use of antibiotics has selected for pathogens for which few or no treatment options remain. This problem is discussed in more detail in Chapter 27. Plasmids replicate using the same enzymatic machinery as that which the cell uses for chromosomal replication, and like chromosomes, most circular plasmids replicate bidirectionally (see Section 4.6).

Students may be interested to learn that many bacteria produce plasmid-encoded proteins called bacteriocins that kill related species, or even different strains of the same species, while at the same time producing proteins that make themselves immune to these toxins.

Plasmids have also been engineered and artificially modified to use in research. The construction of artificial plasmids containing selected genes has played a key role in the exploitation of microorganisms for biotechnological and industrial purposes. The topic of genetic engineering will be covered in Chapter 11.

Use Table 4.2 to describe some of the phenotypes conferred by plasmids on their bacterial hosts, stressing that a given plasmid can be lost from the population if the genes it possesses .

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do not provide a selective advantage to the host. The reasons for this stem from the fact that a bacterium will not expend the energy it takes to maintain an extrachromosomal element and transcribe and translate its genes unless the products of those genes are in some way beneficial. Consequently, a daughter cell produced without the plasmid may grow and reproduce faster, eventually curing the population of the plasmid.

4.4–4.6 | Transmission of Genetic Information: DNA Replication Use Figure 4.11 to summarize the products of DNA replication, stressing the semiconservative process that occurs in both prokaryotes and eukaryotes. The precursor of each new nucleotide to be added to the chain is a deoxyribonucleoside triphosphate. Hydrolysis of two of the phosphates is required by DNA polymerase to drive phosphodiester bond formation at the 3–OH of the preceding nucleotide (Figure 4.12). E. coli contains at least five distinct DNA polymerases (DNA Pol I–V). DNA Pol III functions as the main enzyme in DNA replication. DNA Pol I also plays a crucial role in replication, while the other three polymerases function primarily in DNA repair. Note that the initiation of DNA replication requires a primer. For circular molecules of DNA, the primer is a segment of RNA synthesized by the enzyme primase (Figure 4.13). Students must understand that a primer is necessary because DNA polymerase can begin synthesis only on DNA molecules having a free 3–OH. RNA primers inserted during replication are later removed and replaced with DNA nucleotides, as described below.

Next, discuss the events that occur during the initiation phase at the replication fork and the enzymes involved (Figure 4.14). The origin of replication is an approximately 250-bp sequence that is recognized by the DnaA protein, which binds and opens the helix (Table 4.3).

Two helicase (DnaB) enzymes bind with the help of the DnaC protein, and each attaches to one of the two strands in opposite directions (Figure 4.14 shows one of the two helicase complexes). Finally, single-stranded binding proteins bind to the two strands, stabilizing them and preventing them from re-annealing (Figure 4.15).

Continue your discussion of replication of circular prokaryotic chromosomes by clearly delineating the events involved in leading strand versus lagging strand synthesis (Figures 4.15 and 4.16). Note that the logistical problems inherent in synthesizing the lagging strand away from the replication fork necessitate the generation of Okazaki fragments and a “backstitching” type of mechanism by primase and DNA Pol I. RNA primers are removed by DNA Pol I and replaced with deoxyribonucleotides. The enzyme DNA ligase is required to seal the backbone (add a final phosphodiester bond) between Okazaki fragments on the lagging strand (Figure 4.16). Use Figure 4.17 to show students that there are two replication forks for a circular chromosome, resulting in the theta structure. The problem of excess torsion created by supercoils ahead of the replication forks is resolved by the topoisomerase DNA gyrase, which regulates the degree of supercoiling during replication (Figure 4.18). The proteins involved in replication do not act independently, but they aggregate into a large complex called the replisome (Figure 4.18). Table 4.3 describes the key enzymes involved in DNA replication in Bacteria.

Students need to understand that although DNA polymerases III and I are remarkably efficient and precise in maintaining the fidelity of the DNA sequence during replication, they .

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have an error (mutation) rate of ~10–8 and 10–11 errors per base pair inserted. Consequently, a backup system detects and corrects these errors. Most (but not all) of the errors are corrected by the proofreading capabilities of DNA Pol III and Pol I. Each of these enzymes possesses a 3 → 5 exonuclease activity that is capable of excising a mismatched base and inserting the correct (complementary) base in its place (Figure 4.19). Other mechanisms for reducing errors during DNA replication are discussed in Chapter 10.

4.7 | Transcription Begin your discussion of transcription by reviewing the structural differences between RNA and DNA. Three major types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each of these RNAs is encoded in the chromosome, so remind students that while most genes code for proteins, some code for structural RNA molecules (e.g., tRNA and rRNA molecules) that are not translated into proteins. Note that although the cell also produces other RNAs that mostly function in regulation (Chapter 7), this chapter focuses only on those involved in transcription and translation.

Transcription and translation are often presented as a single topic, and students often get lost in the myriad details surrounding these processes. The approach here allows the instructor to focus student attention on specific events in transcription before proceeding to translation.

The processes discussed are those that occur in Bacteria, using E. coli as the model organism.

Figure 4.20 presents an overview of transcription, focusing on the function of RNA polymerase in this process. The process of transcription can be approached in three stages: initiation, elongation, and termination. Note here that there are significant differences between RNA polymerases from Bacteria, Archaea, and Eukarya (Figure 4.21; archaeal and eukaryotic RNA polymerases are also discussed in Section 4.9). The Bacterial RNA polymerase holoenzyme consists of five different subunits, with the sigma factor subunit conferring promoter recognition specificity. Following initiation of transcription, the sigma subunit dissociates from RNA polymerase holoenzyme, and the remaining RNA polymerase core enzyme carries out elongation. E. coli makes seven different sigma factors that recognize different promoters.

Discuss the interaction of sigma factor with specific recognition sequence contact points within the promoter (–10 and –35 regions; Figure 4.22). Sigma factors can bind tightly to specific promoter sequences called strong promoters or less specifically to weak promoters, thus affecting gene expression via the efficiency and rate of transcription. Inform students that strong promoters are used in genetic engineering to support high expression levels of desired genes (Chapter 11). Table 4.4 lists the common and alternative sigma factors used by E. coli, along with the function of the genes or operons upon which they act.

Following elongation of the RNA transcript, termination of transcription ensues. In Bacteria, this process is controlled by specific DNA sequences downstream of the 3 end of an open reading frame. These sequences, called inverted repeats, often consist of two GC-rich sequences with a nonrepeating sequence between them, allowing for the formation of a stem-loop structure (Figure 4.23). This type of intrinsic termination occurs independently of catalysis by the termination-inducing enzyme Rho and is thus called Rho-independent termi.

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nation. In contrast to this mechanism, Rho-dependent termination requires the specific protein Rho, which binds the newly synthesized RNA molecule and causes both it and RNA polymerase to be released from the DNA. Students should understand the difference between intrinsic terminators and Rho-dependent termination.

4.8 | The Unit of Transcription Emphasize for students the difference between a gene and a transcriptional unit—students often get these concepts confused. Figure 4.24 provides a helpful representation of an rRNA transcriptional unit that can be used to describe all of the elements a single unit contains. Note that an rRNA operon contains all three rRNA genes with spacers (short segments of noncoding DNA) between them. This cotranscription ensures that 16S, 23S, and 5S rRNA molecules are transcribed in equal proportions. Often, one or more tRNA genes are also included within an rRNA operon. Discuss the concept of operons and polycistronic mRNA (Figure 4.25) and the advantages of cotranscribing multiple related genes into a single RNA transcript.

4.9 | Transcription in Archaea and Eukarya Transcription in Archaea shares features with the mechanisms of transcription in both the Bacteria and Eukarya. Recall that the RNA polymerase of Archaea is markedly similar to RNA Pol II of Saccharomyces cerevisiae, a eukaryote (Figure 4.21). As in eukaryotes but unlike Bacteria, the RNA polymerase of Archaea is not sensitive to the antibiotic rifampicin. However, regulation of transcription in Archaea is similar to that in Bacteria (Chapter 7).

Figure 4.26 shows features of an archaeal promoter region, including the 6–8-bp TATA box that is recognized by the transcription factor TATA-binding protein (TBP). Discuss the role(s) of each transcription factor. Little information is available regarding transcription termination signals in Archaea, although some species appear to use inverted repeats, as observed in Bacteria (Section 4.7). Eukaryotic transcription termination often requires termination factor proteins, but these are not related to the Rho protein of Bacteria.

Introns are intervening, noncoding DNA sequences within genes that are excised before producing the mature RNA transcript. As is the case in Bacteria, introns are rare in Archaea, although several tRNA and rRNA genes do possess them. Intron excision in Archaea is catalyzed by a specific endoribonuclease (Figure 4.27). Most eukaryotic transcripts contain introns, however, and they must be processed before they are functionally mature. Figure 4.28 illustrates the structure and mechanism of action of the spliceosome. This macromolecular complex contains small ribonucleoproteins and other proteins that facilitate both the removal of introns from primary transcripts and the splicing of exons to produce the mature mRNA. Discuss each of the steps shown in Figure 4.28a–e.

Next, describe two other unique RNA processing steps: (1) RNA capping and (2) the poly(A) tail (Figure 4.29). Note that both processes take place before intron excision. The 5' guanosine cap is needed during formation of the mRNA-ribosome translation initiation complex, and the poly(A) tail stabilizes the transcript and signals its identity as mRNA to the .

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ribosome. This is in contrast to the function of poly(a) tails on mRNA molecules of chloroplasts and certain bacteria, which are usually interpreted as a signal for degradation of the transcript.

4.10 | Polypeptides, Amino Acids, and the Peptide Bond Proteins are important components in the chemistry of life, serving both structural and catalytic roles. Consequently, a general understanding of protein structure will allow students to better comprehend the tremendous variety of functions that these macromolecules perform in the cell as they proceed through the course. Figure 4.30 gives an excellent representation of amino acid structure and respective side chain (R group) diversity.

Discuss the dehydration synthesis that leads to peptide bond formation (Figure 4.31), and mention here that this reaction is catalyzed within a region of the ribosomal large subunit during translation of an mRNA molecule into protein. The resulting string of amino acids specified by a gene is known as the primary structure of the protein, and the sizes of known proteins range from just 15 to 10,000 amino acids. Stress that proteins can vary in the number, types, and sequence of amino acids, which explains the enormous variety of protein structures possible. For these reasons, many early scientists believed that protein, not DNA, was the hereditary material in living systems.

4.11–4.12 | Translation and the Genetic Code and Transfer RNA Protein synthesis, or translation, is a continuous process in the life of the cell. As in transcription, the various phases of translation can be considered as three discrete steps: initiation, elongation, and termination. Before discussing these details, students need to understand how genetic information in DNA dictates the amino acid sequence of a protein according to the genetic code (Table 4.5). The genetic code uses codons to specify amino acids. Each codon consists of a triplet of three nucleotide bases. Explain to students that a code of three-letter codons made up of four separate bases yields 64 possible codons (43) for encoding the twenty amino acids found universally in living organisms. Thus there is more than one codon for most amino acids. In addition, there is one start codon⎯AUG, which codes for methionine (or N-formylmethionine in Bacteria)⎯and three stop codons: UAA, UAG, and UGA (Table 4.5). These signal the beginning or end of the encoded polypeptide, respectively.

Now is a good time to introduce the role of tRNA, which possesses a complementary anticodon that recognizes a specific codon (or codons) on mRNA and delivers the encoded amino acid (see Figure 4.34). There is usually more than one tRNA for a given amino acid because more than one codon can specify a single amino acid. However, some tRNAs can base pair with more than one codon due to the wobble phenomenon, in which complementary base pairing at the third nucleotide position of the codon is not required (Figure 4.32). For example, the anticodon for one lysyl tRNA (tRNA carrying lysine) can base pair with either AAA or AAG due to the wobble position effect (see Table 4.5).

Every gene needs to have a start signal and a stop signal that is recognized by the translational machinery and that defines the correct open reading frame. Use Figure 4.33 to show .

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that while there are three possible reading frames on each strand in the 5 → 3 direction, there is only one correct frame that specifies the desired polypeptide. It is important to point out that, in prokaryotes, DNA is not isolated from the cytoplasm by a nuclear membrane, and therefore transcription and translation are coupled processes (i.e., they occur concurrently).

As soon as the ribosome-binding site (also called the Shine-Dalgarno sequence) near the 5 end of the growing mRNA transcript becomes accessible, ribosomes can bind and begin translation. Discuss the concept of codon bias and why this is such an important consideration in genetic engineering (see Chapter 11).

Students may be surprised to discover that over 100 amino acids that are not part of the universal genetic code are found in proteins. Most of these amino acids are generated by modification after being inserted into the protein chain. However, two genetically encoded amino acids that are not universally found in living organisms are selenocysteine and pyrrolysine, both of which are encoded by stop codons. In such cases where a stop codon is intended to encode one of these rare amino acids, a recognition sequence just downstream of the codon is employed to indicate its role as a sense rather than nonsense (stop) codon.

Students should also be aware of the importance of secondary structure to the activity of transfer RNA molecules (Figure 4.34). Figure 4.35 nicely illustrates the mode of activity and charging mechanism of tRNAs by aminoacyl tRNA synthetases (Figure 4.35).

4.13 | Protein Synthesis Begin your discussion of protein synthesis with the structure of the ribosome (Figure 4.36).

Note the size of the small (30S) and large (50S) ribosomal subunits of prokaryotes, giving a total ribosome size of 70S. This is smaller than the 80S ribosomes of eukaryotes. Point out that ribosomes are ribonucleoprotein complexes (i.e., the E. coli ribosome contains three rRNA molecules and 52 distinct proteins).

Figure 4.36 shows the three stages of translation: initiation, elongation, and termination.

The initiation stage begins with the attachment of a free 30S ribosomal subunit to the ribosome-binding site on the mRNA transcript. The initiator N-formylmethionine tRNA and several initiation proteins (IF1, IF2, and IF3) then attach, followed by the addition of the large (50S) subunit to form the active 70S ribosome. Describe the events occurring during elongation, including translocation of the ribosome one codon at a time along the mRNA transcript (Figure 4.36) and the eventual termination of translation at the stop codon. The termination event occurs when the ribosome reaches, and stalls, at a stop codon. This is followed by the dissociation of the polypeptide chain by release factor proteins.

Summarize the multiple roles of the ribosome during all stages of protein synthesis.

Explain that multiple ribosomes can translate a single mRNA transcript simultaneously, forming a polysome (Figure 4.37). Also discuss how truncated or defective mRNA molecules that may lack a stop codon can result in stalled ribosomes. This situation is remedied using a small RNA molecule called tmRNA, which is charged with alanine and contains its own stop codon (Figure 4.38). Binding of a release factor protein to the tmRNA stop codon frees the ribosome for use elsewhere.

You may want to conclude your discussion of translation by mentioning the fact that many antibiotics interfere with protein synthesis by binding to the large or small subunit of the .

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ribosome. Because prokaryotic ribosomes differ in structure from eukaryotic ribosomes, these antibiotics are medically useful because of their specificity. Examples include streptomycin, chloramphenicol, and tetracycline, and the mode of action of these and other antibiotics is discussed in Chapter 5.

4.14 | Protein Folding and Secretion Discuss the important chemical interactions that confer secondary structure to a protein, including the formation of α-helices and β-sheets (Figure 4.39). These structures often fold into functional regions, or domains, that are of utmost importance in the proper function of the final protein molecule. For example, transmembrane proteins, such as some sensor kinases involved in signal transduction, have both an extracellular and cytoplasmic face, as well as a domain traversing the hydrophobic interior of the plasma membrane.

Examples of these and other protein domains and their function are discussed throughout the text.

Higher order structure in proteins includes tertiary and quaternary structures. Tertiary structure is determined by both the primary and secondary structure and involves hydrogen bonding, covalent disulfide bonds, hydrophobic interactions, and other atomic interactions (Figure 4.40). Quaternary structure describes a protein composed of two or more polypeptide chains (either identical or nonidentical subunits) held together by either noncovalent interactions (hydrogen bonding, van der Waals forces, or hydrophobic interactions) or covalent linkages (intersubunit disulfide bridges).

Denaturation of a protein often results in a permanent loss of biological activity. This fact should demonstrate to students that the final three-dimensional conformation of a protein as determined by secondary, tertiary, and/or quaternary interactions is not the result of its primary structure but rather is directed by it via the above interactions between amino acids within the polypeptide chain.

Although most proteins fold spontaneously into their active conformation, some require the aid of molecular chaperones (chaperonins). E. coli contains four essential chaperonins that use the energy released by ATP hydrolysis to properly fold proteins: DnaJ, DnaK, GroEL, and GroES (Figure 4.41). The DnaKJ complex functions to slow spontaneous folding of newly synthesized proteins. When left unchecked, this process can result in an increased probability of folding errors. GroEL and GroES ensure the proper folding of only about 2% of proteins synthesized by E. coli. Nevertheless, at least one dozen of this small percentage is essential for cell survival, and therefore these molecular chaperones are indispensable.

Expression of molecular chaperones increases when cells experience heat stress because these proteins also play a key role in refolding partially heat-denatured proteins.

Protein transfer (secretion) across membranes should be mentioned because the process requires protein modification, usually in the form of a hydrophobic signal sequence that is cleaved after passage through the membrane. E. coli employs the Sec system to transport many proteins across the membrane. Using the membrane translocase SecYEG, proteins containing a signal sequence are either bound by the SecA protein and secreted into the external environment or bound by the signal recognition particle (SRP) and inserted into the membrane (Figure 4.42). Using the Sec system, proteins are transported in an unfolded state .

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and fold into their final conformation only after being transported through the membrane. A second transport system in E. coli, the Tat system, allows transport of proteins already folded into their final conformation.

In regards to protein export, gram-negative bacteria have an additional logistical challenge because they have not one, but two membranes (the cytoplasmic and outer membranes) in which to deposit or through which to secrete proteins. Six different secretion systems (types I–VI) have been identified for this purpose. These complexes form protein channels that shuttle the transported protein through the membrane (or membranes) in either a single step (types I, III, IV, and VI) or by a two-step mechanism in which the Sec or Tat system is also employed (types II and V). Students should find it interesting that some pathogenic bacteria use single-step secretion systems called injectisomes (due to their structure and mechanism of action) to directly inject toxins into host cells (Figure 4.43).

Answers to Review Questions 1. The central dogma holds that genetic information is contained in DNA. The flow of information is from DNA → RNA → protein.

2. A nucleotide is a monomer of a nucleic acid containing a nitrogenous base (adenine, guanine, cytosine, thymine, or uracil), one or more molecules of phosphate, and a sugar, either ribose (in RNA) or deoxyribose (in DNA). A nucleoside is a nitrogenous base (adenine, guanine, cytosine, thymine, or uracil) plus a sugar (either ribose or deoxyribose) but lacking phosphate. The pentose sugar in a nucleotide is ribose in RNA and deoxyribose in DNA. 3. Negative supercoiling is inserted in DNA by DNA gyrase, a type II topoisomerase.

4. Linearized DNA is many times longer than the cell it came from because DNA in cells is tightly packaged in supercoiled domains and, in the case of some Archaea and all eukaryotes, also wrapped around protein complexes called histones.

5. The E. coli chromosome is ~4.6 Mbp and contains 4288 potential protein coding sequences. Additional genetic elements that may be found in an E. coli cell include plasmids and viral genomes.

6. R plasmids are extrachromosomal genetic elements in microorganisms that confer resistance to antibiotics and other growth inhibitors. The ability of these large plasmids to carry multiple antibiotic resistance genes and to spread rapidly by cell-to-cell contact makes them a serious medical problem.

7. In reference to DNA, semiconservative refers to the fact that following DNA replication, each of the new DNA molecules contains one parental (old) strand and one progeny (new) strand. In DNA, the two strands are complementary to one another in that, based on the hydrogen bonding patterns dictated by the chemistry of the nitrogenous bases, adenine always pairs with thymine, and guanine always pairs with cytosine. The two strands are also antiparallel, with one strand having a 5 to 3 orientation and its complement running in a 3 to 5 direction.

8. Replication of circular bacterial chromosomes is bidirectional at the origin. When replication is underway, newly formed DNA forces adjacent strands apart, leading to a .

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structure resembling the Greek letter theta. Eventually, as the round of replication is completed, two newly synthesized, semiconservative, circular chromosomes are produced.

9. The rarity of errors in DNA replication is because a cell has two chances to incorporate the correct base at a given site. Complementary base pairing rules offer the initial opportunity. Secondly, because DNA Pol III has 3 → 5 exonuclease activity, any mismatched nucleotide detected during proofreading may be replaced with the correct one.

10. Because tRNA is synthesized by transcription, its genes must have promoters. However, tRNA is not translated, so a start codon as such would not exist. This does not preclude the existence of an AUG sequence on the tRNA. 11. The start and stop signals for transcription are promoters and transcription terminators (Rho-dependent or Rho-independent). For translation, the start begins at a start codon (in association with a ribosome-binding site) and ends with a stop codon.

12. An operon is a cluster of genes that are under the control of a single promoter and are therefore cotranscribed as a single, polycistronic mRNA molecule. This arrangement allows related genes to be expressed in a coordinated fashion.

13. The term primary transcript refers to the RNA molecule that is originally transcribed before the introns are removed to generate the final mRNA, consisting solely of the exons.

A spliceosome catalyzes the removal of introns from the primary transcript of a proteincoding gene in a eukaryote.

14. Amino acids are molecules that contain an —NH2 group (the amino portion) and a —COOH group (the carboxylic acid portion); thus the term amino acid. Refer to Figure 4.30 for the general structure of an amino acid. The R group gives an amino acid its identity. This identity may be as simple as an H atom (as in glycine) or as complicated as the double-ringed R group in tryptophan. The positions of the various R groups influence protein structure through bonding between side chains. Nonpolar side chains tend to congregate at the protein’s core because of hydrophobic interactions. Hydrogen bonds and ionic bonds, though weak, also influence the structure of the protein. The R group of cysteine contains a sulfur atom, which can combine with another cysteine R group, either on the same or another polypeptide chain, to form a disulfide bridge. Such covalent bonds are important in defining and maintaining protein shape.

15. The “wobble” effect refers to the requirement for some tRNAs to form stable base pairs only with the first two positions of the codon, which allows some tRNAs to recognize more than one codon.

16. Aminoacyl-tRNA synthetases must be able to recognize an amino acid and the specific tRNA corresponding to that amino acid. The enzyme must first activate the amino acid with ATP: Amino acid + ATP → aminoacyl–AMP + P–P and then transfer the activated amino acid to the free 3-OH of the terminal adenosine of the tRNA: Aminoacyl–AMP + tRNA → aminoacyl–tRNA + AMP .

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Transfer RNA recognition occurs through the anticodon loop, the D-loop, and the acceptor stem (the 5 end of the molecule).

17. Peptidyl transferase activity appears to be catalyzed by the 23S rRNA component of the ribosome.

18. Chaperonins prevent improper aggregation of newly synthesized proteins and refold proteins that have partially denatured in the cell. Chaperonins are thus one type of heat shock protein, and their synthesis is greatly accelerated when a cell is stressed by excessive heat.

19. Proteins that aid other proteins in assuming their functional final conformation are called molecular chaperones, of which the major examples include the DnaKJ complex and the GroEL/GroES complex. Enzymes that degrade misfolded proteins are called proteases.

20. SRP binds proteins that are destined to be inserted into the membrane but not released on the other side. It delivers proteins to membrane secretion complex and after inserting into the membrane the signal sequence is removed by a protease.

Answers to Application Questions 1. DNA gyrase is a type II topoisomerase that inserts negative supercoils into DNA by making double-strand breaks. This can be prevented by treatment with two classes of antibiotics: a. aminocoumarins (novobiocin), which bind to the ATPase active site resulting in competitive inhibition and thus prevent DNA gyrase from further catalysis, b. quinolones (nalidixic acid and ciprofloxacin), which bind to DNA gyrase to prevent them from DNA decatenation.

2. Both DNA and RNA polymerase catalyze the formation of phosphodiester bonds, using deoxyribonucleotides and ribonucleotides, respectively. Both enzymes synthesize their polynucleotide chain in the 5 → 3 direction. However, unlike DNA polymerase, RNA polymerase can initiate synthesis de novo (not requiring a primer). Because both polymerases require a DNA template, they are more accurately referred to as a “DNAdependent DNA polymerase” and “DNA-dependent RNA polymerase,” respectively.

3. There will be no change in the protein. A difference of one base would be reflected on the 5' Untranslated Region (5' UTR)—sequences on the 5' end of mRNA but not translated into protein. It extends from the transcription start site to just before the ATG translation initiation codon.

4. Mutations are changes in the DNA sequence introduced by errors in DNA replication.

Mutation rates in cells are remarkably low, between 10−8 and 10−11 errors per base pair inserted. This accuracy is possible partly because DNA polymerases get two chances to incorporate the correct base at a given site. The first chance comes when complementary bases are inserted opposite the bases on the template strand by DNA Pol III according to the base-pairing rules, A with T and G with C. The second chance depends upon a second enzymatic activity of both DNA Pol I and Pol III, called proofreading, which occurs if an incorrect base has been inserted because this creates a mismatch in base pairing.

Many organisms have additional mechanisms for reducing errors made during DNA replication that operate after the replication fork has passed by. .

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Mutations mostly occur as a result of the natural selection pressure that paves way to evolution. Hence, sometimes mutations are beneficial, such as the evolution of Biston betularia, the black-bodied peppered moth, by replacing their white counterparts after Industrial Revolution; survival of pathogens by mutating into antibiotic resistant strains; etc.

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CHAPTER

5

Microbial Growth and Growth Control

Summary Discussion of microbial growth in Chapter 5 is a logical follow-up to material presented earlier regarding prokaryotic cell structure and function (Chapter 2) and an overview of nutrition and metabolism (Chapter 3). The principles by which microorganisms regulate and control the replication of their genomes in concert with cell division has important implications for understanding the reproductive and adaptive potential of microorganisms in their environment. Knowledge of the processes governing microbial growth is also important for understanding and developing methods to control the growth of microbial populations, especially those of microorganisms that cause disease.

5.1 | Binary Fission In microbiological terms, growth is defined as an increase in the number of cells. In prokaryotes, cellular growth occurs as binary fission (Figure 5.1), in which a single cell elongates and divides into two identical daughter cells. This process requires a large number of genes and gene products (proteins), as well as a large investment of energy. The amount of time required for a prokaryotic cell to undergo binary fission and develop into two daughter cells is called the generation time. This feature is highly variable between species. Even within a species, the generation time can vary considerably depending upon the physiochemical conditions of the environment (e.g., temperature, pH, and nutritional composition). Under optimal laboratory culture conditions, the fastest generation time among prokaryotic species is 15–20 minutes. To stimulate intellectual interest in the subject of microbial growth, ask your students how a bacterium can have a generation time of 20 minutes if it takes 40 minutes for its DNA polymerase to replicate its chromosome. See the following section for the answer to this conundrum.

5.2 | Fts Proteins and Cell Division Discuss the major proteins that comprise the divisome complex, especially the Fts (Filamentous temperature sensitive) proteins, so named because mutations in E. coli Fts proteins led to a temperature-sensitive filamentous phenotype. FtsZ, which forms the protein ring .

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that determines the division plane in the exact middle of the cell, is of particular significance and is present in all Bacteria, Archaea, mitochondria, and chloroplasts. FtsZ ring formation occurs after DNA replication takes place.

• Discuss the function and mechanism of action of Fts proteins within the divisome during cell division (Figure 5.2). The sequence begins with the formation of the FtsZ ring, followed by the addition of the ZipA anchor protein and FtsA, which recruits FtsI and FtsK. The FtsI protein is required for peptidoglycan synthesis, and because penicillin binds FtsI and inhibits its activity, it is one of several penicillin-binding proteins. FtsK assists in the proper partitioning of chromosomes⎯one into each daughter cell⎯during septum formation. The proteins MinC, D, and E ensure that the divisome, and thus the division plane, forms only at the exact center of the cell (Figure 5.3).

• Students should understand and appreciate the value of basic research into cellular processes, the outcome of which could benefit humankind. For example, the bacterial specificity of Fts proteins, along with their being essential for the cell’s survival, may make them attractive targets for antimicrobial drugs.

• Note that FtsZ shows similarity to the eukaryotic tubulin protein that also plays a role in cell division (Section 2.22).

The text summarizes the coordination of replication and cell division in E. coli in Figure 5.4. The “dilemma” for the cell is that if it takes 40 minutes to replicate its entire chromosome (based upon the speed of DNA Pol) and 20 more minutes for cell division, how is a doubling time of 20 minutes possible? The answer is that multiple rounds of replication are underway simultaneously as the cell is constantly growing and dividing.

5.3 | MreB and Cell Morphology The discovery of actin-like proteins (MreB protein) in prokaryotes is a dramatic example of the evolutionary relatedness of all life on Earth. Cytoskeleton filaments were described for decades as characteristic of eukaryotes only. This finding suggests a possible origin of the eukaryotic cytoskeleton from prokaryotic cells. Discuss the mechanism of MreB in determining cell shape (Figure 5.5a and b). Point out the fact that coccoid cells lack a gene that encodes MreB, and thus the “default” morphology for bacterial cells appears to be a sphere.

The vibrio-shaped bacterium, Caulobacter crescentus, produces the shape-determining protein crescentin in addition to MreB (Figure 5.4c). Crescentin influences the curved shape of vibrio cells, but details of the mechanism are currently not known. Proteins similar to crescentin have also been identified in other helical bacteria, such as Helicobacter, the causative agent of gastric ulcers (see Section 29.10).

Cell division in Archaea is not well understood, but FtsZ-type proteins have been identified in archaeal genomes. However, there is considerable structural homology of bacterial MreB, FtsZ, and crescentin proteins to microfilaments, microtubules, and intermediate filaments, respectively—the major cytoskeletal elements found in eukaryotic cells.

5.4 | Peptidoglycan Biosynthesis .

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Discuss the role of peptidoglycan and autolysins in cell division (Figures 5.6 and 5.8), focusing on how peptidoglycan is synthesized. Students need to understand that for a cell to expand before division, peptidoglycan has to be severed to allow for insertion of the newly synthesized cell wall material. The actual events begin at the FtsZ ring, where autolysins hydrolyze the -1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid residues in peptidoglycan (the same bond attacked by lysosyme; see Figure 3.7). You may want to briefly review the structure of peptidoglycan from Chapter 2. Figure 5.7 shows the peptidoglycan precursor building blocks (the glycan tetrapeptide; see Figure 2.25) attached to the hydrophobic carrier molecule bactoprenol. Bactoprenol carries these precursors across the cytoplasmic membrane and interacts with transglycosylases to insert them into the spaces produced by autolysins (Figure 5.8a).

Discuss the final step in peptidoglycan synthesis: the transpeptidation reaction (Figure 5.8b). Remind students of the structural differences in the peptide bridges produced by gram-negative (E. coli) versus gram-positive (S. aureus) bacteria (see Figure 2.26). In reference to transpeptidation, discuss the antimicrobial activity of penicillin, the first commercially available antibiotic. Point out the fact that because transpeptidation is prevented by penicillin, the continued activity of the naturally produced autolysins and the internal turgor pressure of the cell result in cell lysis. The term penicillin-binding protein often confuses students because it implies that the bacterium produces a protein specifically to bind penicillin. Actually, the lethal activity of penicillin is defined by its ability to form a complex with transpeptidase (FtsI in E. coli), causing it to lose catalytic activity.

5.5 | Quantitative Aspects of Microbial Growth Any student wishing to pursue a career in microbiology or microbial genetics will find that an understanding of the concepts presented in these sections is essential. Therefore, spend considerable time discussing the concepts related to growth and the measurement of growth in these sections. In most microbiology courses, laboratory activities will also demonstrate these concepts. However, you may wish to cover these topics in lecture before the students’ laboratory activities.

Define the generation time of a bacterium, using Figure 5.9 to demonstrate the effects of doubling time on the total cell number. As discussed in Section 5.1, stress the idea that the generation time of bacteria varies between species and varies widely within a single species, depending upon both nutrient and environmental conditions. This leads to the concept of exponential (or logarithmic) growth. Semilogarithmic graphs allow the plotting of very small to very large cell numbers, and the exponential growth phase of a culture can easily be visualized as a straight line (in contrast to the continuous increasing slope of a curved line on an arithmetic scale; Figure 5.9b). Discuss the consequences of exponential growth using the practical example of food spoilage described in the text. To further explore this concept, question students as to the importance of these ideas in relation to foodborne infections caused by pathogenic bacteria.

Students should also have a basic understanding of the mathematics of exponential growth regarding the calculation of generation times and cell numbers (Figure 5.10). These are important tools in microbial genetics and applied aspects of industrial microbiology and the .

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biotechnology industry. In courses for upper-division students, specific growth rate (k) and division rate (v) should be covered.

5.6–5.7 | The Growth Cycle and Continuous Culture Discuss the concept of batch culture with your students, pointing out that bacterial cells cannot remain indefinitely in exponential growth phase. Give the following example from the text: A bacterium that divides every 20 minutes for 48 hours during exponential growth would produce a mass of cells that would weigh 4000 times the weight of Earth! To show this, the calculation would progress in the following manner: 48 h  3 divisions/h = 144 divisions = 2144 cells An average bacterial cell weighs approximately 1 × 10–12 g (a picogram).

Therefore, 2144 cells multiplied by 1 × 10–12 g/cell would equal ~2.23×1028 kg, a mass of cells that is equivalent to about 4000 times the weight of Earth.

Bacterial cells grown in closed vessels (batch cultures) go through a growth curve as shown in Figure 5.11. Discuss the events that take place during the lag, exponential, stationary, and death phases.

Most students will not be familiar with the chemostat, or continuous culture, method for growing microbial cultures (Figure 5.12). Inform students that the batch cultures they work with in the laboratory are closed systems, and as such they display a typical growth curve.

Just as was described for organisms growing in batch culture, the cell density in the chemostat is a function of the amount of the limiting nutrient available (Figure 5.13). In nature, conditions vary dramatically from those in laboratory culture with respect to nutrient concentrations. The chemostat is thus a useful tool for looking at microbial growth rates in real-world scenarios in which both substrate concentrations and the accumulation of waste products can fluctuate dramatically (Figure 5.14). Give examples of potential experimental uses of chemostats in the biotechnology and pharmaceutical industries, as well as in basic research.

5.8–5.10 | Measuring Microbial Growth Many of the concepts in these sections are covered in the laboratory portion of most microbiology courses. Regardless, it is beneficial to preview these techniques in lecture so that students understand the advantages and disadvantages of each method before their application in the laboratory. Total cell numbers can be determined by direct microscopic counts (Figure 5.15); however, this technique will not allow one to distinguish an intact nonviable cell from a viable cell. In addition to the problem of obtaining reliable counts at high cell densities in stationary phase, the total cell number obtained will invariably be higher than the viable cell number. By contrast, viable cell counts are determined by plating liquid cell cultures, either by the spread-plate or pour-plate method, usually after first performing a serial dilution (Figures 5.16 and 5.17). Because only viable cells will form colonies on agar media, .

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an estimation of living cells/ml can be obtained using this method, assuming the solid medium employed is suitable for reliable growth of the organism.

A third technique for measuring microbial growth is the turbidimetric method, in which a spectrophotometer is used to estimate cell numbers based on the density of a liquid culture (Figure 5.18). Readings are given as optical density (OD) at a given wavelength, and a growth curve can be generated over time. To relate actual cell numbers to OD units, a standard curve comparing the two must be made. If another culture medium or growth condition is employed, a new standard curve should be obtained for that condition to equate OD units to actual cell numbers. As part of this discussion, you should note and discuss the limitations and sources of error in all three methods of measuring microbial growth. [For a discussion of the interference of biofilms in obtaining accurate turbidimetric measurements of growth, see the feature box Explore the Microbial World: Stick or Swim.]

5.11–5.13 | Effect of Temperature on Microbial Growth The remaining sections of Chapter 5 pertain to environmental effects on microbial growth and how these may be used to control microbial growth. Based on information presented in these sections, convey to students that the genetic inventory of a given bacterium allows for a specific degree of adaptation when encountering a particular environmental stimulus (e.g., a change in pH or temperature). Though limited, the range of tolerance of a cell to a particular environmental factor can be impressive. For example, E. coli is capable of growth between 8°C and 48°C. These factors are also linked to microbial diversity; future chapters will describe extreme environments and their microbial inhabitants.

Every microorganism has a cardinal temperature profile that defines its minimum, optimum, and maximum temperature for growth (Figure 5.19). If one compares the temperature optima of microorganisms from a diversity of environments, four classes can be defined: (1) psychrophiles, (2) mesophiles, (3) thermophiles, and (4) hyperthermophiles (Figure 5.20).

Describe the general characteristics of each class of organisms before discussing the molecular adaptations that allow for existence within a temperature extreme. The strength of the biochemistry background of your students should determine the extent of detail provided in this regard. Students having a fundamental understanding of the structure and function of macromolecules, especially those of proteins and lipids, will more readily comprehend these concepts.

Discuss with students the fact that extremophiles show extensive adaptation to their environments; they do not merely tolerate the extreme condition but actually require it to remain viable. Contrast some of the adaptations possessed by psychrophilic bacteria (Figures 5.21 and 5.22) with those of thermophilic bacteria (Figures 5.23 and 5.24). Discuss the concept of cold-active and heat-active enzymes, many of which have useful properties that are exploited for use in biotechnology and industrial microbiology. In addition, the concept of membrane fluidity as a function of lipid composition, mentioned in the Chapter 2 material on cell membranes, is revisited here in a discussion of the membrane lipids of heatadapted microorganisms. Students will likely find Table 5.1 interesting, as it highlights the remarkable heat tolerance of hyperthermophilic Archaea compared to all other forms of life.

Remind the students that the heat stability of the membranes of these microorganisms is due .

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in large part to their possession of a lipid monolayer rather than a typical lipid bilayer (Section 2.7), which melts much more readily.

5.14–5.16 | Other Environmental Effects on Microbial Growth These sections describe microbial growth given variations in pH, osmotic stress, and oxygen.

Students will be intrigued by the ability of some microorganisms to grow at or below pH 1 or at pH 10 or higher (Figure 5.25 and Table 5.2). However, most will not have considered the problems each organism faces in generating ATP and maintaining a near neutral internal pH. Challenge students to consider a couple of scenarios to investigate outside of class: • Alkaliphiles live bathed in a sea of hydroxyl ions but can still generate a PMF (i.e., pump protons outside the membrane). How can this charge separation be established under these conditions to drive the synthesis of ATP? • Acidophiles, on the other hand, live in a sea of protons. What are the bioenergetic advantages and disadvantages of this environment for these organisms?

Salt has been used as a preservative for thousands of years. Ask students how salt can preserve meats and inhibit microbial growth by affecting the water activity of substances (Table 5.3). Despite the antimicrobial properties of high salt concentrations, some microorganisms either tolerate or absolutely require high salinity conditions for life. Organisms that can tolerate relatively high salt concentrations but do not require them for growth are halotolerant, such as species of Staphylococcus (Figure 5.26). Organisms that require high salt conditions are halophilic (Figure 5.26), and they deal with the osmotic stress of living in high salt environments by synthesizing compatible cytoplasmic solutes (Table 5.4).

Explain to students that extremophiles often require more than one extreme condition for growth and viability. Examples would be those organisms that thrive in the following environments: • Acidic hot springs (high temperature + low pH) = a thermophilic acidophile • Deep-sea hydrothermal vents (high temperature + high pressure) = a hyperthermophilic barophile • Saline soda lakes (high salt concentration + high pH) = a halophilic akaliphile • Antarctic saline lakes (low temperature + high salt concentration) = a psychrophilic halophile Quizzing students on the classification of extremophiles from a variety of different extreme environments provides an enjoyable review of key terms from this lecture.

Spend sufficient time on the topic of oxygen and microorganisms. Most microorganisms must deal with fluctuating levels of oxygen during growth, with some organisms requiring its presence, others tolerating its presence, and some absolutely requiring its absence for survival (Figures 5.27 and 5.28; Table 5.5). Most introductory microbiology courses will have a laboratory activity addressing this topic. However, students should learn the ways that living cells eliminate toxic derivatives of oxygen that are formed during the reduction of O2 (Figure 5.29). For example, aerobic organisms contain superoxide dismutase (SOD) in addition to catalase (Figures 5.30a, c, and d and 5.31). However, strict anaerobes lack SOD and thus are .

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rapidly killed in the presence of O2. A similar enzyme, superoxide reductase, has been found in obligately anaerobic Archaea that reduces superoxide to peroxide without producing O2 as a byproduct (Figure 5.30e). A peroxidase-like enzyme then reduces peroxide to water, again avoiding the formation of O2 (Figure 5.30b).

5.17–5.19 | Control of Microbial Growth In vitro methods of controlling microbial growth can be differentiated between physical methods (e.g., heat, radiation, and filter sterilization; Sections 5.17 and 5.18) and chemical methods (e.g., antiseptics, disinfectants, and sterilants; Section 5.19), and we will consider the physical methods first. Heat sterilization is probably the most common method used by microbiologists to eliminate microorganisms and includes dry heat, incineration, and autoclave sterilization. Use Figure 5.32 to discuss the kinetics of temperature on cell viability and the concept of decimal reduction time (D) as a quantitative tool for evaluating the effect of heat on a specific organism’s viability. A faster and more convenient method is to determine an organism’s thermal death time. Inform your students that this measurement varies depending on the species, and remind them that endospores are naturally much more resistant to heat exposure. Briefly review the structure of endospores (Section 2.16), whose components are responsible for their impressive heat resistance. In particular, discuss how water is retained and partitioned into a gel-like state from the interactions of Ca2+-dipicolinic acid and small acid-soluble spore proteins (SASPs). This is a good preface to a discussion of the autoclave and the kinetics of sterilization by steam heat under pressure (Figure 5.33). It is important for students to understand that autoclaves kill cells by heat, not by pressure. The temperature required to penetrate and kill endospores (121C) is well above the boiling point of water at 1 atm, and therefore pressure is increased to prevent the boiling of culture media at this temperature. Also note that the pasteurization process does not sterilize; it is a method used to reduce the number of microorganisms in a liquid, but it does not eliminate them completely.

Discuss the types of radiation commonly used to control microbial growth (microwave, ultraviolet, and ionizing) in some detail (Section 5.18; Figure 5.34). Include in your discussion the effects of radiation exposure on the viability of microorganisms (Figure 5.35). Provide examples of the radiation sensitivity of various microorganisms (Table 5.6), pointing out that the inactivation of viruses generally requires a stronger dose of radiation than does killing cells. Although not included in the table, you may wish to point out the impressive radiation resistance of endospores compared to vegetative cells, noting the difference between the killing dose for E. coli O157:H7 (3,600 Gy) and that for Clostridium botulinum endospores (39,600 Gy!).

Many medical and laboratory products are sterilized by radiation, such as tissue grafts, antibiotics and vaccines, and prepackaged, disposable supplies (e.g., syringes, pipettes, and needles). In addition, ionizing radiation as a means of sterilization is an accepted technology for decontaminating foods, such as fruit and poultry, in many countries, but it is banned in others. Many groups hotly protest its use. A discussion of the use of radiation to sterilize .

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foods and medical equipment might impress upon students the need to be informed when discussing controversial topics.

Another effective sterilization method is the use of filters. Filters are used to remove microorganisms from liquids and gases. Depth filters (Figure 5.36a), such as high-efficiency particulate air (HEPA) filters (as used in laminar flow hoods; Figure 5.34), are most commonly employed for this purpose. If heat-sensitive liquids containing larger particles are to be sterilized, depth filters are often used as prefilters to remove larger particles, whereas membrane filters containing tiny pores are used to remove microbial cells from the liquid (Figures 5.36b and c, 5.37, and 5.38).

Finally, discuss chemical methods of controlling microbial growth (Section 5.19), including the microbial actions of the three basic types of antimicrobial agents: bacteriostatic, bacteriocidal, and bacteriolytic (Figure 5.39). Explain that depending on the type of microorganism or pathogen to be controlled, these terms can also be applied to fungi (e.g., fungicidal) and viruses (e.g., viricidal). Also describe the details of the techniques for measuring antimicrobial activity, including minimum inhibitory concentration (MIC) and the disk diffusion technique (Figures 5.40 and 5.41, respectively). Identify some of the common industrial uses of antimicrobial chemicals, and explain the differences between antiseptics (or germicides), disinfectants, sanitizers, and sterilants (Table 5.7). A little additional time spent discussing Table 5.7 will help students link these agents to their effects on microbial cell structure and chemistry, which was covered in detail in Chapter 2. It is useful to mention that most antiseptics and germicides are used in handwashing, which is an important component of the 12-step program devised by the Centers for Disease Control and Prevention (CDC) to minimize the spread of antimicrobial drug-resistant pathogenic strains.

Answers to Review Questions 1. Cell division is a complex process, and the rate of division can vary widely depending on the physical and nutritional characteristics of a cell’s immediate environment. Cell division requires several proteins referred to as Fts proteins. Fts proteins interact to form an apparatus called the divisome, the key protein of which is FtsZ. FtsZ shows structural similarities to the eukaryotic tubulin protein, and its synthesis results in ring formation during cell elongation before division. Depolymerization of FtsZ occurs as cell constriction begins at the septum. Min proteins, especially MinE, are involved in directing FtsZ to the cell midpoint.

2. Replication of circular genomes of Escherichia coli is bidirectional from the origin of replication. During bidirectional replication, synthesis occurs in both a leading and a lagging fashion on each template strand, and this allows DNA to replicate as rapidly as possible. Cells of E. coli growing at doubling times shorter than 40 min contain multiple DNA replication forks. That is, a new round of DNA replication begins before the last round has been completed, and therefore, some genes are present in more than one copy.

This ensures that at generation times shorter than the time required to replicate the genome (a process that occurs at a constant maximal speed), each daughter cell receives a complete copy of the genome at the time of septum formation.

3. The protein MreB is the key shape-determining protein in prokaryotes, forming an actinlike cytoskeleton. MreB forms spiral-shaped bands just inside the cytoplasmic membrane .

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and defines the cell’s shape. Mutations in E. coli MreB protein would result in a spherical shape in the mutant. Support for this can be found in the fact that spherical cells, such as Staphylococcus, lack a gene encoding an MreB-like protein.

4. Bactoprenol transports peptidoglycan precursors across the cytoplasmic membrane by rendering them sufficiently hydrophobic to pass through the membrane interior. Once in the periplasm, bactoprenol interacts with enzymes called transglycosylases, which insert cell wall precursors into the growing point of the cell wall and catalyze glycosidic bond formation.

5. The specific growth rate constant (k) is the change in cell number or cell mass per unit time. The generation time (g) is the time required for the formation of two cells from one. Thus, g is the time required for a population of cells to double (during exponential growth).

6. The growth cycle of a population of cells consists of four phases: lag, logarithmic (exponential), stationary, and death. The lag phase is the period of time in which cells are synthesizing necessary enzymes to begin exponential growth. The exponential phase is the period of maximal growth rate in which the cells are in their healthiest state. At stationary phase, cell growth levels off as nutrients are consumed and toxic metabolic byproducts build up in the batch culture. Finally, the death phase is characterized by an increasing number of nonviable cells that may also undergo lysis.

7. A chemostat can regulate growth rate and cell numbers independently by independently adjusting the nutrient concentration and the dilution rate. Over a broad range of dilution rates, the bacterial concentration in the chemostat remains relatively constant. Thus, as the dilution rate increases, the growth rate also increases. At a constant dilution rate, increasing the limiting substrate will also increase the growth rate and cell yield (to a point).

8. Total microbial cell counts, such as those obtained from microscopic methods of enumeration, include both viable (living) and intact but nonviable (dead) cells, whereas viable cell counts include only live cells.

9. Turbidity, as measured by a spectrophotometer, can be used to estimate cell numbers because cells scatter light. An increase in cell density results in an increase in light scattered (to a point). The amount of light absorbed (or transmitted) will be directly proportional to cell number during exponential growth.

10. In relation to their growth temperature optima, microorganisms can be divided into four broad classes: psychrophiles, with low temperature optima; mesophiles, with midrange temperature optima; thermophiles, with high temperature optima; and hyperthermophiles, with very high temperature optima.

11. Psychrophiles have an optimal growth temperature of −15°C or lower, a maximum growth temperature below 20°C and a minimal growth temperature at 0°C or lower. Psychrotolerant microorganisms grow at 0°C but have optima of 20–40°C. The adaptive features include heat stable proteins and enzymes; increased ionic bonds between basic and acidic amino acids and their often highly hydrophobic interiors; protective solutes such as di-inositol phosphate, diglycerol phosphate, and mannosyl.

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glycerate; a lipid monolayer (which prevents the membrane from melting at high growth temperatures); and ether linkages.

12. An alkaliphile is an organism that has a growth pH optimum of 8 or higher. Alkaliphilic microorganisms are typically found in highly alkaline habitats, such as soda lakes and high-carbonate soils. Certain alkaliphiles they produce hydrolytic exoenzymes, such as proteases and lipases. The exoenzymes of alkaliphiles function well at alkaline pH; these enzymes are produced commercially on a large scale and added as supplements to laundry detergents to remove protein and fat stains from clothing.

13. Compatible solutes are water soluble organic molecules (sugars, alcohols, amino acid derivatives) that maintain homeostasis when organism is transferred from high to low aw.

Glycerol.

Examples: Glycine betaine, sucrose and trehalose, and dimethylsulfoniopropionate. Glycerol is the most common compatible solute in xerophilic fungi, which grows at the lowest water activities known.

14. A facultative aerobe is an organism that can grow in either the presence or absence of O2.

Obligate anaerobes are organisms that are inhibited or killed by O2. Examples of obligate anaerobes are Methanobacterium formicicum and Clostridium. As oxygen is fatal to anaerobes, unlike aerobes, their culture medium should be completely devoid of oxygen. A chemical called a reducing agent is added to the culture medium to remove traces of O2 by reducing it to water (H2O). An example is thioglycolate, which is present in thioglycolate broth, a medium commonly used to test an organism’s requirements for O2.

15. Superoxide dismutase is the most important free-radical scavenging enzyme of aerobes.

Catalases are porphyrin-containing proteins, while superoxide dismutases are metalcontaining proteins, the metals being copper and zinc, manganese, or iron.

16. Decimal reduction time is the time required for a tenfold reduction in the viability of a microbial population at a given temperature. Thermal death time is the time required to kill all cells in a culture at a given temperature. The presence of bacterial endospores would greatly affect both of these values. Because of the heat stability of endospores, both the decimal reduction time and the thermal death time would increase for endospores, and it may be necessary to increase the temperatures used to obtain these values. For example, the decimal reduction time for vegetative cells of Clostridium botulinum may be one minute at 105C, but this temperature would be insufficient to kill endospores of this bacterium.

17. Autoclaves use steam heat under pressure to sterilize materials. Whereas water boils at 100C at atmospheric pressure, liquids in an autoclave can reach 121C (or more) without boiling by increasing the pressure in the chamber. Because of the remarkable heat stability of endospores, the elevated temperatures obtained in an autoclave are required for sterilization of materials such as culture media. Boiling at atmospheric pressure does not destroy endospores.

18. Ionizing radiation can produce electrons, hydroxyl radicals, and hydride radicals, all of which can degrade and/or permanently alter the macromolecules essential for life, including nucleic acids, proteins, and lipids. .

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19. Membrane filters mounted to a syringe, pump, or vacuum apparatus are commonly used to sterilize heat-sensitive liquids.

20. A series of broth tubes containing increasing concentrations of the bacteriocidal substance is prepared; the concentrations should run from 0% to considerably above the concentration causing complete inhibition of growth. All tubes are inoculated with a uniform quantity of the chosen Escherichia coli strain. After incubation, often overnight, the series is examined for growth. The minimum inhibitory concentration (MIC) of the bacteriocide is the concentration in the tube containing the lowest concentration of bacteriocide that shows no growth of the organism, as determined by the turbidity of the culture.

21. Agents called disinfectants directly target pathogens, although all microorganisms and endospores may not be eliminated. They are corrosive in nature (like phenol) and hence used only on inanimate objects. Antiseptics are chemicals that kill or inhibit the growth of microorganisms but are sufficiently nontoxic to animals to be applied to living tissues.

Sterilants (also called sterilizers or sporicides) destroy all microorganisms, including endospores. Chemical sterilants are used for decontamination or sterilization in situations where it is impractical to use heat or radiation. Cold sterilization is a process by which gases such as ethylene oxide, formaldehyde, or peroxyacetic acid are used to treat objects within an enclosed device. Sodium hypochlorite or amylphenol is used for instruments that cannot withstand high temperatures or gas.

Several factors affect the efficacy of any chemical antimicrobial agent. For example, many antimicrobial agents are bound and inactivated by organic matter; thus, disinfecting a kitchen countertop littered with spilled foods is more difficult than disinfecting a clean countertop. Furthermore, bacteria often form biofilms, covering surfaces of tissue or medical devices with layers of microbial cells embedded in polysaccharides. Biofilms may slow or even completely prevent penetration of antimicrobial agents, reducing or negating their effectiveness.

Answers to Application Questions 1. Make a slurry out of the soil sample by dissolving it in distilled water. Boil it and then allow it to cool. Pour it onto a nutrient agar plate. Endospores—being heat resistant—will grow. a. A total count of microbial numbers in a culture or natural sample can be done by simply observing and enumerating the cells present. The most common total count method is the microscopic cell count using the Petroff–Hausser counting chamber. b. Viable counting is done by using serial dilutions followed by the spread-plate method or the pour-plate method.

2. Enzymes in cells of E. coli would quickly denature at 110°C, causing the cessation of metabolism, DNA replication, transcription, and translation. In addition, membrane lipids would be compromised in E. coli cells at such a high temperature, and death of the cells would soon follow. Pyrolobus fumarii contains enzymes and other macromolecules that function optimally at temperatures above 100C. A temperature of 40C would be much too cool for proper catalytic activity and membrane flexibility in P. fumarii. Metabolic .

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reactions in a hyperthermophile would occur too slowly at 40C to support growth. The macromolecules of E. coli are optimally adapted to temperatures near 40C, whereas those of P. fumarii are adapted to temperatures near 110C. 3. Formaldehyde is a bacteriocidal agent that binds tightly to the cellular target and kills the cell. Pine oils and betadine are bacteriostatic; they typically inhibit important biochemical processes such as protein synthesis, and bind relatively weakly. If the agent is removed, the cells can resume growing. Benzalkonium chloride, triclosan, and penicillin are bacteriolytic agents; they kill cells by lysing them and releasing their cytoplasmic contents.

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CHAPTER

6

Microbial Genomics

Summary Genomics is the sequencing, mapping, and comparison of the genomes of organisms. It has become an extremely useful and informative subdiscipline in the biological sciences. Chapter 6 presents information concerning microbial genomic analyses, including the genomics of bacterial virulence, the effects of horizontal (lateral) gene flow on genome evolution, and the field of metagenomics. The major advances in molecular technology⎯from shotgun sequencing to second-, third-, and now fourth-generation sequencing to powerful computational bioinformatics⎯ have made it possible to sequence and analyze large, complex genomes. The focus here is on the analysis, function, regulation, and evolution of microbial genomes, as well as some of the interesting outcomes of genome sequencing projects.

6.1 | Introduction to Genomics This chapter presents students with current applications of technologies recently developed from fundamental research in bacterial genetics (see Chapters 10 and 11). Inform your students that in vitro techniques developed during the 1970s usually pertained to single genes or small segments of a genome and were time and labor intensive. Improvements in DNA sequencing and data analysis capabilities now allow one to analyze entire genomes or even multiple genomes from natural samples. Provide a brief historical perspective on genome sequencing. The first genome to be sequenced occurred in 1976 and was that of MS2, an RNA virus whose genome contains only 3569 nucleotides. That was followed a year later by the sequencing of the first DNA genome from the single-stranded DNA virus X174 (see Section 9.3). A group led by Fred Sanger, who pioneered a new dideoxynucleotide DNA sequencing technique (see Section 6.2), carried out this work. The first cellular genome (the 1,830,137-bp genome of Haemophilus influenzae) was sequenced many years later in 1995 at The Institute for Genomic Research (TIGR). Since that time, the genomes from thousands of other organisms, including humans and many other animals, have been sequenced. Students may be interested to learn that the largest genomes (in terms of the number of genes) thus far sequenced are not those of animal species but rather belong to some higher plants (black cottonwood and rice) and two protozoans (Paramecium tetraurelia and Trichomonas vaginalis). By comparison, the human genome, though much larger in terms of total numbers of base pairs, has several thousand fewer genes than these organisms (see Section 6.6). The first diploid human genome was completed and released in late 2007. .

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Provide students with the website for an up-to-date database of genome sequencing projects that have either been completed or are in progress (http://www.genomesonline.org/).

Table 6.1 provides a list of selected prokaryotic genomes, including a number of important pathogens. Hyperthermophiles and other extremophilic microorganisms are also featured, as they may have important uses in the biotechnology industry. Because the cost of genome sequencing has dropped dramatically in recent years, genomes from a variety of organisms of interest have been or are being sequenced, regardless of whether they have medical or biotechnological significance.

6.2 | Sequencing Genomes The various “generations” of DNA sequencing (Table 6.2) refer to instances in which major technological advancements lead to higher throughput and lower costs. Discuss the generations of DNA sequencing beginning with the first generation, the Sanger dideoxy method (Figures 6.1 and 6.2), stressing the fact that most sequencing is now automated and can be performed rapidly and on a massive scale.

Discuss the methods used to obtain genomic DNA sequences: restriction digest of the DNA, cloning of fragments and generation of a genomic library, and shotgun sequencing of the library clones. Depending upon the molecular background of the students, topics concerning sequencing and annotating genomes may be somewhat abstract and confusing. Explain that once random DNA fragments (called contigs) have been sequenced via the shotgun approach, the fragments are assembled using overlapping regions. It is most desirable to have approximately ten replicate sequences (a 10-fold coverage) for any given region of the genome so that errors can be virtually eliminated. For example, if 9 out of 10 replicates indicate that cytosine is present at a particular locus on the DNA strand, then the consensus DNA sequence would designate a “C” at that point in the sequence.

Sequencing technology has advanced quickly, and new and much faster DNA sequencing technologies have emerged. Second-generation sequencing techniques, including 454 pyrosequencing and Illumina/Solexa sequencing, require the use of massively parallel methods in which miniaturization and high-throughput, computerized detection methods are key (Figure 6.3). Whether you choose to cover these methods in detail will depend on the scope of your course, but students should be aware that these techniques allow DNA sequencing that is 100 times faster than Sanger dideoxy sequencing. The entire procedure, from picking colonies to isolating DNA and sequencing, is done using sophisticated robotics.

The primary innovation of third-generation sequencing is that it allows single molecules of DNA to be sequenced. This is done using either automated microscopic analysis of fluorescently labeled nucleotides (HeliScope single molecule sequencing) or differentially labeled nucleotides that emit flashes of light as they polymerize in a “zero-mode waveguide” nanocontainer (single-molecule real-time sequencing). Instead of optically detecting labeled nucleotides, fourth-generation ion torrent sequencing measures the release of a proton with each addition of a complementary nucleotide to a template strand of DNA (Figure 6.4a). Nanopore sequencing technology measures the change in electrical current across a transmembrane protein nanopore that separates double-stranded DNA into single-stranded DNA .

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(Figure 6.4b). Both of these methods are extremely fast, with ion torrent technology capable of sequencing up to 3 billion base pairs in a single day!

As mentioned above, a computer carries out the task of assembling random DNA fragments into a single molecule by analyzing overlaps in the fragments (Figure 6.5). However, there are often gaps in draft genome sequence data that need to be filled. The polymerase chain reaction (PCR; see Section 11.3) can be used to generate missing spans of DNA by targeting sequences that flank these gaps. While this technique ultimately provides a highly desirable closed genome with no missing sections of DNA, it is a time-consuming process that requires much effort and, therefore, adds significant cost to sequencing projects.

6.3 | Bioinformatics and Annotating Genomes After the sequence fragments of a genome are assembled, ask your students what remains to be done with this long string of nucleotide sequence data. The answer is that the genome must now be annotated, which means that it needs to be “deciphered” to reveal the genes present.

Merely sequencing a genome tells a scientist nothing unless protein-encoding genes (or putative protein-encoding genes) are identified. The first step in annotation is to search for open reading frames (ORFs), sequences of DNA that can be translated to individual polypeptides.

Computer programs are used to predict potential ORFs by (1) looking for start and stop codons in all possible reading frames (Section 4.11); (2) identifying ribosomal binding sites near start codons; (3) calculating whether the potential ORF shows codon bias (Table 6.3); and (4) by searching for regions of the DNA sequence that are homologous (share a similar sequence) to known ORFs in other organisms (Figure 6.6).

Point out to students that in most genomes, a relatively high percentage of ORFs — in some cases up to 40% — are uncharacterized. The gene products of these ORFs are usually referred to as hypothetical proteins since their functions have not been identified. Still other genes encode RNA molecules that are not translated (e.g., tRNAs, rRNA, and several types of regulatory RNA molecules that are difficult to detect using conventional autoannotation programs). Use this opportunity for a quick molecular biology review by asking your students to name as many types of noncoding RNA molecules as they can. To pique student interest in bioinformatics and genome annotation, you may wish to visit websites where major sequencing projects are detailed (for example, see the Department of Energy’s Joint Genome Institute web page; www.jgi.doe.gov).

6.4 | Genome Size and Content Discuss the size range of prokaryotic genomes, noting that a strong correlation exists between genome size and ORF content (~1,000 ORFs/megabase; Figure 6.7). This is not the case in eukaryotes, in which most of the genome consists of noncoding DNA (see Table 6.5). Ask your students how many genes are required for life. The genomic requirements of even the smallest free-living cells, which typically encode about 1400 genes, are greater than those of parasitic or endosymbiotic cells, which rely on their hosts for certain metabolic needs and thus can have fewer ORFs. One of the smallest bacterial genomes to have been sequenced to .

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date is from the insect endosymbiont Hodgkinia cicadicola, containing about 144 kbp and a mere 169 ORFs (Figure 6.8 and Table 6.1). By contrast, the largest bacterial genome sequenced is from the gliding myxobacterium Sorangium cellulosum at just over 13 Mbp (Table 6.1).

Impress upon students that the complement of genes possessed by an organism defines its biology. A good example is the obligate parasite Treponema pallidum, the causative agent of syphilis (Sections 14.20 and 29.12). This parasite lacks genes for amino acid biosynthesis and, as might be predicted, it obtains amino acids by degrading host peptides for survival.

One could also predict that hyperthermophiles must contain certain thermostable enzymes, and thus bioinformatics analyses of the genes and metabolic pathways in these organisms can identify possible biotechnological applications. Figure 6.9 shows how one can use genome information to construct a summary model of metabolic pathways and transport systems predicted to be present in an organism.

When one compares genes from a number of prokaryotes, certain trends appear. For example, metabolic genes are generally the most abundant ORFs of known function. Use Figure 6.10 and Table 6.4 to evaluate gene categories as a function of genome size. Note that the smaller the genome, the greater the percentage of genes pertaining to DNA replication and protein synthesis. In contrast, the percentage of transcriptional regulatory genes increases with genome size. Because protein synthesis is an essential process in all living systems, the percentage of genes related to this process would be expected to be higher in small genomes than large ones, the latter of which contain more genes with nonessential functions.

6.5 | Genomes of Organelles Review the endosymbiotic theory with your students, and remind them that the mitochondria and chloroplast organelles found in eukaryotes contain their own genomic DNA. Note also that these present-day organelles are closely related to prokaryotes (Section 17.1).

The chloroplast genome consists of a small circular DNA molecule (~120 kbp–160 kbp) that contains two inverted repeats (6 bp–76 bp) (Figure 6.11). It should be logical to students that chloroplasts contain genes for photosynthesis and autotrophy, in addition to rRNA genes for their ribosomes, which are 70S, as in prokaryotes. Point out that some genes for photosynthesis are encoded in the nuclear genome of the eukaryotic cell, suggesting an evolutionary migration long after a stable endosymbiosis was established.

The mitochondrial genome primarily encodes proteins for oxidative phosphorylation, in addition to the rRNAs and tRNAs required for protein synthesis within the organelle.

Figure 6.12 shows a map of the human mitochondrial genome, which consists of 16,569 bp and encodes only 13 proteins, 22 tRNAs, and 2 rRNAs. Inform the students that although the proteomes of most mitochondria consist of several hundred proteins, only a relatively small percentage of these proteins is synthesized within the organelle itself (Figure 6.3). Instead, these proteins are made in the cytoplasm using cellular (80S) ribosomes and transcripts produced by genes located in the nucleus. The proteins are then transported into the mitochondria to perform their functions. Give an overview of the differences in mitochondrial

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genomes, including the wide range in the number of protein-encoding sequences found in them (as few as 3 to as many as 62), using the representative examples presented in the text.

In biology, it seems there are always exceptions to the rules. It may interest students to know that the “universal” genetic code is not truly universal. A few organisms and some mitochondria use variations of the genetic code in that certain codons produce abnormal results during translation. For example, in some cases, a stop codon may act as a sense codon, resulting in the incorporation of an amino acid rather than halting translation. This is the case in animal and yeast mitochondria, where UGA, normally a stop codon, encodes tryptophan.

This section also addresses symbiont bacteria, which often have tiny genomes (Figure 6.14). In some cases, they contain fewer genes than organelles and even some viruses. As one might guess, these bacteria are not free-living cells but instead are completely dependent upon their hosts for survival; some host organisms even contain multiple symbionts within the same cell (Figure 6.15). In return for a sheltered habitat, these degenerate bacteria provide essential metabolites, such as vitamins and amino acids, to the host cell.

Considering this mutualistic dependency, one could make a strong case that these symbionts should actually be considered organelles. However, unlike mitochondria, for example, bacterial symbionts are found in cells of only certain tissues of the host organism. In addition, symbiont proteins are encoded and synthesized entirely within the symbionts themselves, and there is no evidence for the transfer of essential genetic material from the symbiont to the host cell nucleus.

6.6 | Eukaryotic Microbial Genomes The genomes of a large number of microbial eukaryotes and higher eukaryotes have now been sequenced. Focus your discussion on the eukaryotic microbial genomes shown in Table 6.5. Note that a higher chromosome number does not correlate directly with the number of protein-encoding genes, as students often predict. As a clear example of this fact, point out that the haploid genomes of humans and mice have ~25,000 protein-encoding genes compiled into 23 chromosomes, whereas those of the single-celled protozoan Trichomonas vaginalis contain 60,000 genes in only 6 chromosomes.

Among single-celled eukaryotes, the yeast S. cerevisiae is most widely used as a model organism, and therefore it is useful to discuss aspects of its genome in some detail. Discuss some of the key points presented by the authors. An interesting question that may arise is, “What is the minimum number of genes yeasts need for survival and reproduction?” Point out that the use of knockout mutations (Section 11.5) indicates that only about 900 ORFs are essential in S. cerevisiae, while ~3100 are clearly dispensable. Also point out that, in general, smaller eukaryotic genomes contain fewer introns, and, as in prokaryotes, some eukaryotic microbial genomes possess only a few or no introns (Figure 6.16). Note also that although introns are present in the yeast genome, there are only 225 of them, and they are all located near the 5' end of the gene. In contrast, higher eukaryotes, such as the fruit fly Drosophila melanogaster and the worm Caenorhabditis elegans, have an average of four to five introns per gene. Complete your discussion of eukaryotic microbial genomes by summarizing significant observations from other eukaryotic microorganisms (e.g., Trichomonas, Dictyostelium, Entamoeba histolytica, and Trypanosoma brucei). .

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6.7–6.9 | Functional Genomics This section introduces students to the variety of “ome” and “omics” terms that are now in common usage, and the list can be bewildering at first. Table 6.6 nicely summarizes the key “omics” terms that biology students are likely to come across in most of their coursework.

Students may initially show apprehension toward this list of words, but simplify things for them by explaining that the suffix “ome” in each case refers to the total complement of whatever parameter is described by the prefix of each word. A proteome, therefore, would be all of the proteins encoded by a cell’s genetic material.

It is important for students to understand that because not all genes are constitutively expressed, the mere presence of a gene within a genome does not mean that the gene will be transcribed under a given set of conditions. The use of microarrays allows one to analyze gene expression (transcription) of an entire genome under specific conditions, and the resulting complement of RNA produced under these conditions is referred to as a transcriptome.

Discuss the technology and procedures employed in the construction and use of gene chips (Figure 6.17). The DNA (e.g., synthesized oligonucleotides or cDNA) immobilized on the chip can be hybridized to mRNA extracted from cells grown under a specific set of conditions. Stress that the mRNA must first be amplified, which is usually accomplished by converting the mRNA to cDNA using reverse transcriptase (see Section 11.3), followed by amplification of the cDNA via PCR. An alternate method uses T7 RNA polymerase to generate cRNA. The cDNA or cRNA can then be hybridized to the chip.

Discuss whole genome arrays, which contain DNA segments that cover the entire genome.

The human genome chip contains 47,000 human transcripts with room to add 6500 additional oligonucleotides for clinical diagnosis (Figure 6.18a). Figure 6.18b shows the results from microarray analysis of a portion of the yeast genome. Discuss the meaning of the fluorescent hybridization signal differences observed on the chip with the students. Gene expression can also be measured using high-throughput sequencing technologies (Section 6.2) in a technique called RNA-Seq. In this method, a cell’s entire transcriptome under a given set of growth conditions is sequenced, and this provides both qualitative and quantitative gene expression data (Figure 6.19). Continue your discussion with the various applications of DNA chips and RNA-Seq technologies (e.g., global gene expression and microorganism identification in the food industry and in clinical medicine).

The pioneering technology for the burgeoning field of proteomics (and also translatomics, which looks only at the proteins present in a cell at a given time rather than the entire complement of proteins encoded by a genome) was two-dimensional polyacrylamide gel electrophoresis (Figure 6.20). Several newer methods still require elaborate genetic techniques, such as transposon and site-directed mutagenesis, mass spectrometry, and genomic library construction, in addition to extensive computer analysis capabilities. However, the technology is advancing rapidly, and high-pressure liquid chromatography (HPLC) is now routinely used to separate protein mixtures. Separated proteins can then be digested and the peptides identified using mass spectrometry. If the genome sequence of an organism has been obtained, then proteomic data can be screened against genome sequence data from other organisms in a process known as comparative genomics. These techniques are carried out using powerful computer programs that search extensive sequence databases. Usually, the protein sequence rather than the DNA sequence is used for comparative genomics studies because changes in .

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DNA sequence do not necessarily result in a changed protein (Section 4.11). Proteins having greater than 50% amino acid sequence identity can be expected to have similar functions since they often have conserved domains that bind the same ligand or perform the same catalytic activity (Figure 6.21).

The interactome refers to the complement of the various molecular interactions between proteins (or between proteins and other macromolecules, such as DNA and RNA) in an organism. Because of the complexity and inherent difficulty of illustrating the entire interactome of an organism, such data are usually reduced to specific aspects of the cell, such as a particular biosynthetic pathway or nutrient uptake mechanism. Figure 6.22 shows both the core interactome that regulates motility in Campylobacter jejuni (Figure 6.22a) and a comprehensive interactome that includes peripheral proteins that interact with core chemotaxis proteins (Figure 6.22b).

The metabolome is defined as the complete set of metabolic intermediates and small molecules produced in an organism. The complexity of determining the identity of these metabolites is the reason that this field has lagged behind genomics and proteomics. The most promising new tools for metabolomic studies are those developed as variants of mass spectrometry, such as MALDI–TOF mass spectrometry (Figure 6.23). These high-resolution techniques are extremely sensitive and enhance the ability to obtain the correct molecular formula for any small molecule.

All of the “omics” discussed in these sections can be combined to obtain a comprehensive understanding of an organism, community, or even an entire ecosystem in an integrated approach called systems biology (Figure 6.24). Using this methodology, emergent properties of an organism (or community, etc.) are revealed over time as “omics” studies provide new data to compile into a comprehensive analysis. The limiting factor in such studies is computing power.

The amount of data that can now be generated for just a single organism is immense, so to apply these techniques to entire ecosystems will require massive computing power.

6.10 | Metagenomics The focus of metagenomics, also called environmental genomics, is to characterize microbial diversity in a particular environment by analyzing pooled DNA or RNA from an environmental sample without actually culturing and isolating individual organisms. Impress upon your students that the majority of microorganisms in an environmental sample have never been isolated in pure culture, yet their presence can be ascertained with metagenomic analysis. Using today’s technology, it is even possible to carry out genomic, transcriptomic, and proteomic analyses on single cells isolated from a natural sample (see Explore the Microbial World: Genomics, One Cell at a Time).

Metagenomic sequencing projects have revealed that extreme environments generally have low levels of biodiversity, whereas more hospitable and nutrient-rich environments show vast species diversity. It may also interest students to know that most of the genes found in natural environments do not belong to cells but are instead derived from viruses. Nevertheless, one of the greatest benefits of metagenomic studies is that it allows one to fully explore the microbial diversity and composition of natural samples in a way that is simply not possible using traditional culturing methods (Figures 6.25 and 6.26). .

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6.11 | Gene Families, Duplications, and Deletions Discuss the concept of gene families with students, pointing out that each “family” consists of genes that are homologous⎯they show a high sequence similarity due to a common evolutionary history. Stress to students that although these data may provide important information on the evolutionary history of an organism, the process of horizontal (lateral) gene transfer, discussed next, makes these analyses more difficult to interpret. Differentiate between paralogs, genes whose similarity is the result of a gene duplication event at some point during an organism’s evolution, and orthologs, genes similar to those found in another organism because the two species share a common ancestor (Figures 6.27 and 6.28).

6.12 | Horizontal Gene Transfer and Genome Stability Horizontal (or lateral) gene transfer (HGT) refers to DNA exchange between cells by a mechanism other than the usual vertical inheritance of genetic material from parent cell to daughter cell (Figure 6.29). As will be discussed in detail in Chapter 10, HGT in prokaryotes occurs through any of at least three mechanisms: transformation, transduction, and conjugation. The horizontal transfer of genes can often complicate phylogenetic analyses of organisms in that it can be difficult to detect unless large genetic differences exist between participating organisms. Of course, if genome sequence data are available, then this can facilitate detection of horizontal transfer between organisms. Point out the example in the text, wherein eukaryotic genes have been found in the human bacterial pathogens Chlamydia and Rickettsia.

A type of exaggerated HGT has also recently been developed in vitro in which whole bacterial genomes have been artificially synthesized and transferred into a suitable host cell.

The process requires a complex series of molecular techniques, including oligonucleotide synthesis, assembly via recombination, cloning of bacterial artificial chromosomes into E. coli, transformation into the yeast S. cerevisiae, and finally, insertion into the host cell (a strain of Mycoplasma, in this case).

Mobile DNA elements, such as plasmids, integrated viruses, and transposons, play important roles in facilitating the evolution of genomes (Figure 6.30). Transposons can transfer genes between organisms and are capable of creating large rearrangements within bacterial chromosomes. Bacteria that experience frequent stressful conditions, including those within human sites of infection, tend to have active chromosome rearrangements with many transposons and insertion sequence elements. Conversely, bacteria that exist in comparatively stable evolutionary niches tend to have lost most of these elements.

6.13 | Core Genome versus Pan Genome Distinguish between the core genome and the pan genome of a prokaryotic species. The core genome consists of genetic material shared by all strains of a species, whereas the pan genome includes all other DNA specific to a particular strain, including genes contained in .

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transposable elements, chromosomal islands, viral genomes, and plasmids (Figures 6.31 and 6.32).

The study of core versus pan genomes has revealed the existence in some strains of clusters of specialized genes that are not essential components of the chromosome. These gene clusters, called chromosomal islands, can add significant size to a cell’s genome and also impart new genetic capabilities to the cell that may alter its phenotype. Many virulence genes are clustered in specific types of chromosomal islands called pathogenicity islands (PAIs).

A comparison of the genetic map of the nonpathogenic laboratory E. coli strain K-12 with that of the urinary tract pathogen, E. coli strain 536, and a second pathogenic strain, E. coli strain 073, shows the presence of PAIs in each pathogenic strain that are not present in strain K-12 (Figure 6.33).

Answers to Review Questions 1. Genomics is the discipline of mapping, sequencing, analyzing, and comparing genomes.

The first bacterial genome sequenced was the chromosome of Haemophilus influenza, published in 1995.

2. In the Sanger procedure the sequence is determined by making a copy of the original single-stranded DNA using the enzyme DNA polymerase. This enzyme adds deoxyribonucleoside triphosphates to a growing DNA chain. However, in Sanger sequencing, small amounts of the corresponding dideoxyribonucleotide are included in each of four incubation mixtures, one for each of the four bases—adenine, guanine, cytosine, and thymin.

The dideoxy analog is a specific chain-termination reagent, and because it lacks a 3′hydroxyl, it prevents further elongation of the chain after its insertion.

3. Codon bias is the relative proportions of different codons encoding the same amino acid.

It varies in different organisms. Some genes encode RNA molecules that are not translated. Such genes therefore lack start codons and may well have multiple stop codons within the gene. In addition, these genes lack codon bias; consequently, they will not be recognized by programs that search for ORFs. Some noncoding RNAs are easy to detect because they are well characterized and are highly conserved. These include tRNAs and rRNAs. 4. Genome size refers to the number of base pairs comprising the genetic constitution of an organism. In prokaryotes, each megabase pair (Mbp) of DNA can be expected to encode about 1000 ORFs (i.e., approximately 1 ORF/kbp), and this ratio is consistent regardless of genome size. In other words, in prokaryotes, the larger the genome, the more ORFs one can expect the genome to contain.

5. The percentages of genes related to protein synthesis and DNA replication are highest in organisms with small genomes, whereas in larger genomes, the percentage of transcriptional regulatory genes is greatest.

6. Chloroplast genomes are much larger than mitochondrial genomes. Chloroplast genes commonly contain self-splicing introns. Mitochondrial genomes mainly encode proteins involved in oxidative phosphorylation. The number of protein-encoding genes can vary greatly in mitochondria, from less than 5 to more than 60. In addition, mitochondria have .

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a much simpler genetic code, probably resulting from selection pressures of a smaller genome.

7. We reach this conclusion after systematically inactivating each gene in turn with knockout mutations. Knockout mutations cannot normally be obtained in essential genes in a haploid organism. However, yeast can be grown in both diploid and haploid states. By generating knockout mutations in diploid cells and then investigating whether they can also exist in haploid cells, it is possible to determine whether a particular gene is essential for cell viability. Using knockout mutations, it has been shown that around 900 yeast ORFs (17%) are essential.

8. Genome refers to the entire complement of an organism’s genes, and genomics describes the discipline of mapping, sequencing, analyzing, and comparing genomes. Proteome refers to all of the proteins encoded by an organism’s genome, whereas all the proteins present in a cell or organism at any one time is a translatome. Proteomics, therefore, describes the large-scale study of the structure, function, and regulation of proteins of an organism. The transcriptome refers to the complement of all RNA produced by an organism under a specific set of conditions.

9. A 2D gel of a cell’s proteins shows the number of proteins present in that cell under a particular condition. Each spot on the gel represents a polypeptide that can be identified by biochemical or genetic means. The protein present at each spot in the gel can be excised and identified using modern techniques, such as MALDI–TOF mass spectrometry. Once deciphered, the structural features of a protein give clues as to its function.

Additional insights can be inferred from the presence or absence of a given protein in the gel (i.e., the patterns of gene expression) depending upon the conditions in which the cells were grown.

10. The study of metabolomics has been lagging behind proteomics because of the immense chemical diversity and often transient nature of small metabolites present in a cell. This presents enormous technical challenges for improving the resolution of detection and thus the identification of these molecules.

11. The approach of systems biology is to compile the immense amounts of data obtained from “omics” studies and then develop a computational model of an organism or system under study, with the expectation being that its behaviors and/or properties can be predicted under a given set of conditions.

12. Gene expression can be measured in uncultured bacteria (e.g., bacteria present in environmental or clinical samples) using microarrays and RNA-Seq technologies.

13. Several environments have been surveyed by large-scale metagenome sequencing projects. Extreme environments, such as acidic runoff waters from mines, tend to have low species diversity. Consequently it has been possible to isolate community DNA and assemble much of it into nearly complete individual genomes. Even if complete genomes cannot be assembled, much useful information can be derived from metagenomic surveys. For example, environments can be analyzed for the presence and distribution of different taxonomic groups of bacteria.

14. An example of a gene family that contains both paralogous and orthologous genes is the one encoding lactate dehydrogenase (LDH) enzymes. They are paralogous in humans .

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and orthologous in the lactic acid bacterium Lactobacillus. Isoenzymes are encoded variants of paralogous genes that are structurally distinct yet highly related carrying out the same enzymatic reaction.

15. Evolutionary studies based upon rRNA analyses use rRNA genes as a molecular chronometer. Evolutionary distance is calculated as the percentage of nonidentical sequences between any two organisms. This procedure relies on the assumption that the mutation rate of rRNA genes is similar among all organisms, and that those mutations that become fixed in a given organism are passed down over time to progeny cells. Horizontal gene transfer between unrelated organisms can introduce genetic changes that do not reflect the cell’s endogenous mutation rate and thus make interpretation of sequence similarities based upon rRNA sequences difficult or misleading. Clues leading to the detection of horizontally transferred genes include the presence of proteins that are found only in distantly related organisms, an abnormal G+C ratio in certain genes that does not match that found in the majority of the genome, and a codon bias that is inconsistent with that used in the rest of the genome.

16. Horizontal gene transfer refers to transfer from one cell to another by means other than the usual (vertical) inheritance process in which the genome is transferred from mother cell to daughter cell. Horizontal gene transfers can be detected in genomes once the genes have been annotated. The presence of genes that encode proteins typically found only in distantly related species is one signal that the genes originated from horizontal transfer.

However, another clue to horizontally transferred genes is the presence of a stretch of DNA whose guanosine/cytosine (GC) content or codon bias differs significantly from the rest of the genome. An example of horizontal transfer exists with the organism Thermotoga maritima, a species of Bacteria, which was shown to contain over 400 genes (greater than 20% of its genome) of archaeal origin. Of these genes, 81 were found in discrete clusters. This strongly suggests that they were obtained by horizontal gene transfer, presumably from thermophilic Archaea that share the hot environments inhabited by Thermotoga.

17. Chromosomal islands, such as pathogenicity islands, are flanked by inverted repeats, suggesting that they were acquired, and are transmitted, via transposition. Some also carry genes for integrase. In addition, the G+C content of these islands often differs from that of the host chromosome, and some strains of a species may possess a chromosomal island whereas others do not.

18. A pathogenicity island is a type of chromosomal island (a relatively large chromosomal insert of foreign origin) that contains genes that confer pathogenic properties to a cell.

The transfer of pathogenicity islands can convert a nonpathogenic strain of a species to a virulent strain.

Answers to Application Questions 1. The major challenges of obtaining complete sequences of eukaryotic genomes include that they have multiple chromosomes that often contain long runs of repetitive DNA (it is very difficult to accurately sequence and assemble these regions); they often have large numbers of noncoding RNAs; their genes usually contain introns that can comprise .

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a considerably higher percentage of the genome than exons; and they contain organelles and sometimes also nucleomorphs that have their own genomes.

2. Genomics is a key to understanding early life forms and may eventually help answer the most fundamental of all questions in biology. The genomes of many pathogens have been sequenced. In some cases multiple strains of a pathogen that vary in virulence have been compared in the hope of revealing which genes are medically relevant. The genome is an organism’s entire complement of genetic information, including genes that encode proteins, RNAs, and regulatory sequences, and any noncoding DNA that may be present.

The genome sequence of an organism not only reveals its genes but also yields important clues to how the organism functions and its evolutionary history. Because chromosomes from many different microorganisms have already been sequenced, such comparisons can be easily done, and the results are often surprising. Comparative genomics shows that many genes have arisen by duplication of other genes. Evolution dictates the transfer of genetic traits from one generation to the next. Horizontal gene transfer is detectable by comparative genomics.

Functional genomics, which deals with gene expression (transcription) and the function of the final gene product, encompasses not only the complete sequencing of the DNA and identification of all the genes but also the study of gene expression at a genome-wide level. Computational tools are used to store and analyze the sequences and the structures of biological macromolecules. The metabolome is the complete set of metabolic intermediates produced by an organism. Systems biology uses data from genomics, transcriptomics, etc. (including microarrays and DNA chips) to build computer models of molecular activities and interactions in cells.

The traditional approach to studying gene expression was to focus on a single gene or group of related genes. In the genomics era, the expression of all or most of an organism’s genes can be examined in a single experiment.

Advances in genomics rely heavily on improvements in molecular technology and computing power. Major advances include the automation of DNA sequencing, the miniaturization of analytical procedures, and the development of powerful computational methods for analysis of DNA and protein sequences. New advances arrive every year and have driven down the cost and increased the speed at which genomes are analyzed.

3. Homologous sequences are paralogous if they were separated by a gene duplication event. If a gene in an organism is duplicated to occupy two different positions in the same genome, then the two copies are paralogous. Paralogous genes often belong to the same species, but this is not necessary. For example, the hemoglobin gene of humans and the myoglobin gene of chimpanzees are paralogs. Lineage-specific gene duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving their functional connectivity with other genomes. Orthologs and paralogs can be identified by combining the results obtained from both comparative and functional genomics.

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CHAPTER

7

Metabolic Regulation

Summary Chapters 4 and 6 covered the manner in which cells store and transmit genetic information, including how genetic information dictates the production of proteins. In Chapter 7, the authors present mechanisms employed by cells for the regulation of gene expression, including cell sensing and signal transduction systems and global regulatory mechanisms that coordinate the expression of gene networks. Several new or updated figures have been added to this chapter, and sections concerning protein regulation mechanisms have been imported from other chapters to improve flow and enhance student comprehension.

7.1 | Major Modes of Regulation Use Figure 7.1 to provide an overview of gene expression and to provide a preview of protein regulation mechanisms. Review the anatomy of a gene and its mRNA transcript: the promoter, the transcriptional start and termination sites, the 5 and 3 untranslated regions (UTRs) that flank the ribosome-binding site and stop codon, and the structural gene sequence (cistron) itself. Point out that cells use two major methods to regulate metabolism. First, cells control the production of a gene product (i.e., the amount of protein present) by regulating either transcription of the gene or translation of the mRNA synthesized from the gene. Alternatively, cells can regulate the activity of an already synthesized gene product (e.g., an enzyme) using a variety of posttranslational modifications. Figure 7.2 provides a striking example of the use of fluorescent labeling to show localized expression and activity of proteins that facilitate endosporulation in Bacillus.

7.2 | DNA-Binding Proteins The primary focus of Chapter 7 is on transcriptional regulation in prokaryotes. The turnover rates of prokaryotic mRNA are rapid. The degradation of mRNA molecules after only a few minutes allows for quick responses to changes in environmental conditions, including nutrient availability. The ability to quickly initiate transcription of a gene or operon depends upon specific DNA-binding proteins, and most of these proteins interact with specific DNA sequences (Figure 7.3). Using the foundation provided by material in Chapter 4 concerning nucleic acid and protein structure, describe how proteins are able to interact with nucleic acids in either a sequence-specific or sequence-independent (nonspecific) fashion. Histone proteins (Section 2.20) provide a good example of nonspecific binding, whereas RNA poly.

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merase (Section 4.7) is an enzyme that binds DNA only at sequence-specific sites. Point out to your students that many DNA-binding proteins are homodimers, with a domain from each monomer interacting with a specific region (often an inverted repeat) within the DNA major groove (Figure 7.3). Describe the different motifs found in DNA-binding proteins discussed in the text: (1) helix-turn-helix (Figure 7.4), (2) zinc finger, and (3) leucine zipper. Finally, it is important to stress that DNA-binding proteins can either activate or block transcription, resulting in positive or negative regulation, respectively, as discussed in the following sections.

7.3–7.4 | Negative Control: Repression and Induction; and Positive Control: Activation Ask students to think about why and how a cell needs to regulate transcription. In your discussion, clearly differentiate between negative control of transcription, which uses repressor proteins for both enzyme repression and induction, and positive control of transcription, which uses activator proteins to control gene expression. The key point is that in negative control, a repressor protein brings about repression of mRNA synthesis, whereas in positive control, an activator protein initiates transcription by activating the RNA polymerase.

Because the synthesis of enzymes necessary for specific biosynthetic pathways is an energetically expensive process, these enzymes are often produced only when a specific nutrient is not already available to the cell. Using Figure 7.5, show that cells can “sense” and respond to the introduction of the amino acid arginine to the growth medium by preventing the transcription of genes whose products control arginine biosynthesis. Note that this transcriptional control is specific and does not affect the synthesis of other proteins in the cell. How does this occur? It is important to challenge your students to come up with possible mechanisms so that the topics of signal transduction and cell-to-cell communication of how cells sense their environment will be understood, not merely memorized. The discussion of cellular metabolism (Chapter 3) introduced the idea that the cell can sense its redox state⎯the relative levels of reduced versus oxidized forms of an essential molecule. Similarly, here the cell is sensing the presence of excess arginine molecules through chemical interactions. If the pool of arginine molecules is larger than is required for continued protein synthesis, then some of these excess molecules will occupy an allosteric site on the repressor protein that controls arginine biosynthesis and deactivates it (see Figure 7.7).

• Stress the idea that regulation is tied to the cell’s ability to sense concentrations of certain molecules (e.g., ATP/ADP ratios, NAD+/NADH ratios, glutamate/-ketoglutarate ratios, etc.), and when a ratio becomes lopsided, the molecule in excess can affect the activity of a key enzyme (and transcription of the genes involved).

• Using Figure 7.6, demonstrate the process of enzyme induction using the example of -galactosidase in a similar manner as above.

• The molecular mechanisms of enzyme repression and induction are shown in Figures 7.7 and 7.8, respectively.

• Discuss maltose catabolism in E. coli as an example of positive control (Figure 7.9).

Figure 7.10 shows the interaction of a positive regulatory protein with DNA, in which binding of the activator protein bends the DNA in such a way as to allow RNA polymerase to make the correct contacts with the promoter. Activator proteins can also directly bind .

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RNA polymerase to facilitate transcription initiation (Figure 7.11). Include in your discussion the concept of operons versus regulons. In the case of the maltose regulon in E. coli, genes for maltose utilization are spread throughout the chromosome in several operons, and each of these contains a binding site for the maltose activator protein (Figure 7.12).

Students must have a clear understanding of these examples of negative (repression and induction) and positive control of transcription before presenting the material on global regulatory networks.

7.5 | Global Control and the lac Operon Discuss the classic example of global regulatory mechanisms using catabolite repression of the lac operon in E. coli. Note that one of the consequences of catabolite repression is two exponential growth phases, a situation referred to as diauxic growth (Figure 7.13). This phenomenon is observed because the carbon source that supports the fastest growth rate (i.e., provides the most energy per mole of substrate) is preferentially used over other usable carbon sources. The synthesis of enzymes that allow for catabolism of secondary carbon substrates, such as lactose in E. coli, takes place only when the preferred carbon source is exhausted.

Interestingly, catabolite repression of the lac operon is both positively controlled by the activator protein CRP (cyclic AMP receptor protein) and negatively controlled by the LacI repressor protein. The binding of cyclic AMP (cAMP; Figure 7.14) to CRP allows it to bind to a specific sequence near the lac promoter, which in turn allows RNA polymerase to bind to the promoter. However, for transcription of the lac operon to occur, the repressor protein LacI must also be released from the operator. This occurs in the presence of the inducer lactose.

Thus, the release of catabolite repression of the lac operon requires the presence of both cAMP and the inducer molecule lactose. Note that the concentration of cAMP in the cell is inversely proportional to that of glucose (i.e., high levels of glucose result in low concentrations of cAMP). Figure 7.15 presents a good overview of regulation of the lac operon.

7.6 | Transcription Controls in Archaea Despite the similarity between the mechanisms of replication and transcription in Archaea and Eukarya, the regulation of transcription in Archaea is more similar to that of Bacteria than Eukarya. Relatively few repressor or activator proteins have been characterized in detail from Archaea. Repressor proteins in Archaea generally work by blocking the binding of RNA polymerase or by blocking the TBP and TFB transcription initiation proteins (Section 4.9). Discuss the well-characterized nitrogen metabolism repression system in the methanogen Methanococcus maripaludis. In this example of negative regulation, the repressor protein NrpR represses genes that function in nitrogen assimilation when adequate amounts of organic nitrogen are already available to the cell (Figure 7.16). The inducer of transcription in this case is -ketoglutarate, which is present in high concentrations in the cell when fixed nitrogen levels become limiting.

The protein SurR in the hyperthermophile Pyrococcus furiosus functions as both a repressor and activator. When S0 is present, genes that enable growth by anaerobic respiration (S0 reduction) are induced. When S0 is absent, hydrogenase genes become activated, and growth via fermentation ensues. .

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7.7–7.8 | Two-Component Regulatory Systems and Regulationof Chemotaxis Describe the roles of the sensor kinase and response regulator in two-component signal transduction systems (Figure 7.17). Stress to students that an environmental signal triggers the genetic response in the cell. Sensor kinases are typically membrane-spanning proteins that receive the external signal and undergo autophosphorylation through ATP hydrolysis. The sensor kinase then phosphorylates the response regulator, which is usually a DNA-binding protein. Figure 7.18 illustrates a two-component system that controls the expression of porin proteins in the outer membrane of E. coli in response to changes in osmotic pressure. Some two-component systems have multiple regulators, such as the Nar regulatory system, which controls anaerobic growth by respiration of nitrate and/or nitrite (Section 13.17). Table 7.1 provides several examples of two-component systems for discussion.

Use the example of chemotaxis in E. coli to describe the modified two-component signal transduction systems that regulate chemotaxis in prokaryotes (Figure 7.19). Note that while there are several taxes of microorganisms that use specific sensor proteins, including chemotaxis, phototaxis (movement toward light), and aerotaxis (movement toward oxygen), many of the same cytoplasmic Che proteins are employed in each mechanism of flagellar control.

7.9–7.10 | Quorum Sensing and Other Global Control Networks In recent years, a great amount of research interest concerning sensing and signal transduction control systems has revolved around the mechanism of quorum sensing, which was first described in the bioluminescence of the bacterium Aliivibrio fischeri (Figures 7.20 and 7.21).

The key concept for the student is that multiple genes are turned on in a cell densitydependent manner, and this involves a DNA-binding transcriptional activator protein that is activated by a class of small organic molecules known as autoinducers. Several different classes of autoinducers have been identified, the first being acyl homoserine lactones (AHLs, Figure 7.20). AHL is produced by each bacterium at a relatively low level and diffuses freely out of the cell, accumulating in the medium as culture density increases.

At high cell densities, AHL is produced at a high enough concentration to induce its corresponding activator protein among the cell population, resulting in the transcription of specific sets of genes.

Similar quorum-sensing systems have now been described for many pathogenic bacteria (e.g., Shiga-toxin producing E.coli, Staphylococcus aureus, and Pseudomonas aeruginosa), where the activation of transcription produces gene products related to virulence (Figure 7.22) or biofilm formation (Figure 7.23). Several other quorum-sensing systems have been identified in bacteria, some of which use small peptides as the signaling molecule, as observed in gram-positive bacteria. There is some evidence for quorum sensing in Archaea, but the data are not definitive at this time. Quorum sensing has also been identified in some microbial eukaryotes, such as the yeast Saccharomyces cerevisiae, which produces specific aromatic alcohols that control the transition between growth as single cells or elongated filaments. .

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Table 7.2 lists examples of other global control systems. Discuss the classic heat shock system from E. coli (Figure 7.24), focusing on the heat shock proteins (Hsp70, Hsp60, and Hsp10) and alternative sigma () factors, especially RpoH. The Hsp proteins described in this section were discussed earlier, as they are in fact the chaperonins DnaK, GroEL, and GroES that were covered in Section 4.14. Point out that heat shock proteins are activated not only from exposure to excessive heat, but also by other stresses the cell may encounter, such as ultraviolet radiation and exposure to destructive chemicals.

7.11–7.12 | Sporulation in Bacillus and Caulobacter Differentiation Discuss sporulation in the model organism Bacillus subtilis in some detail, stressing the five sensor kinases that monitor environmental conditions that may require endospore formation for survival (e.g., desiccation, high cell densities, starvation, extreme temperatures, etc.).

Although this system resembles a two-component signal transduction system, it is considerably more complex. Once sporulation is triggered, four different sigma factors control endospore formation (Figure 7.25). Give the example of “cellular cannibalism” that precedes endospore formation by cells in which the sporulation factor Spo0A has already been activated. To ensure that enough nutrients are available to carry out endosporulation, cells with activated Spo0A secrete a toxic protein that lyses neighboring cells in which Spo0A is not yet active.

The toxin-secreting cells protect themselves from their own toxin by simultaneously producing an antitoxin!

Caulobacter is a fascinating example of a bacterium that can undergo extensive cellular differentiation. Single Caulobacter cells create daughter cells that, despite being genetically identical, perform different functions and express different sets of genes. Discuss the Caulobacter life cycle (Figure 7.26) in some detail, being sure to indicate the major role of swarmer cells (dissemination) versus stalked cells (reproduction) and the three major global regulators (DnaA, GcrA, and CtrA) that control the different phases of the life cycle. Because a complete genome sequence is available, and the organism itself is amenable to genetic manipulation, Caulobacter is a model organism for studying cell development.

7.13 | Nitrogen Fixation, Nitrogenase, and Heterocyst Formation Explain to students that because nitrogen fixation is such an energy demanding process, regulatory controls for the expression of nitrogenase genes must be efficient. Discuss the genetics of N2 fixation by describing the nif regulon in the well-studied Klebsiella pneumoniae (Figure 7.27). Mention the key regulatory aspects of the process, including the activities of the positive regulator NifA, which activates transcription of nif genes and is itself regulated

by NtrC, and the negative regulator NifL, which suppresses expression of nif genes in the presence of O2, Also mention the posttranslational ammonia switch-off mechanism possessed by some nitrogen-fixing bacteria.

This section also discusses heterocysts in cyanobacteria, which students should find interesting. These differentiated cells forgo oxygenic photosynthesis (they lack photosystem II) to spe.

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cialize in nitrogen fixation. Because of the oxygen-sensitivity of nitrogenase, heterocysts have a thickened envelope to minimize diffusion of O2 into the cell. Heterocysts tend to be evenly dispersed in cyanobacterial filaments, and a network of intercellular channels allows diffusion of small molecules between the cells of the filament (Figure 7.28a and b). Vegetative cells in the filament specialize in oxygenic photosynthesis (and therefore carbon fixation), whereas heterocysts carry out nitrogen fixation; the intercellular channels allow for the exchange of fixed carbon from vegetative cells for fixed nitrogen from the heterocysts (Figure 7.28b).

Heterocysts form when levels of fixed nitrogen are insufficient for growth and metabolism of the cells. Such conditions cause the intracellular glutamate concentration to become

limiting and levels of -ketoglutarate to become elevated (review Figure 3.28a). The accumulation of -ketoglutarate activates the global regulator NtcA, which causes expression of hetR. In turn, the HetR protein activates a suite of genes whose products lead to heterocyst formation (Figure 7.28c). To ensure their even distribution in the filament, formed heterocysts produce a small protein called PatS, which diffuses into neighboring cells in the filament through the intercellular channels (Figure 7.28b). PatS apparently inhibits heterocyst formation in nearby cells by inactivating HetR.

7.14–7.16 | Regulatory RNAs: Small RNAs and Antisense RNA; Riboswitches; and Attenuation Although this chapter has focused on transcriptional control of protein synthesis, the activity of gene products can also be controlled at the level of translation. Consequently, these sections of Chapter 7 discuss gene regulation using RNA-based mechanisms, as opposed to the protein-based systems (i.e., repressors and activators) described earlier. One mechanism uses small regulatory RNA molecules (sRNAs) called antisense RNAs that undergo complementary base pairing with mRNA transcripts and either prevent or stimulate their translation, depending on the structure of the mRNA itself or on the binding efficiency of ribonuclease (Figure 7.29). A similar sRNA regulatory system uses trans-RNAs, which are encoded in the intergenic regions of a chromosome. The RNA chaperone Hfq often facilitates the binding of trans-RNAs to their target sequences (Figure 7.30).

Section 7.15 introduces riboswitches to the students. These RNA molecules resemble repressor and activator proteins in that they regulate gene expression by binding metabolites, such as amino acids, based on their secondary structure (Figure 7.31). The three-dimensional conformation of riboswitches varies depending on whether the metabolite is bound.

Riboswitches are often used as a sort of feedback inhibition mechanism (see Section 7.17).

For example, when sufficient amounts of a product of a metabolic pathway, such as an amino acid, are present, translation of the mRNA is blocked due to internal complementary base pairing that renders the ribosome-binding site inaccessible (Figure 7.31). Make sure students are aware that riboswitches are not a common mechanism of regulation, being found only in some bacteria and a few plants and fungi.

Although catabolite repression may be thought of as a “coarse” control, attenuation represents a “fine” control that is common in amino acid biosynthesis. Attenuation is possible only in prokaryotes because transcription and translation are coupled (they occur simultane.

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ously). This is important because in attenuation, it is translation that actually regulates transcription. Discuss the regulatory details involved in attenuation, including how the concentration of a metabolite determines the pattern of stem loop formation in the mRNA transcript (Figures 7.32 and 7.33). As with ribozymes (catalytic RNAs) and riboswitches, emphasize once again the key role of secondary structure in RNA molecules in controlling transcription by attenuation.

7.17–7.18 | Regulation of Enzymes and Other Proteins The regulatory mechanisms discussed in this section deal with the control of activity of enzymes that already exist in the cell. Cells can control the amount of a protein synthesized, but in many cases they can also control the activity of enzymes. Allosteric enzymes provide instant and exquisite control of biochemical pathways. Such regulation is common in both prokaryotes and eukaryotes and occurs via feedback inhibition, a process in which the end product of a biochemical pathway inhibits activity of the first enzyme in the pathway, thereby preventing further production of the end product (Figure 7.34a). Students should become familiar with the concept of allostery (Figure 7.34b) and its role in the regulation of both catabolic and anabolic pathways in the cell.

Many biosynthetic pathways, such as those for synthesis of tyrosine, tryptophan, and phenylalanine, are regulated by isoenzymes. Figure 7.34c shows that although isoenzymes catalyze the same step in a biochemical pathway, they are feedback-inhibited by different end products, allowing for an incremental reduction in the productivity of the pathway.

As an example of the dynamic nature and complexity of enzymes, use Figure 7.35 to demonstrate how the multisubunit enzyme glutamine synthetase is regulated by covalent modification via stepwise adenylation, an example of posttranslation regulation. This is an excellent example of how the activity of some enzymes can be “turned up” or “turned down” gradually, thus providing a more finely tuned level of control than a simple on/off switch, as in feedback inhibition.

Answers to Review Questions 1. The level of protein synthesis in a cell can be controlled either at the level of transcription of genes or at the level of translation of mRNA transcripts.

2. Double-stranded DNA has a different molecular conformation than does single-stranded DNA. Many DNA-binding proteins, such as those with the helix-turn-helix motif, bind to the major groove in double-stranded DNA. Because single-stranded DNA does not have this three-dimensional feature, binding cannot occur.

3. Biosynthetic operons normally require only negative control for effective regulation because the cell needs only to know when there is “enough” of that biosynthetic product for growth. Catabolic operons, on the other hand, have to be regulated both positively and negatively because their transcription must be controlled by the presence of a specific substrate, such as lactose, and by the absence of other substrates that might be used preferentially, such as glucose.

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4. An operon is a series of structural genes under the control of a shared regulatory site and transcribed into a single RNA molecule. A regulon is a series of related operons that are under the control of a single regulatory protein.

5. Regulatory mechanisms that respond to environmental signals by regulating the expression of many different genes are called global control systems. Catabolite repression is a mechanism of global control that controls the use of carbon sources if more than one is present. In E. coli and other organisms for which glucose is the best energy source, catabolite repression prevents expression of most other catabolic operons as long as glucose is present. Dozens of catabolic operons are affected, including those for lactose, maltose, a host of other sugars, and most other commonly used carbon and energy sources for E. coli. Cyclic AMP is a key molecule in many metabolic control systems, both in prokaryotes and eukaryotes. When glucose enters the cell, the cyclic AMP level is lowered, CRP cannot bind DNA, and RNA polymerase fails to bind to the promoters of operons subject to catabolite repression. Catabolite repression is an indirect result of the presence of a better energy source (glucose); the direct cause of catabolite repression is a low level of cyclic AMP. Other regulatory nucleotides include cyclic guanosine monophosphate, cyclic di-GMP, and guanosine tetraphosphate.

6. Archaeal repressor proteins inhibit transcription by either blocking the binding of RNA polymerase directly or blocking the binding of TBP (TATA-binding protein) and TFB (transcription factor B), both of which allow RNA polymerase to bind to the promoter.

7. Signal transduction contains two parts (hence the term two-component regulatory systems), consisting of sensor kinase protein in cytoplasmic membrane and a response regulator protein in cytoplasm. Sensor kinases detect a signal from the environment and phosphorylate themselves (a process called autophosphorylation) at a specific histidine residue. The phosphate is then transferred from the sensor to another protein inside the cell, the response regulator. The latter is typically a DNA-binding protein that regulates transcription in either a positive or a negative fashion. For example, in Escherichia coli almost 50 different two-component systems are present. One of them is responsible for porin regulation depending on the environmental osmotic pressure. Here the sensor kinase is EnvZ and response regulator is OmpR. Some signal transduction systems have more than one regulatory element and their activities can quickly become quite complex.

For example, the Ntr regulatory system in many Bacteria that regulates nitrogen assimilation is a multiple regulators system.

8. Sensory proteins called methyl-accepting chemotaxis proteins (MCPs) make contact with the cytoplasmic proteins CheA and CheW. CheA is the sensor kinase for chemotaxis.

When an MCP binds a chemical, it changes conformation and, with help from CheW, leads to the autophosphorylation of CheA to form CheA-P. Attractants decrease the rate of autophosphorylation, whereas repellents increase this rate. CheA-P then passes the phosphate to CheY (forming CheY-P); this is the response regulator that controls flagellar rotation.

9. Quorum sensing is a regulatory system that monitors the population level and controls gene expression based on cell density. Species that employ quorum sensing synthesize a specific signal molecule called an autoinducer. This molecule usually diffuses freely across the cell envelope in either direction. Because of this, the autoinducer reaches high concentrations inside the cell only if there are many cells nearby, each making the same .

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autoinducer. Inside the cell, the autoinducer binds to a specific transcriptional activator protein or a sensor kinase of a two-component system, ultimately triggering transcription of specific genes.

10. During the heat shock response, E. coli expresses three major classes of heat shock proteins: Hsp70 (DnaK), Hsp60 (GroEL), and Hsp10 (GroES). The role of each of these proteins is to prevent or correct misfolded proteins, with Hsp60 and Hsp10 acting as molecular chaperones and Hsp70 acting to prevent aggregation of newly synthesized proteins, stabilize denatured proteins, and indirectly enhance the transcription rate of heat shock genes.

11. At least four alternative sigma factors control endospore development in Bacillus: F, G, E, and K. The former two sigma factors activate specific genes inside the developing endospore, whereas E and K activate genes in the mother cell surrounding the endospore. Endosporulation is triggered by external signals that cause the release of F from SpoIIAB, which activates F and begins a cascade in which the other sigma factors initiate transcription of endosporulation-specific genes.

12. DnaA binds the origin of replication of the chromosome of stalked Caulobacter cells to initiate DNA replication during the S (synthesis) phase of the life cycle.

13. Nitrogen fixation is the process of reducing N2 to NH3 for use in biological molecules.

Nitrogenase is composed of two proteins, dinitrogenase and dinitrogenase reductase.

Anabaena is able to fix nitrogen even though they produce oxygen during photosynthesis. To avoid this incompatibility, these cyanobacteria undergo a developmental process that forms dedicated nitrogen-fixing cells called heterocysts at regular intervals along a filament. The chemoorganotrophic bacterium Klebsiella pneumonia does not form heterocysts. The genes for nitrogen fixation form a regulon called the nif regulon, which spans 24 kilobase pairs of DNA and contains 20 genes arranged in operons such that genes whose products have similar functions are cotranscribed. In the presence of sufficient O2, NifL FAD is oxidized and the protein can then repress transcription of other nif genes; this prevents synthesis of the oxygen-labile nitrogenase.

14. Regulation of translation by sRNAs requires the synthesis of a noncoding transcript that contains a section of nucleotide sequence that can undergo complementary base pairing with the mRNA transcript to be regulated. A riboswitch, on the other hand, is an mRNA domain having secondary structure that is altered upon binding a specific metabolite in such a way that the ribosome-binding site becomes blocked, thereby preventing translation of the mRNA itself. Riboswitches occur upstream of the coding sequence to be translated and are, therefore, part of an mRNA transcript that regulates its own translation.

15. Transcriptional attenuation is a regulatory mechanism that occurs after initiation of transcription but before it is completed; that is, transcription is prematurely terminated.

It functions essentially to reduce the number of transcripts from an operon (see Figure 7.33). This type of regulation has not been found in eukaryotes because it requires the coupling of transcription and translation, which cannot happen in eukaryotes.

16. Regulation of DAHP synthesis occurs via independent feedback inhibition of the three DAHP synthase isoenzymes. Each possesses an allosteric site for one of the amino acid end products of the pathway (phenylalanine, tyrosine, or tryptophan). Glutamine .

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synthetase, on the other hand, is regulated by covalent modification of one or more of its 12 subunits.

17. The most common covalent modification to affect protein activity is phosphorylation.

Answers to Application Questions 1. If the regulatory protein-binding region of a promoter under negative control were deleted, one would expect constitutive production of the operonal gene products. If, on the other hand, a positive control region were deleted, one would expect no production of the operonal transcript since there would be no place for the activator protein to bind.

2. If a promoter closely matches the DNA consensus sequence, one would expect RNA polymerase to bind to that promoter and induce transcription in the absence of other factors. Therefore, promoters under positive control would be expected to be quite different.

3. Nitrogen fixation is repressed by O2 and by fixed forms of nitrogen. A major part of this regulation occurs in the expression of nif structural genes, whose transcription is activated by the positive regulator NifA. By contrast, NifL is a negative regulator of nif gene expression and contains a molecule of FAD, an O2 sensor. In the presence of sufficient O2, NifL FAD is oxidized and the protein can then repress transcription of other nif genes; this prevents synthesis of the oxygen-labile nitrogenase. With a faulty NifL, this mechanism would not materialize and the nitrogenase synthesized by the cell would be wasted.

4. One consequence of catabolite repression is that it may lead to two exponential growth phases, a situation called diauxic growth. If two usable energy sources are available, the cells grow first on the better energy source. Growth stops when the better source is depleted, but then, following a lag period, it resumes on the other energy source. For E. coli grown on a mixture of glucose and lactose, the cells grow more rapidly on the former as it is the superior energy source. The proteins of the lac operon, including the enzyme βgalactosidase, are required for using lactose and are induced in its presence. But the synthesis of these proteins is also subject to catabolite repression. As long as glucose is present, the lac operon is not expressed and lactose is not used. However, when glucose is depleted, catabolite repression is abolished, the lac operon is expressed, and the cells grow on lactose.

5. Amino acid biosynthetic operons under control by attenuation and negative control are logical if one considers the role of each in the cell’s response to the extra- and intracellular environment. Keeping in mind that transcription and translation are coupled processes in bacteria, the precursors for the synthesis of amino acids can fluctuate rapidly and dramatically in nature. Negative control tells the cell there is enough of a particular amino acid, so it can turn down or turn off transcription of the operon, and attenuation can respond while the operon has been induced and transcription is in process when the required metabolites rise or fall rapidly.

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6. Both eukaryotic and prokaryotic transcription and translation are sub-cellular processes that follow the central dogma. Transcription of both prokaryotic and eukaryotic mRNA requires RNA polymerase. The prokaryotic RNA polymerase synthesises mRNA, rRNA, and tRNA, when compared to the eukaryotes which require RNA polymerases I, II, and III for synthesizing mRNA, rRNA, and tRNA respectively.

In prokaryotic cells transcription and translation are coupled as there is no nucleus to separate the processes. However, in eukaryotic cells transcription occurs in the nucleus, and translation occurs in the cytoplasm; thus, they are spatially and temporally separated. The TATA box and several initiation factors are required for eukaryotic transcription, unlike that of prokaryotes. Transcription of prokaryotes is polycistronic and they have multiple Shine-Dalgarno sequences; in contrast, it is moncistronic in eukaryotes and they have a single start site. In prokaryotes, 23S, 16S, and 5S rRNA are formed in a single primary transcript while in eukaryotes the 28S, 18S, 5.8S and 5S rRNA are formed in two primary transcripts. In eukaryotes the first amino acid is methionine, unlike prokaryotes, which have N-formylmethionine.

Eukaryotes utilize several initiation factors as compared to prokaryotes. The 5′ end of mRNA is readily available to ribosomes in prokaryotes for translation. In contrast, the mRNA is processed by RNA splicing, 5' end capping, and addition of a polyA tail, and transported to the cytoplasm in eukaryotes prior to translation. Eukaryotic elongation factors are EF1α and EF1βγ, while that of the prokaryotes are EF-Tu and EF-Ts. The spatial differentiation in the eukaryotes that rules these cellular processes make it more complicated and less efficient than found in prokaryotes.

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CHAPTER

8

Viruses and Virology

Summary Viruses are obligate, nonliving, intracellular parasites that are the most numerous microscopic entities on Earth, infecting all types of cellular organisms. This chapter provides an overview of the nature of viruses and the discipline of virology. Viruses are discussed in more detail in later chapters dealing with viral genomics and diversity (Chapter 9), genetic engineering (Chapter 11), and human diseases caused by viruses (Chapter 29).

8.1–8.4 | The Nature of Viruses An amazing diversity of viruses exists, and they can infect all types of organisms, including bacteria. Students need to be aware from the start that viruses require enzymes contained in a host cell for self-replication. This leads to a distinction between the intracellular and extracellular states of the virus. The intracellular form is active, whereas the extracellular form, called a virion, is metabolically inert. Some animal viruses, such as poliovirus, have the ability to skip the extracellular form and move from cell to cell directly by causing the fusion of neighboring cells in the host organism.

The basic structure of a virus is nucleic acid surrounded by a protein shell called the capsid. The complete complex of nucleic acid and protein in the virion is called the nucleocapsid. Some viruses possess a membrane that surrounds the nucleocapsid (enveloped viruses) (see Figure 8.5), whereas others do not (naked viruses) (see Figure 8.1). The presence of an envelope is more typical of animal viruses, and the envelope phospholipids are extracted from the host cell as viral particles exit the cell. The most structurally complex viruses are bacteriophages, viruses that infect bacteria. These viruses often have separate head and tail protein fibers that give the virus an elaborate morphology (see Figure 8.20).

Figure 8.2 demonstrates the genomic diversity of viral genomes, which can consist of DNA or RNA (or both at different stages of the viral replication cycle) that in either case can be double-stranded or single-stranded. Note that RNA viruses are the exception to the central dogma of information flow (DNA → RNA → protein) because RNA rather than DNA serves as the template for the replication of the viral genome. Despite this, information in the RNA genome still encodes the production of specific proteins.

Some viruses have only one kind of protein in their capsid, the structural subunits of which are called capsomeres. Viral capsids self-assemble in one of two symmetrical forms, which correspond to the basic shape of the virus: helical (rod-shaped; Figure 8.3) or icosahedral (spherical; Figure 8.4). Despite the very small volume typical of viral capsids, some viruses .

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carry enzymes important for infection and replication. For example, some bacteriophages contain lysozyme to break down peptidoglycan in the bacterial cell wall (Section 2.10). In addition, some RNA viruses, called retroviruses (e.g., HIV), carry an RNA-dependent DNA polymerase (reverse transcriptase) that converts viral RNA to an intermediate DNA molecule.

The viral life cycle can be communicated by reducing this continuous process into discrete, recognizable steps: (1) attachment to a susceptible host cell; (2) penetration of the viral genome (or the intact nucleocapsid); (3) synthesis of viral nucleic acid and proteins using the host cell’s biosynthetic machinery; (4) assembly of new virions inside the host cell (viral maturation); and (5) release of new virus particles (Figure 8.6). The one-step growth curve (Figure 8.7) provides a superficial view of only steps (1) and (5) unless biochemical analysis and electron microscopy are employed.

To study viruses in the laboratory, one has to be able to effectively culture the relevant host cell, which could be eukaryotic (plant, animal, protist, etc.) or prokaryotic, depending on the virus studied. These cultures can be difficult to grow and are easily contaminated. The ideal viral host cell used in laboratory research can be subcultured and grown indefinitely as a permanent cell line. However, many host cells are primary cell cultures that are capable of growth for only a few days and cannot be transferred.

Quantification of virus particles is an important tool in virology. The preferred approach in determining the number of virion particles per unit volume (the titer) is to measure effects on the host following inoculation (other methods, such as electron microscopy, can be expensive and impractical). The plaque assay allows one to easily calculate the number of infectious particles, or plaque-forming units (PFUs), in the original sample (Figure 8.8). Plaques can also be obtained from animal cell culture systems in a similar manner (Figure 8.9). However, stress to students the fact that plating efficiency (the efficiency in which viruses infect host cells) is always lower than direct electron microscopy counts. Although plating efficiency can often exceed 50% in bacteriophages, it may only be 0.1%–1.0% in some animal viruses. This concept is important for the study of viruses in the laboratory. The quantification of viruses is often included as a laboratory exercise in general microbiology courses, usually using bacteriophage T4 and/or tobacco mosaic virus (TMV).

8.5–8.8 | Bacteriophage Life Cycles Although several diverse bacteriophages that infect E. coli have been well studied (Table 8.1), these sections focus mostly on the bacteriophages T4 and lambda. Section 8.5 addresses attachment and penetration of phage T4. The receptors for viral attachment are normal surface components of the host cell such as pili, flagella, nutrient-uptake proteins, bacterial cell membrane components, animal cell glycoproteins, or lipoproteins (Figure 8.10). Penetration of the bacterial host by phage particles requires the activity of a lysozyme-like enzyme due to the presence of peptidoglycan in the cell wall. Use the analogy of the phage as a hypodermic syringe packed with nucleic acid—the sharp point of the needle represents the lytic enzyme in the phage tail (Figure 8.11).

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Section 8.6 presents the major characteristics of the T4 coliphage genome, which is relatively large and more complex than the genomes of many other viruses. For example, the T4 genome encodes its own DNA polymerase, along with seven other proteins required to assemble a DNA replisome complex that carries out phage-specific DNA replication. Explain the interesting mechanism of concatemer formation (a molecule of DNA that contains repetitive sequences linked in series) and circular permutation during the replication of the T4 genome (Figure 8.12a), being sure students understand that this is a characteristic of some viruses and not a feature of the replication of cells.

Successful double-stranded (ds) DNA viruses must be capable of overcoming host restriction by making their own genetic material resistant to enzymatic attack by restriction endonucleases (see Section 11.1). This is usually accomplished by modifying restriction sites via methylation or glucosylation or by encoding proteins that prevent activity of host restriction enzymes. T4 surmounts the attack of host restriction enzymes by incorporating a unique base, 5-hydroxymethylcytosine, in place of cytosine (Figure 8.12b). The glucosylated hydroxyl group of 5-hydroxymethylcytosine is resistant to restriction enzymes produced by the host. Virus-encoded enzymes produced soon after infection catalyze the synthesis of this base.

The presence of 5-hydroxymethylcytosine in T4 genetic material was noted early, although the role of its glucosylation in rendering it resistant to restriction endonucleases was not then known.

T4 contains more genetic information than most other phages, encoding more than 20 new proteins plus new tRNAs early in the infection. The T4 genome contains early, middle, and late proteins whose expression is sequentially regulated following infection of a bacterial cell (Figure 8.13). Early and middle proteins are enzymes needed for DNA replication, whereas late proteins are used for packaging the DNA and forming the phage lysozyme essential for virion liberation. A point to emphasize is that throughout the T4 replication cycle, phageencoded, anti-sigma factor proteins deactivate the host RNA polymerase sigma factor, resulting in a situation in which only phage promoters are recognized.

The packaging of the replicated T4 genome into an already assembled capsid is a process that should interest students. The mechanism uses a tiny molecular motor (encoded by phage genes) to actively pump the T4 genome into the phage head at the expense of host ATP (Figure 8.14). After the phage head is filled and pressurized with DNA, the motor detaches to allow the head to be sealed and the phage tail assembly to attach. The entire replication process takes only about 25 minutes (Figure 8.13), at which time over 100 mature virions are released from the lysed host cell.

Although T4 is strictly virulent, some viruses, called temperate viruses, have two possible outcomes upon infection of a host cell: a lytic phase or a lysogenic phase (Figure 8.15). The lysogenic phase of bacteriophages involves insertion of the viral genome into the host genome without the expression of virulent genes, creating a prophage (see Figure 8.17a).

Discuss the well-characterized temperate bacteriophage lambda. The lambda strain used in laboratory research looks similar to tailed phages but lacks tail fibers (Figure 8.16). The lytic pathway of lambda requires replication by the rolling circle method (Figure 8.17b), which generates long concatemers of DNA that are cut and packaged into phage heads, as observed in phage T4. Use Figure 8.18 to summarize the complex lytic versus lysogenic life cycle of lambda, which largely depends on the prevailing concentrations and activities of two re.

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pressor proteins, cI protein and Cro. The lambda life cycle represents the best example of a genetic switch between competing genetic functions. Temperate phages are important because viral genetic material becomes integrated into the host genome. You may find it useful to spend considerable time discussing lysogeny and lambda, as these topics provide an opportunity to preview the use of lambda in the field of genetic engineering. Because lambda possesses nonessential DNA that can be replaced by other genes of interest, it represents an excellent genetic tool for use as a cloning vector. This topic will be discussed in detail in Section 10.7.

8.9–8.11 | Viral Diversity and Ecology These sections describe the incredible diversity of viruses and provide a good overview of this important topic if you do not plan to cover Chapter 9 in your course. Section 8.9 provides an overview of bacteriophages (Figure 8.19). Although most viruses that infect bacteria resemble T4 in that they are nonenveloped, complex (i.e., have both head and tail proteins; Figure 8.20), and contain dsDNA genomes, examples of bacteriophages from the full range of genome types are known, including those with ssDNA or ssRNA genomes. Examples of the main classes of bacteriophages are shown in Figure 8.19 to approximate scale.

RNA viruses that synthesize complementary strands of RNA from their RNA genomes produce an enzyme called RNA replicase, which is an RNA-dependent RNA polymerase.

The gene encoding this enzyme is unique to such viruses because cells do not synthesize RNA from an RNA template. In addition, many small DNA and RNA bacteriophages feature overlapping genes. The ability to produce more than one polypeptide from a single open reading frame helps them to overcome the space constraints imposed by the extremely small volumes of their nucleocapsids.

Students are often especially interested in animal viruses because of their importance in diseases, such as influenza and AIDS (see Table 8.2). A brief review of the differences in the site of transcription and translation in prokaryotic and eukaryotic cells as well as the marked differences in their gene arrangement is appropriate here. The consequences of an encounter between an animal cell and a virus depend upon whether the host cell is permissive. Permissive host cells possess both appropriate surface receptors and a suitable intracellular milieu.

Viral replication proceeds in an uninhibited fashion in such cells. A brief overview of the diversity of animal viruses is shown in Figure 8.21. Note that all genomic classes (ssRNA, dsRNA, ssDNA, and dsDNA) are represented. It is also evident that the majority of animal viruses are enveloped. This is likely a result of the fact that animal cells have no cell wall, and therefore a membrane surrounding the viral nucleocapsid likely eases cell penetration and release of virions through the host cell. Figure 8.22 shows the possible consequences of viral infection of animal cells. Most animal viruses cause disease via host cell lysis. However, persistent (slow release of virions without host cell lysis) and latent (virus DNA present but inactive) infections also occur. While the former can continue indefinitely, the latter infection eventually leads to cell lysis for a variety of reasons, such as host immune deficiency due to high levels of stress, poor nutrition, or insufficient sleep. Other consequences of animal virus infection include cell fusions, which allow viruses to invade adjacent cells without

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moving through the extracellular environment, and host cell transformation, which induces tumor formation.

Among all animal viruses, retroviruses are unique in terms of their mode of replication.

Recognition of reverse information flow (RNA → DNA) was a landmark discovery in retroviruses, providing a starting point for understanding and, hopefully, eventually controlling virally induced cancer and AIDS. The unique genome of retroviruses consists of two identical (+) ssRNA molecules paired to specific cellular tRNAs that function as primers for reverse transcription. In addition to reverse transcriptase, other enzymes carried by retroviruses are integrase, which allows for incorporation of the virus genome into the host genome, and a specific protease, which is essential for the processing of viral proteins. Whereas one could deliver an entire course on retroviruses alone, the discussion in this chapter is restricted to Figures 8.23 (retrovirus structure and function) and 8.24 (retrovirus replication). The result of viral invasion of the host cell is reverse transcription and integration of the virus dsDNA genome into the host cell genome to create a provirus. Transcription of provirus DNA yields new copies of (+) ssRNA genomes, which are encapsidated and obtain their envelope coating upon virion release. Host cells may undergo transformation to become cancer cells (if the viral genome has the oncogene that codes for the proteins involved in cell transformation). In some cases, retroviruses may be viewed as agents for transferring genes between cells and as useful tools in gene therapy.

For the final section of the chapter, inform students that the number of prokaryotic cells on Earth (~1030) far exceeds the number of eukaryotic cells, yet the number of viruses is about tenfold higher than prokaryotic cells (Figure 8.25). It is estimated that 5%–50% of the bacterial population in seawater is killed daily by bacteriophages, and the majority of these are dsDNA viruses (RNA phages are rare by comparison). Protozoa ingest much of the remaining percentage of bacterial cells. However, the enormous biotic potential of bacteria prevents their numbers from dwindling and keeps the population more or less in balance. A relatively new realization is the huge quantity of Archaea in planktonic (open ocean) waters, including members of the phylum Thaumarcheaota. Some, or possibly many, of the dsDNA viruses in these waters may actually infect the archaeal population rather than bacteria.

Viruses, particularly bacteriophages, contain most of the genetic diversity of the biosphere.

Many viruses in nature are important facilitators of horizontal gene transfer and therefore greatly affect (and accelerate) bacterial evolution. Any given environment has its own viral metagenome, the sum total of all viral genes contained within a particular environment. Metagenomic studies indicate that a huge diversity of viruses exists, and the majority of gene sequences obtained show no significant similarity to any previously characterized virus genes.

Answers to Review Questions 1. Views of how to define the term “virus” have been and remain conflicted. Wildy (1984) jestingly notes that Lwoff’s 1957 view that “Viruses should be considered as viruses because viruses are viruses” is not so bad! A more serious definition might be as follows: A virus consists of appropriately packaged genetic material of DNA or RNA that can enter a susceptible host cell and modify the genetic state of that cell such that new virus particles can be produced and released. This definition excludes subviral entities, such as defective viruses, which do not contain sufficient genetic information to produce mature .

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virus particles, viroids (infectious nucleic acids that do not require packaging), and prions (infectious proteins that contain no nucleic acid).

2. In contrast to cells, viral genomes can contain the minimum amount of information necessary to code for their structural and nucleic acid components, and in some cases, only a few viral genes permit their replication in a host cell. The genomes of viruses are therefore generally much smaller than those of cells. In addition to dsDNA, which is the genetic material in all cells, viral genomes can be composed of ssDNA, dsRNA, or ssRNA, depending on the virus.

3. Viruses of all types require a genome of either DNA or RNA, which can be either double- or single-stranded. Surrounding the genetic material is a shell-like protein coat called a capsid. Although some viruses are composed of nothing more than this, other viruses possess a membrane envelope and/or carry specific enzymes with them that are used for their replication.

4. The dynamics of viral growth curves as shown in Figure 8.7 differ from bacterial growth curves in that the entire burst size of virus particles is released more or less simultaneously as the host cell lyses. This is the “one-step” growth curve of viruses. Newly released virions then attach to new host cells and proliferate in the same manner, resulting in a stepped rather than smooth growth curve.

5. Animal viruses are cultivated in tissue cultures, which are cells obtained from an animal organ and grown in sterile glass or plastic vessels containing an appropriate culture medium. Tissue culture media are often highly complex, containing a wide assortment of nutrients including blood serum and antimicrobial agents to prevent bacterial contamination.

6. When a virus infects host cells growing on a flat surface, a zone of cell lysis called a plaque forms and appears as a clear area in the lawn of host cells. With bacteriophages, plaques may be obtained when virions are mixed into a small volume of molten agar containing host bacteria that is spread on the surface of an agar medium. During incubation, the bacteria grow and form a turbid layer (lawn) that is visible to the naked eye. However, wherever a successful viral infection has occurred, cells are lysed, forming a plaque.

By counting the number of plaques, one can calculate the titer of the virus sample (often expressed as “plaque-forming units” per milliliter). This method is called the plaque assay.

7. The attachment of bacteriophage T4 to cells of E. coli depends on the presence of specific viral receptors⎯carbohydrates in the lipopolysaccharide layer of the cell’s outer membrane.

8. MS2; ϕX174; M13, f1, and fd; Lambda; T7 and T3; T4; and Mu are some bacteriophages of Escherichia coli. Bacteriophage T4 consists of an icosahedral head, within which the viral linear double-stranded DNA is folded, and a long, complex tail, which ends in a series of tail fibers and tail pins that contact the cell surface. T4 DNA enters the cytoplasm of the E. coli cell through a tail tube in a fashion resembling that of injection by a syringe, while the T4 capsid remains outside the cell. DNA inside bacteriophage heads is under high pressure, and because the interior of a bacterial cell is also under pressure from osmotic forces, the phage DNA injection process takes several minutes to complete. .

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9. Chromosomes of higher organisms and genomes of bacteria contain the same genes in the same order in cells of different individuals of the same species. This is also true of many virus genomes, but not all. Sometimes a population of virions of a single virus contain genomes with the same set of genes but arranged in a different order. This phenomenon is called circular permutation.

10. Early genes are transcribed soon after infection and produce proteins necessary for the replication of the viral nucleic acid. Late genes are expressed later and produce the proteins of the virus coat. Phage T4 controls transcription of its genes by sequentially modifying the specificity of the host RNA polymerase. In combination with a phageencoded sigma factor, the modified host RNA polymerase specifically recognizes late gene promoters (70 has already been deactivated at this point by a phage-encoded antisigma factor).

11. Temperate bacteriophages are those that are capable of initiating a lysogenic pathway, thereby becoming a prophage via integration into the host cell’s genome. Lambda is a well-studied temperate phage that infects cells of E. coli.

12. Bacteriophage shapes include icosahedral, helical, and complex, which is a combination of icosahedral and helical shapes in the form of viruses that have a head-and-tail configuration. The latter morphology is the most common in nature.

13. The diversity of viral genomes is remarkable and includes the following categories and examples: • Single-stranded (ss) RNA: positive-strand (phage MS2, poliovirus, coronaviruses, rhinoviruses, hepatitis A virus), negative-strand (rhabdoviruses [rabies], influenza virus) • Double-stranded (ds) RNA: reoviruses (rotavirus) • ssDNA: bacteriophages M13 and X174 • dsDNA: herpesvirus, Epstein-Barr virus, pox viruses, adenoviruses 14. Animal viruses can contain any of all of the known possibilities of genome configurations, including dsDNA, ssDNA, dsRNA, (+) ssRNA, or (−) ssRNA. 15. Retrovirus genomes consist of two identical strands of (+) ssRNA. Upon infection of a host cell, one of the strands reverts to a dsDNA form using the unique enzyme reverse transcriptase. Although hepadnaviruses also contain reverse transcriptase, their virions carry DNA rather than RNA. 16. The total number of viruses in seawater is estimated to be an order of magnitude higher than the number of prokaryotes in seawater.

17. Although viruses that infect Bacteria and Archaea usually lyse their host cells, this activity does serve to stabilize the overall populations of these hosts. In addition, infection by temperate viruses, which results in a lysogenic state, often imparts new genetic capabilities to the host cell that may be beneficial, a phenomenon called phage conversion. Lastly, bacteriophages facilitate horizontal gene transfer in prokaryotes through the process of transduction, and this contributes to the amazing adaptability of prokaryotes.

Answers to Application Questions .

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1. Viruses use host cell biosynthetic machinery to replicate themselves, and thus, actively growing cells containing sufficient levels of essential biosynthetic enzymes are required for viral replication. Because the host population will eventually reach a stationary (nongrowth) phase due to their increasingly unfavorable environment (a result of cell crowding, nutrient limitation, pH fluctuations, etc.), plaque size will no longer increase because there will be no metabolically active host cells to support virion production.

2. The native host RNA polymerase generally reads early promoters on the viral genome, whereas a virus-encoded or, as in the case of bacteriophage T4, virus-modified host RNA polymerase often recognizes late promoters. Accordingly, one would expect early viral promoters to have similar sequences to host promoters, whereas late promoters would be significantly different. Efficient replication of the virus depends upon the successful co-opting of the host RNA polymerase, altering it such that it is essentially working for the virus rather than the host cell. Therefore, the presence of an altered RNA polymerase that recognizes late viral promoters, but not host promoters, greatly accelerates and ensures the success of virus replication.

3. Some double-stranded DNA bacterial viruses, although able to catalyze a virulent cycle, can also infect their host and establish a long-term stable relationship. These viruses are called temperate viruses. Temperate viruses enter into a state called lysogeny. In this state, most virus genes are not transcribed and instead, the virus genome is replicated in synchrony with the host chromosome and passed to daughter cells at cell division. The lysogenic state may confer new genetic properties on the bacterial host cell, a condition called lysogenic conversion. Hence, temperate viruses are usually DNA viruses.

4. Positive-strand RNA bacteriophages require an RNA-dependent RNA polymerase (i.e., RNA replicase) since RNA is synthesized from an RNA template. This enzyme first produces minus strands from the positive strand genomic RNA, then additional plus strands are synthesized from the newly made minus strands. The plus strands can be used as mRNAs or packaged as the genetic material in new virus particles. Because a retrovirus also contains positive-strand ssRNA, the genomic RNA has the same sequence configuration as the mRNA, as in any positive-strand RNA virus. In the case of the retroviruses, however, three enzymes are required, including reverse transcriptase, a retroviral protease, and integrase. This is because the replication cycle of a retrovirus requires integration into the host chromosome as DNA rather than RNA, and mRNA transcripts are then made from the DNA.

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CHAPTER

9

Viral Genomes and Diversity

Summary Whereas Chapter 8 provided a broad overview of the principles of virology, Chapter 9 progresses deeper into the study of viruses by giving special attention to the genomic and replicative diversity of individual virus types. Impress upon students that although viruses are nonliving entities that possess no independent metabolisms of their own, they infect all organism types on the planet, adapting to their host’s lifestyle and, in many cases, affecting events in the life cycle of the host. Students may be impressed with the tremendous diversity of viral genomes and the complex mechanisms they employ for replication. You may want to present this chapter in an overview format in an introductory microbiology course, or it can be used as supplemental material in an upper division course. Whatever the case, it is important that students be exposed to viruses at some level, especially considering their use as tools in molecular biology and their effects on living organisms as they parasitize cells and appropriate their metabolic resources.

9.1–9.2 | Viral Genomes and Evolution Viral genomes exist in a wide range of sizes, from less than 2 kb to at least 1.25 Mb, which is larger than the genomes of some prokaryotes, but smaller than all documented eukaryotic genomes (Figure 9.1). The largest viral genomes belong to DNA viruses, and examples of both linear and circular viral genomes are known (Table 9.1).

When a virus infects a suitable host cell, virus-specific proteins must be synthesized and the genome replicated, and thus virus-specific mRNA must be produced. The central role of virus-specific mRNA, originally depicted by virologist David Baltimore during the early 1970s, provides a framework for introducing the diversity of ways in which viral mRNA synthesis may be achieved, including the unusual information flow mediated by retroviruses.

Discuss the Baltimore Classification Scheme, which recognizes seven classes of viruses based upon the relationship of the viral genome to its mRNA (Figure 9.2). Discuss the fact that single-stranded (ss) DNA viruses (class II) synthesize a complementary DNA strand to produce double-stranded (ds) DNA before producing viral mRNA (Figure 9.2a). The dsDNA intermediate can then use host enzymes for transcription and to generate the viral genome (the DNA strand not retained as the genetic material gets degraded). A more complete description of the nature of ssDNA phages is provided in Section 9.3.

For RNA viruses (classes III–VI), the challenges of replication are quite different. For students to understand these concepts, discuss the standard nomenclature used in virology f .

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or positive- and negative-strand RNA viruses (Figure 9.2b). Emphasize in particular that because mRNA is of the plus (+) configuration (by convention), single-stranded viral genomes having the same orientation as their mRNA are positive-strand viruses, whereas single-stranded viral genomes that are complimentary to their mRNA are negative-strand viruses. Next, discuss the key requirements and strategies employed by each of the class III–VI viruses to replicate their genomes and transcribe their genes. Be sure to indicate the distinction of retroviruses (class VI), which use reverse transcriptase to create complementary (c) DNA from their ssRNA genomes, as discussed in detail in Section 9.11. Note that partially dsDNA viruses (class VII), such as hepatitis B virus, also use reverse transcriptase, although for a different purpose than that of retroviruses (see Section 9.11). Also point out that some viruses closely regulate the transcription of some genes to produce early proteins (for nucleic acid replication) and late proteins (for the virus coat).

The intriguing evolutionary history of informational molecules, in particular the possible viral origin of DNA as the genetic material in cells, is also discussed in this chapter in Section 9.2. Discuss the hypothesis of a virus-mediated transition from RNA as the genetic material in cells to DNA (Figure 9.3a). Use this opportunity to emphasize the superior structural stability of the DNA double helix by asking your students what major advantage DNA molecules have over RNA molecules as the genetic material. Students interested in evolutionary biology will also appreciate a discussion regarding a potential explanation for the biochemical similarities observed between members of the Archaea and Eukarya. Similar DNA viruses may have infected ancestral cells of these domains, whereas a different DNA virus having a distinct complement of enzymes could have infected ancestral Bacteria (Figure 9.3b).

The final topic of this section is viral phylogenetics, a field of study that is hindered by the lack of a suitably conserved, universal gene for sequence comparisons. However, members of some viral families, such as the nucleocytoplasmic large DNA viruses (NCLDV), do show enough similarity to construct a reliable phylogeny (Figure 9.4). This particular family includes pox viruses, iridoviruses, and the giant Mimivirus. With a diameter of nearly 0.75 m and a genome of 1.2 Mb, Mimivirus is the largest known virus.

9.3–9.7 | Viruses with DNA Genomes These sections cover the diversity and replication of viruses that have DNA as their genetic material. The following points summarize the major characteristics of these viruses.

• ssDNA bacteriophages: Single-stranded DNA phages are either icosahedral or filamentous, although the DNA genome in both types is circular. Their extremely small size suggests greater economy in reading genetic information and marked dependence upon host cell DNA replication machinery. Coliphage X174 has been thoroughly studied, and like phage MS2 (Section 9.8), it contains overlapping genes (Figure 9.5). Moreover, only cell-encoded proteins are involved in the conversion of viral (+) DNA into a doublestranded (+) replicative form. The atypical nature of the initiation of replication with a single-stranded closed circle should be noted and the efficiency of the small genome emphasized. Rolling circle replication (Figure 9.6), as occurs in phage X174, also occurs .

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in many other viruses and plasmids. The unique features of filamentous ssDNA phages, such as M13, should be mentioned since these viruses contain several desirable traits as cloning and sequencing vehicles. These include (1) the retention of cellular integrity by the producer cell (i.e., the virus buds from the host cell without lysis; Figure 9.7), (2) single-stranded DNA to simplify methodology, and (3) intergenic space in which to insert desirable genes. The major features of bacteriophages X174 and M13 are summarized below.

The major features of phage X174 include the following: • small (25 nm in diameter) with icosahedral symmetry • circular genome of 5386 nucleotides • complementary DNA strand required before transcription can occur The major features of phage M13 include the following: • 6 nm in diameter  480 nm long with helical symmetry • circular genome of 6408 nucleotides • leaves cell without lysis • useful as a cloning vector • dsDNA bacteriophages: Double-stranded DNA phages include T4 (Sections 8.5−8.7); lambda (Section 8.8); and T7 and Mu, both discussed in Section 9.4. The dsDNA phage T7 genome has been completely sequenced and its replication cycle studied in detail (Figure 9.8). Genetic informational economy is achieved by gene overlapping and internal frameshifts. Phage T7 uses host RNA polymerase and translation machinery to rapidly synthesize some early proteins, including a protein that inhibits the production of restriction enzymes in the host. Other early proteins include T7 RNA polymerase and two proteins that inactivate the host RNA polymerase, thereby preventing further transcription of host genes. The host cell is devastated—even the nucleotides of degraded host DNA are used for phage DNA synthesis.

The major features of phage T7 include the following: • icosahedral head (45 nm in diameter) with small tail • linear genome 39,936 base pairs with gene overlaps • proteins necessary for replication and transcription are encoded by T7 • replication cycle requiring 25 minutes • dsDNA transposable bacteriophage Mu: This temperate and transposable coliphage generates mutations in its host by integrating itself into the host genome, and thus phage Mu is useful to generate bacterial mutants. The Mu genome contains an invertible region whose orientation determines the host range of the virus, with potential hosts including various strains of Escherichia coli and several other species of enteric bacteria. The integration of the Mu genome into the host genome is necessary for either lytic or lysogenic propagation, which requires the activity of a Mu transposase. Unless the Mu repressor protein is made in sufficient quantities, the phage will enter the lytic pathway.

The major features of phage Mu include the following: .

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• large, with icosahedral head and helical tail having six tail fibers (Figure 9.9a) • linear genome of ~39 kbp, including a length of host DNA attached at each end • useful as a mutagen in microbial genetics • complex replication cycle with a transposition mechanism (Figure 9.9b) • Viruses of Archaea: Using a metagenomics approach, RNA archaeal viruses have recently been identified, but they have not yet been characterized or cultured in the laboratory. Therefore, focus your discussion on the DNA viruses that infect Crenarchaeota (Figure 9.10a, b, and d) and Euryarchaeota (Figure 9.10c). First discuss viruses that infect Sulfolobus (SSV and SIFV) and Pyrococcus (PAV1), which are highly diverse and morphologically unusual. SSV (Figure 9.10a) is spindle-shaped and extremely resistant to heat and low pH. The genome of SSV is circular dsDNA (~15 kbp), rather than the linear arrangement of all known bacterial dsDNA viruses. Another Sulfolobus virus (SIFV) is a linear dsDNA, filamentous virus (Figure 9.10b). The euryarchaeote virus PAV1 has a circular dsDNA genome of ~18 kbp and buds from Pyrococcus without lysing the cell (Figure 9.10c). The circular dsDNA (~68 kbp) virus ATV is either lytic or lysogenic and infects the hyperthermophile Acidianus convivator. Interestingly, ATV is the first virus discovered to show independent structural development outside of its host. ATV has a lemon shape when first released from the cell, but it soon develops tubular tails at each end of the virus particle (Figure 9.10d).

• dsDNA animal viruses—Pox Viruses: Pox viruses are among the largest and most complex viruses known. Unique among DNA viruses, pox viruses replicate their genomes in the cytoplasm rather than the nucleus. Pox viruses are large enough to be seen under the light microscope at up to ~400 nm in diameter. Although the virions are nonenveloped, they are encased in a protein lattice that, along with a dumbbell-shaped nucleocapsid, is visible in transmission electron micrographs (Figure 9.11). Discuss the replication mechanism of vaccinia virus, a close relative of the smallpox virus. Explain the use of vaccinia in preparing the smallpox vaccine, which has effectively eradicated the disease, and in designing genetically engineered, recombinant vaccines.

• dsDNA animal viruses—Adenoviruses: Adenoviruses are a group of linear dsDNA viruses with icosahedral symmetry that generally cause mild respiratory infections (Figure 9.12a). The genomes of adenoviruses average about 36 kbp in length. They have a terminal protein attached to the 5 end with inverted terminal repeats of several hundred nucleotides, both of which are necessary for virus replication (Figure 9.12b). Adenoviruses are especially interesting because of the unique cyclizing mechanism by which they avoid lagging strand DNA synthesis. Discuss the replication events of adenoviruses shown in Figure 9.12b.

• dsDNA animal viruses—Polyomavirus SV40: Polyomaviruses, such as SV40, induce tumors in animals. SV40 is a nonenveloped, icosahedral (45 nm in diameter) virion with a circular, supercoiled, dsDNA genome of 5243 bp (Figure 9.13). The transformation of infected cells into tumor cells can result from the integration of the virus genome into the genome of a nonpermissive host cell, essentially forming a provirus (infection of permissive host cells results in the usual viral multiplication and host cell lysis). Subse.

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quent transcription and translation of polyomavirus genes produces tumor-inducing viral proteins that can inactivate host proteins that control cell division (Figure 9.13b).

• dsDNA animal viruses—Herpesviruses: Herpesviruses deserve detailed consideration because of their involvement in a variety of human disease states, including as a prevalent STD and in tumor formation. Herpesviruses are the causative agents of fever blisters, genital herpes, chickenpox, and shingles. Epstein-Barr, a tumorigenic virus, causes Burkitt’s lymphoma, a form of cancer common in children of tropical regions. This same virus can also cause infectious mononucleosis. The widespread herpesvirus cytomegalovirus (CMV) is present in most adults of at least 40 years of age in the United States.

Although CMV infection is asymptomatic in most cases, it can cause serious disease in immunocompromised individuals that is often manifested as pneumonia, retinitis, or gastrointestinal disease. The large size and morphological complexity of herpesviruses correlates with their coding complexity (Figure 9.14). As a representative example, herpes simplex I virus is enveloped (~150 nm in diameter), has icosahedral symmetry, contains a genome of one large, linear dsDNA molecule of 152,260 bp, and has a fibrous layer outside the capsid called the tegument, a structure that is unique to herpesviruses. Discuss the life cycle replication events of herpesviruses shown in Figure 9.14, which include the synthesis of intermediate early, delayed early, and late proteins.

9.8–9.11 | Viruses with RNA Genomes These sections discuss the diversity and replication of viruses that have RNA as their genetic material. The following points summarize the major characteristics of these viruses.

• RNA bacteriophages: All RNA bacteriophages are small and icosahedral, and the complete nucleotide sequences of some are known. Some, such as coliphage MS2, infect only cells of E. coli capable of forming a conjugation pilus, which serves as their site of attachment and subsequent infection (Figure 9.15a). Once infection has occurred, the events of MS2 multiplication are well-known. Like DNA bacteriophage X174 (Section 9.3), the genome of phage MS2 possesses overlapping genes, in which parts of the nucleotide sequence are read twice by varying both direction and reading frame (Figure 9.15b). This arrangement permits this small virus to have sufficient information for structural and enzymatic requirements. Emphasize to students that the viral RNA genome itself acts as mRNA that is regulated by controlling the access of ribosomes to the start sites on the viral RNA. The infecting RNA is translated into at least four proteins on the host ribosome. One protein, RNA replicase, is a composite protein of both viral and host polypeptides, thereby appropriating host proteins to viral replicase function. RNA replicase synthesizes the (–) RNA strand using the infecting RNA genome as a template; the positive-strand RNA is then synthesized from the minus strand (Figure 9.15c). The maturation protein gene is translated from the nascent form of the (+) RNA as replication occurs. The major protein formed is the coat protein.

The major features of phage MS2 include the following: • small (~25 nm in diameter) with icosahedral symmetry • linear genome of 3569 nucleotides .

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• infects only cells that form a conjugation pilus, to which it attaches • (+) strand RNA genome in virion functions as mRNA • Positive-strand RNA animal viruses: The picornaviruses are discussed first, using poliovirus as the example (Figure 9.16a and b). Structure-function relationships in this virus have been defined in quite specific biochemical terms. Poliovirus provides a good example of posttranslational cleavage (common in animal viruses) of a polyprotein encoded by a monocistronic genome (Figure 9.16c). Discuss the unusual aspect of using VPg, a tiny protein of only 22 amino acids, as a primer for RNA synthesis, as well as the replication and life cycle of poliovirus. Coronaviruses are also ssRNA viruses, but they are considerably larger than poliovirus and lack VPg protein (Figure 9.17a). Their mechanism of replication is also different from poliovirus. Monocistronic mRNAs are transcribed from negativestrand RNA, which is made by RNA replicase⎯the only protein translated from the positive-strand RNA genome. Therefore, unlike for poliovirus, a polyprotein is not produced.

Discuss the replication and life cycle of a coronavirus (Figure 9.17b).

The major features of poliovirus include the following: • very small (30 nm in diameter) with icosahedral symmetry • linear ssRNA genome of 7433 nucleotides • VPg protein covalently attached to 5 end of genome • poly(A) tail present on 3 end of genome • ssRNA genome serves as mRNA • a polyprotein is produced and then cleaved into individual viral proteins The major features of coronaviruses include the following: • enveloped and spherical with glycoprotein surface spikes • largest RNA genome (29,700 nucleotides) • contains a 5 methylated cap and a 3 poly(A) tail • mRNA is transcribed from negative-strand RNA produced by RNA replicase • causes respiratory infections (e.g., SARS) • Negative-strand RNA viruses: The next logical extension of animal virus diversity introduces the negative-stranded RNA viruses using two examples: rhabdoviruses and orthomyxoviruses. The presence of a lipid envelope (obtained from the host cell) in these viruses requires biosynthetic strategies not considered previously, especially for the synthesis of envelope proteins. Remind students that negative-strand RNA cannot act directly as mRNA, and thus a positive strand must be synthesized by RNA replicase. Rabies virus is a rhabdovirus of major importance considering that over 30,000 cases of human rabies, which is usually fatal, occur worldwide every year (Figure 9.18a). Rhabdoviruses that infect crop plants are also known, and therefore these viruses are of economic importance, as well. Discuss the replication of negative-strand RNA viruses (Figure 9.18b). The epidemiological importance of orthomyxoviruses, including the important human pathogen influenza virus, provides a rationale for going into detail here (Figure 9.19). Influenza virus contains a segmented RNA genome, exhibits pleomorphism (having no definite shape), .

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and forms an important surface protein called hemagglutinin, against which antibodies are made for the production of vaccines. These features emphasize why knowledge of the details of viral replication and virion liberation is essential for understanding the role of antigenic shifts (the reassortment of genes between different but related strains) in the control and prevention of epidemics.

The major features of rabies virus include the following: • bullet-shaped with helical symmetry and of moderate size (70 nm in diameter  175 nm long) • envelope present • linear genome of negative-strand RNA • the causative agent of rabies The major features of influenza virus include the following: • nucleocapsid of helical symmetry (6 nm–9 nm in diameter  60 nm long) • pleomorphic • segmented genome of eight linear ssRNA molecules • envelope present • the causative agent of respiratory influenza • Double-stranded RNA viruses: The reovirus group provides an example of doublestranded RNA viruses (Figure 9.20). Reoviruses are relatively small (60 nm–80 nm in diameter), nonenveloped viruses having a double shell of icosahedral symmetry. The genome of reoviruses contains 10–12 molecules of linear dsRNA, and mRNA is transcribed from the negative strand using virus-encoded RNA-dependent RNA polymerase.

Reoviruses initiate infection by binding to a cell receptor that is vital for normal cell function, a trait that renders cellular defense difficult. Upon entering the cell, reoviruses exploit enzymes present in lysosomes to carry out uncoating of the outer shell only, followed by the release of the nucleocapsid into the cytoplasm. To protect the dsRNA viral genome from host enzymes that would destroy it, viral replication events occur within the nucleocapsid (Figure 9.20c). Although you may choose to reserve discussion of the complex replication process of reoviruses for advanced courses, a brief discussion of rotavirus is warranted since it is the most common cause of life-threatening diarrhea in infants 6–24 months of age. Rotavirus is a major cause of infant mortality in developing countries.

• Reverse transcriptase RNA viruses (retroviruses as example): The general principles of retrovirus structure, function, and replication were considered in Section 8.10, and the information included here is designed to complement that section and provide further details of retrovirus activity. This section provides a more detailed treatment of the remarkable activity of the key enzyme reverse transcriptase, which forms dsDNA from ssRNA and is diagrammed in Figure 9.21. In addition, a detailed description of translation of retroviral mRNA and processing of retroviral polyproteins is provided in Figure 9.22. Note that the key enzymes in retroviral replication are reverse transcriptase, integrase, and protease. All retroviruses require multiple protease processing steps to produce mature viral proteins.

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Because of this, protease inhibitors have considerable potential as treatments for retroviral infection, including HIV-1, the causative agent of AIDS. • Reverse transcriptase DNA viruses (hepadnaviruses as example): Hepadnaviruses, including the causative agent of human hepatitis B, are an unusual group of viruses that infect cells of the liver. The hepatitis B virus particles are of an irregular rod shape (Figure 9.23a). The DNA genome of hepatitis B is partially double-stranded, and it is among the smallest of all known virus genomes at a mere 3.4 kb (Figure 9.23b). Not unexpectedly for such a small genome, all of the genes overlap. The replication mechanism of this virus is also unusual in that the host RNA polymerase produces the transcript⎯a single, genomelength mRNA molecule⎯from which reverse transcriptase replicates the DNA genome.

Discuss the life cycle of hepadnaviruses, including their unusual genome structure shown in Figure 9.23.

9.12–9.13 | Subviral Agents Point out to students that viroids and prions are exceedingly unusual in that they are essentially single molecules that have infectious properties and, therefore, have an even simpler structure than viruses. Viroids are infectious, circular ssRNA molecules that infect only plant cells (Figures 9.24–9.26). Consisting of fewer than 300 nucleotides in some cases, they are the smallest known pathogens. Prions, on the other hand, are infectious proteins that consist of a misfolded form of the glycoprotein PrPC, which is primarily found in neurons of animals. The pathogenic conformation of the protein, PrPSc, is capable of converting normal PrPC proteins to the infectious conformation, wherein they accumulate, aggregate, and eventually destroy the cell (Figure 9.27). Neurological symptoms ensue with continued destruction of nervous tissue. Prion diseases are referred to as transmissible spongiform encephalopathies, with the best-known examples being sheep scrapie (the first prion disease discovered), bovine spongiform encephalopathy (i.e., “mad cow disease”), and kuru and variant Creutzfeldt-Jakob disease in humans.

Answers to Review Questions 1. DNA viruses exist along a huge gradient from the tiny circovirus with its 1.75-kilobase single-stranded genome to the 1.25-megabase-pair double stranded DNA genome of Megavirus.

2. The diversity of viral genomes is remarkable and includes the following classes: • Class I (dsDNA): viral mRNA produced by transcription of the minus strand; replication is classical semiconservative.

• Class II (+ ssDNA): viral mRNA produced by transcription of the minus strand of a dsDNA intermediate; replication is classical semiconservative, with the (−) strand discarded.

• Class III (dsRNA): viral mRNA produced by transcription of the minus strand; RNA replicase makes a (+) strand RNA, and this is transcribed to provide the complementary (−) strand. .

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• Class IV (+ ssRNA): the genome is used directly as viral mRNA; RNA replicase makes a (−) strand RNA, and this is transcribed to provide the complementary (+) strand genome.

• Class V (− ssRNA): viral mRNA produced by transcription of the minus strand; RNA replicase makes a (+) strand RNA, and this is transcribed to provide the complementary (−) strand genome.

• Class VI (+ ssRNA retrovirus): reverse transcriptase makes dsDNA, and viral mRNA, as well as new (+) ssRNA genomes, are produced by transcription of the minus strand of the dsDNA. • Class VII (partially dsDNA): transcription of the minus strand of the dsDNA genome produces an RNA intermediate that serves as viral mRNA and is also used by reverse transcriptase to produce the DNA genome.

3. According to one hypothesis about the transition from RNA to DNA, RNA viruses first evolved DNA as a modification mechanism to protect their genomes from cellular ribonucleases that could destroy them. Subsequently, DNA viruses would have had to evolve their own DNA replication machinery to replicate their genomes. It is further hypothesized that DNA viruses infected the ancestors of the three cellular domains. Gradually, by genetic exchange with the DNA viral genomes, each group of cells obtained the machinery necessary to replicate DNA and eventually converted their genomes from RNA-based to DNA-based chemistry. Furthermore, cells with RNA genomes that were not infected

by DNA viruses never evolved DNA genomes, and Darwinian selection would have eventually driven these less fit cells extinct. It is conceivable that an enzyme like reverse transcriptase was a key to the conversion of RNA into DNA, just as it is in retroviruses today.

4. Overlapping genes are coding sequences for one protein that include the start and/or stop codons for other protein-encoding genes. Overlapping genes are common in viruses with small genomes where coding capacity is limiting, yet the genes must be maintained for faithful replication and evolutionary success. Examples of viruses containing overlapping genes include bacteriophages MS2, X174, and T7, as well as polyomavirus SV40 and hepatitis B virus.

5. Transcription in ssDNA phage X174 uses the replicative form (dsDNA) as a template.

Polycistronic mRNA molecules are produced, and several proteins are made using mRNAs transcribed from different reading frames on the DNA template (overlapping genes). In this way, many different proteins can be produced from a tiny genome of just 5386 bp.

6. The DNA sequence in each T7 virion is identical because T7 endonuclease cuts the concatemer at specific sequences. This differs from the situation in phage T4, where DNA concatemers are processed using a “headful mechanism” that generates circularly permuted genomes.

7. Mu replicates as a transposable element, and the genome can be integrated somewhat randomly into the host genome, potentially inactivating genes and creating mutations.

Mu carries host DNA at each end of its genome obtained from where a given particle .

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integrated. Mu requires the synthesis and activity of the gene A product, a transposase, for integration into the host chromosome.

8. The genome of archaeal virus ATV is circular dsDNA, as opposed to the linear dsDNA genome of bacteriophage T7. In addition, virus ATV has an unusual spindle morphology unlike the head and tail morphology of phage T7. Finally, virus ATV provides the first example of virus development outside of a host cell. ATV virions change from a lemon shape upon release from the host cell to an elongated form having tubes that extend from each pole. This form may help stabilize the virions in their extremely inhospitable environment (85C; pH 1.5).

9. Attached to the 5ʹ end of adenoviral genomic DNA is a protein called the adenoviral terminal protein, which is essential for replication of the DNA. The complementary DNA strands also have inverted terminal repeats that play a role in the replication process. Following infection, the adenoviral nucleocapsid is released into the host cell nucleus, and transcription of the early genes proceeds by activity of the host RNA polymerase. Most early transcripts encode important replication proteins such as the terminal protein and a viral DNA polymerase. Replication of the adenoviral genome begins at either end of the linear DNA genome and the terminal protein facilitates this process because it contains a covalently bound cytosine that serves as a primer for DNA polymerase.

10. Adenoviruses are unique in that they are able to replicate their linear dsDNA genomes continuously; the formation of a lagging strand is not required.

11. Both are double-stranded DNA viruses.

12. Poliovirus causes polio and is one of the smallest of all viruses with an icosahedral structure containing the minimum 60 morphological units per virion. Coronaviruses cause respiratory infections in humans and other animals, including about 15% of common colds and SARS, an occasionally fatal infection of the lower respiratory tract in humans.

Coronavirus virions are enveloped and contain club-shaped glycoprotein spikes on their surfaces. These give the virus the appearance of having a “crown” (corona is Latin for crown) and are the largest of any known RNA viruses, about 30 kilobases.

13. The VPg protein of poliovirus is covalently attached to the 5 end of the viral RNA. Along with the stem loop structures that can form at the 5 end, the VPg protein forms a complex resembling the 5 cap in eukaryotic mRNA. Coronavirus genomes are positive-strand RNA also, but they have a 5 methylated cap and 3 poly(A)-tail and so can function directly as mRNA. However, genome replication and the synthesis of most proteins occur through transcription and translation of a negative-strand RNA template, made by the activity of RNA replicase.

14. Poliovirus has a positive-strand RNA genome, whereas influenza virus has a negativestrand RNA genome. In addition, the poliovirus genome consists of a single RNA molecule, whereas influenza viruses have genomes consisting of several discrete RNA segments.

15. Rabies virus is a negative-strand RNA virus, which means that the RNA genome cannot directly serve as mRNA for translation of viral genes. Therefore, the rabies virus must first synthesize a complementary (plus) strand using the virus-encoded RNA-dependent .

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RNA polymerase (RNA replicase). By contrast, poliovirus has a positive-strand RNA genome that can serve directly as viral mRNA. 16. Reoviruses, such as rotavirus, influenza viruses, and phage MS2 all contain linear RNA genomes. However, reovirus genomes are double-stranded RNA, influenza viruses have negative-strand RNA genomes, and MS2 has a positive-strand RNA genome. Unlike the MS2 genome, which is a single RNA molecule, reoviruses and influenza viruses have segmented genomes.

17. Retroviruses have a positive-strand RNA genome, but they replicate through a dsDNA intermediate that integrates into a host cell chromosome. This DNA intermediate can only be synthesized from the RNA template using virally encoded reverse transcriptase.

Hepadnaviruses have a partially double-stranded DNA genome. New copies of their DNA genomes are synthesized directly from an RNA intermediate made by the host cell’s RNA polymerase. As observed in retroviruses, the synthesis of DNA from an RNA template requires reverse transcriptase.

18. Viroids are small, circular, single-stranded RNA molecules that are the smallest known pathogens. Unlike viruses, viroids have no capsid. Viruses are genetic elements consisting of either DNA or RNA surrounded by a protein coat and characterized by an extracellular state. While both are agents of infectious disease, diseases caused by viroids appear to be restricted to plants only.

19. A prion is an infectious protein whose extracellular form contains no nucleic acid.

Viruses are genetic elements consisting of either DNA or RNA surrounded by a protein coat and characterized by an extracellular state. However, both are agents of infectious disease in animals.

20. Viroids and prions are both agents of infectious disease and, by having less complexity than viruses, are considered subviral entities. Viroids are infectious nucleic acids (specifically, RNA molecules) lacking proteins. Prions are infectious proteins lacking nucleic acids. Lastly, viroids cause plant diseases, prions cause animal diseases.

Answers to Application Questions 1. Bacteriophage MS2 is a positive-strand RNA virus that, upon entry into the host, immediately translates its RNA replicase protein required to synthesize a minus strand. The minus strand serves as a template for synthesis of multiple copies of the plus strand.

From this point, there is differential expression and translation of viral genes involving extensive secondary structure, blocking the start codon of several genes. The coat protein start codon is the most accessible to the ribosome at this point. As the population of coat protein increases, these molecules interact around the AUG start codon for RNA replicase, thus acting as a repressor for its synthesis. The overlapping of the lysis gene with the replicase and coat protein genes ensures that the lysis gene is not translated before viral replication and capsid production have reached a level sufficient to ensure assembly of complete virions.

2. The continuous replication of linear dsDNA in adenoviruses can occur without violating the “rule” that all DNA synthesis occurs in the 5 → 3 direction because the two strands .

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are replicated asynchronously. After the minus strand is synthesized, it assumes a cyclic loop formation due to hybridization of inverted terminal repeats. A new complementary strand is then synthesized beginning at the 5 end.

3. Viroids are infectious RNA molecules that differ from viruses in that they lack protein.

Viroids are small, circular, single-stranded RNA molecules that are the smallest known pathogens. Viroid RNA does not encode proteins and thus the viroid is totally dependent on its host for replication. Plants have several RNA polymerases, one of which has RNA replicase activity, and this is the enzyme that replicates the viroid.

The extracellular form of a viroid is naked RNA; there is no protein capsid of any kind. Although the viroid RNA is a single-stranded, covalently closed circle, its extensive secondary structure makes it resemble a hairpin-shaped double-stranded molecule with closed ends. This apparently makes the viroid sufficiently stable to exist outside the host cell.

Most disease symptoms are growth related, and it is believed that viroids mimic or in some way interfere with plant small regulatory RNAs. In fact, viroids could themselves be derived from regulatory RNAs that have evolved away from carrying out beneficial roles in the cell to inducing destructive events. Viroids are known to yield small interfering RNAs (siRNAs) as a side product during replication. It has been proposed that these siRNAs may then function by way of the RNA interference silencing pathway to suppress the expression of plant genes that show some homology to the viroid RNA, and in this way induce disease symptoms.

4. Reoviruses are unique in that they have segmented genomes containing 10–12 molecules of linear dsRNA, most of which encode only a single protein. During reovirus replication, the RNA replicase activity must be confined to the nucleocapsid to avoid host enzymes that quickly degrade dsRNA molecules. In one sense, reovirus replication within the nucleocapsid is analogous to host DNA replication within the nucleus. In both cases, the genomes are contained and replicated within a structure that is isolated from the cytoplasm, and in both cases, mRNA molecules are removed to the cytoplasm to be translated. They contrast with one another in that the genetic material of the virus is dsRNA, which is transcribed by RNA replicase, whereas that of the cell is dsDNA, which is transcribed by RNA polymerase. Although reovirus genomes consist of double-stranded RNA, their replication is not semiconservative for two reasons: (1) positive-strand mRNAs are made only from the negative strand and (2) the synthesis of double-stranded genomes in newly assembled progeny viruses is accomplished using only the positive strand as a template.

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CHAPTER

10

Genetics of Bacteria and Archaea

Summary This chapter continues the discourse of molecular biology of microorganisms by introducing students to prokaryotic genetics. A quick review of material in Chapter 4 pertaining to DNA structure and the arrangement of genetic information may be a helpful introduction to these lectures. Chapter 10 devotes the first several sections to a discussion of DNA mutations. The remaining sections provide an extensive consideration of genetic exchange mechanisms found in Bacteria and Archaea.

10.1 | Mutations and Mutants Genetic change in a genome can be brought about by either mutation or genetic recombination (Section 10.5), with the latter potentially resulting in especially large changes to the genome. Impress upon your students that these two molecular phenomena drive the evolutionary process. Develop the concept of mutations by discussing how they change an organism’s genotype (the nucleotide sequence of the genome) but may or may not change the phenotype (the observable properties of the mutant). Unless previously discussed, this would be a good time to describe the convention in bacterial genetics for writing the genotype for a specific gene as three lowercase letters followed by an uppercase letter, all in italics. By contrast, the protein product of a gene is generally written using the same four letters, except that the first letter is also capitalized, and italics are not used. As an example, one of the genes in the lactose degradation pathway in E. coli would be written as lacZ (genotype), whereas its corresponding product would be written as the LacZ protein (phenotype).

Discuss mutant isolation techniques, including the use of replica plating for the screening of selectable and nonselectable mutations (Figures 10.1–10.2). Note that although screening is more tedious than selection, there are several clever methods one can employ to increase the efficiency of mutant isolation. A particularly cunning and widely used technique for isolating auxotrophs (mutants that require some nutrient for growth) is penicillin selection.

This is a type of negative selection in which only parent cells (prototrophs) capable of growth in a nutrient-deficient medium are killed by the antibiotic added to the medium. Since auxotrophs in the culture are not growing in the absence of the nutrient, they are unaffected by the presence of penicillin. Following incubation, cells are washed free of penicillin and placed in a complete growth medium. This process increases the ratio of auxotrophs to prototrophs, thereby making the screening process more straightforward and increasing the chances of .

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successful isolation of auxotrophic mutants. Discuss the various common classes of mutants and strategies for their selection and isolation, as shown in Table 10.1.

10.2 | Molecular Basis of Mutation Students must understand the molecular basis of mutations, which can be either spontaneous or induced by environmental factors or human intervention (e.g., by exposure to radiation or certain chemicals). Mutations that change only one base are called point mutations, and these occur from base-pair substitutions, deletions, or additions in DNA. If these mutations occur within the coding region of a gene, a defective protein may result. Review the concept of genetic code degeneracy (Section 4.11 and Table 4.5) to remind students that not all mutations within a gene result in a change in the amino acid sequence of its protein. Here you can discuss the three possible outcomes of base-pair substitutions: missense, nonsense, and silent mutations (Figure 10.3). Differentiate between the types of base-pair substitutions in point mutations: a transition is the substitution of one purine (A or G) for another purine or one pyrimidine (C or T) for another pyrimidine, whereas a transversion is the substitution of a purine for a pyrimidine, or vice versa.

Mutations resulting in the loss or gain of a base pair cause what are referred to as frameshifts, and these can have serious consequences. Single or double base-pair insertions or deletions change the reading frame, and thus change the primary structure (amino acid sequence) of a protein significantly. However, three base-pair insertions or deletions result in the addition or deletion of an amino acid in the protein and may not be as serious as other frameshifts. Use Figure 10.4 to develop this concept clearly for students, as these changes can be lethal and cannot be restored by back mutation as with single base-pair substitutions. Other large-scale mutations can be the result of either errors in genetic recombination or the activity of transposable elements (see Sections 6.12 and 10.11).

10.3 | Reversions and Mutation Rates It is important that students understand that point mutations may revert, and they often do so at high frequencies. These reversions (or back mutations) may restore the original nucleotide sequence at the same site, thereby creating a true revertant, or the reversion may occur at a second site in the DNA molecule. Second-site revertants can suppress the effects of the original mutation and restore the wild-type phenotype. Suppressor mutations can occur in the same gene as the original mutation or in another gene whose product is then able to nullify the original mutation (Figure 10.5). A third possibility is that a suppressor mutation in another gene can lead to the production of an alternative enzyme that performs the same function as the product of the original mutated gene.

Many introductory microbiology courses include a laboratory activity in which the Ames test is used to demonstrate the mutagenicity of a chemical (Figure 10.6). Although not all mutagens are carcinogenic, the correlation of mutagenicity with carcinogenicity is high.

Because animal studies are expensive and not done unless necessary, the Ames test has .

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become a good screening tool for the identification of mutagenic substances before they are used in animal test studies for evaluation of possible carcinogenicity.

Explain to the students that mutations occur at spontaneous frequencies in the DNA, depending upon the mechanism involved, and most of these errors are corrected by the proofreading abilities of DNA polymerase (see Section 4.6) and by DNA repair systems, which dramatically reduce the number of mutations fixed in a genome (~10–9 to 10–10). For example, the spontaneous rate of mutation is ~10–6 to 10–7 per kilobase pair (the average length of a gene) during one DNA replication cycle. Consequently, in a culture at 108 cells/ml, one can expect that every milliliter of culture will contain a mutation in any given gene. If one has a selection method for the gene in question, then it is possible to preferentially isolate the cell with the desired mutation. However, the use of mutagenic techniques can increase the mutation rate over the background spontaneous rate such that the number of mutants generated is increased, thereby producing a larger number of cells with the desired mutation (see Section 10.4).

Students may be interested to learn that the mutation rate of viruses having RNA genomes is estimated to be about one thousandfold higher than those having DNA genomes. This has important medical consequences since high mutation rates complicate the development of effective chemotherapeutic drugs. Although some RNA polymerases have proofreading abilities similar to DNA polymerases I and III, there are no RNA repair enzymes. New strains of influenza virus that seem to appear each winter often arise because of a high mutation rate that produces a new genetic strain of the parental virus. Note also that HIV, the causative agent of AIDS, has a high mutation rate that directly affects its virulence.

10.4 | Mutagenesis Discuss the various types of natural and artificial mutagens (Table 10.2) and the mechanisms by which cells deal with them. A relevant example is nucleotide base analogs, which resemble purines or pyrimidines structurally but do not base pair properly (Figure 10.7). If these analogs are incorporated into the DNA during replication, the mutation frequency at the site of incorporation increases. Other chemical mutagens induce chemical modifications (Table 10.2). For example, the alkylating agent nitrosoguanidine can induce base-pair substitutions even in nonreplicating DNA, and thus is a powerful mutagen. Another chemical mutagen group, the acridines, consists of intercalating agents that insert between base pairs and generate frameshifts via insertions or deletions. Ethidium bromide, which is often used to visualize DNA in electrophoresis gels, is an intercalating agent.

Note that in addition to chemical mutagens, both ionizing and nonionizing radiation are also effective at generating mutations in DNA. Ionizing radiation consists of very highenergy, short wavelength rays, including X-rays, gamma rays, and cosmic rays (Figure 10.8).

Mutations resulting from ionizing radiation occur indirectly through the generation of chemical free radicals that react with DNA and other macromolecules. Ultraviolet (UV) radiation is a type of nonionizing radiation widely used in mutagenesis laboratories. Nucleic acids absorb UV radiation strongly at 260 nm, and this results in the formation of pyrimidine dimers⎯ abnormal covalent bonds between adjacent pyrimidine bases (C or T) on the same strand of .

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DNA. Dimer formation interferes with the function of DNA replicating enzymes, particularly DNA polymerase.

Point out to your students that for a mutagen to induce permanent changes in the DNA, these changes have to be “fixed” in the genetic material through the process of DNA replication. Mutations that persist after replication are passed down vertically to daughter cells and thus become heritable. However, many mutations are repaired before replication occurs using a variety of mechanisms in the cell. DNA repair mechanisms for minor mutations are mostly error-free, but sites of major DNA damage often must be repaired using the error-prone SOS repair system. Use Figure 10.9 to show how DNA damage is “sensed” by the cell and how the cell then responds by inducing (derepressing) several genes simultaneously. This is an example of the global control regulatory systems presented in Chapter 7.

Note that although the spontaneous mutation rate is similar in organisms from Bacteria to Archaea, the mutation rate of an organism can be changed, and mutants with hyperactive DNA repair systems have been selected in the laboratory. Share with your students the interesting example of Deinococcus radiodurans, a naturally occurring bacterium with an unusually proficient DNA repair system (see Chapter 15). This species is 20 times more resistant to UV radiation and 200 times more resistant to ionizing radiation than is E. coli.

Note also that in contrast to hyperaccuracy in DNA repair, there are also strains that have a hypermutation rate, usually due to a deficiency in proofreading ability in DNA polymerase III, which confers a selective advantage in their environment. These organisms are referred to as mutator strains.

10.5 | Genetic Recombination Begin your discussion of gene transfer in prokaryotes by giving a brief overview of the three methods of genetic exchange: (1) transformation (Section 10.6), (2) transduction (Section 10.7), and (3) conjugation (Sections 10.8 and 10.9) (Figure 10.10). As stated in the text, regardless of the method of transfer of DNA, there are three possible “fates” that await it: (1) degradation by restriction enzymes, (2) replication by itself, and (3) recombination into the host genome.

Often one of the more difficult concepts for students studying molecular biology is genetic recombination. Spend considerable time and effort helping students follow the process and understand its significance. This section focuses on homologous recombination, which occurs when two distinct DNA molecules that share nearly the same sequence over an extended length base pair with one another. This is the crossing over phenomenon of classical genetics. Discuss the key role of the RecA protein in homologous recombination (Figure 10.11). Although all proteins in the process are not shown in the figure, note the important steps: strand invasion, heteroduplex formation (the Holliday junction), and resolution of duplexes via resolvase proteins. Detection of rare recombinants is possible only if there is an observable phenotype in the recombinant that is distinct from the parent cell (Figure 10.12). Note that this technique is possible only with prokaryotic cells due to the large number of cells required to select for a recombination event.

Point out to the students that RecA-like proteins have been identified in all three domains of life, suggesting the importance of homologous recombination in the evolutionary process. .

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Also note that recombination in eukaryotes involves entire chromosomes, which is not the case for prokaryotes.

Complementation is a condition in which a functional, wild-type gene introduced into an auxotrophic cell as part of an extrachromosomal element (e.g., a plasmid) compensates for a corresponding mutated gene and restores the cell’s prototrophic phenotype. Because two copies of the gene are present (one wild-type and one mutated), the cell is said to be merodiploid (partially diploid). Complementation is a useful method for identifying affected genes following mutagenesis.

10.6–10.7 | Transformation and Transduction Several techniques are available for introducing DNA into bacterial cells. Some of these occur naturally, whereas others are artificially induced. Natural transformation systems have been identified in Bacteria and some Archaea, and these have been modified to increase the efficiency of DNA uptake and expression for use in laboratory research. A landmark event in the study of biology was the discovery of transformation in bacteria. You might wish to discuss Frederick Griffith’s experiments in the late 1920s with smooth (encapsulated) and rough pneumococcus (Streptococcus pneumoniae), the results of which provided the first evidence for a “transforming principle” that could convert a harmless cell into an encapsulated, pathogenic form. You can stress the significance of this work by explaining to students how it prompted a series of experiments that led to the identification of DNA as the genetic material, culminating with the 1953 announcement of the structure of DNA by James Watson and Francis Crick. These early studies eventually led to the development of modern molecular biology and biotechnology.

Discuss the concept of competence in transformation and the fact that integration of DNA into the host genome requires homologous recombination, discussed in the previous section.

The uptake of DNA by competent cells requires special proteins: (1) a membrane-spanning DNA-binding protein, (2) a cell wall autolysin, and (3) nucleases. Cells vary widely in their efficiency of DNA transformation, and high-efficiency transformation systems are rare in Bacteria, with relatively few genera being easily transformed (e.g., Bacillus, Streptococcus, and Thermus). Interestingly, the model bacterium and workhorse of molecular biology, E. coli, has an inefficient natural transformation system. However, competency in E. coli can be improved artificially by treatment of cells with concentrated calcium and magnesium salts for several minutes in the cold. Cells can also be transformed by electroporation in which cells mixed with DNA are subjected to pulses of an electrical current to temporarily increase membrane permeability. The sizes of DNA fragments a cell can take up vary, and as in Streptococcus pneumoniae, DNA is initially bound as double-stranded molecules before being converted into single-stranded DNA during uptake. Naturally transformed DNA is usually integrated into the chromosome of the recipient via recombination mediated by RecA protein (see Section 10.5). Figure 10.13 presents the transformation and integration of DNA from a donor cell into a gram-positive bacterium.

Transduction is the virus-mediated transfer of DNA (including some host genes) from one cell to another. Compare and contrast generalized versus specialized transduction mecha.

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nisms in some detail. In generalized transduction, host DNA is accidentally packaged inside the viral capsid rather than viral DNA, resulting in the formation of a transducing particle that contains no viral DNA but can still transfer genetic material to a recipient cell (Figure 10.14). In contrast, specialized transduction results in the transfer of specific regions of the host genome that are integrated into the viral genome, replacing some viral genes. Discuss the details of the phage lambda specialized transduction mechanism, shown in Figure 10.15.

Note that the phenotype of a host cell can change following lysogenization by a transducing phage in a process called phage conversion. One of the more interesting examples of this process is the conversion of a nontoxin-producing strain of Corynebacterium diphtheriae to the toxin-producing, pathogenic strain following phage  lysogenization.

10.8–10.9 | Conjugation; The Formation of Hfr Strains andChromosome Mobilization Bacterial conjugation is a mechanism by which genetic material is transferred from a donor cell to a recipient cell through direct contact. The process was originally discovered in E. coli by observing the mobilization of chromosomal DNA by an integrated F (fertility) plasmid (Section 10.9). Using Figure 10.16, point out the salient genetic features of the F plasmid.

Indicate both the numerous transposable elements that allow for integration of the F plasmid into the host chromosome and the tra region, which allows for assembly of the sex pilus needed to establish a mating pair (Figure 10.17). Note that during DNA transfer, DNA synthesis occurs using a rolling circle replication process rather than by the usual bidirectional method (Figure 10.18). E. coli cells can carry the F plasmid in a nonintegrated form (creating an F+ strain) or as a form that is integrated into the genome (creating an Hfr strain) (Figures 10.19 and 10.20). Because conjugation between an Hfr cell and an F– recipient is interrupted before complete transfer of the chromosome, the recipient cell does not acquire the entire F plasmid and therefore does not become an Hfr donor itself (Figure 10.21). However, the recipient cell will acquire a new genotype (and likely a new phenotype, as well) if the transferred DNA successfully recombines with the recipient chromosome.

Because of its ability to integrate into the host chromosome, the F plasmid is an episome.

The F plasmid and chromosome each carry multiple insertion sequences (Section 10.11), and therefore homologous recombination can generate multiple types of Hfr strains, depending on where integration into the chromosome occurs (Figure 10.22a). Because of this, different Hfr strains transfer genes in different orders to the recipient cell (Figure 10.22b). This property allowed for accurate mapping of the E. coli genome before the age of DNA sequencing using an interrupted mating strategy. Hfr strains have been very useful in genetic crosses, wherein the donor strain (the prototroph) is sensitive to an antibiotic while the recipient strain is resistant to the antibiotic but auxotrophic for a particular nutrient.

10.10 | Horizontal Gene Transfer in Archaea .

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Molecular procedures for studying genetics in Archaea are far less developed than those for studying Bacteria. While the study of the genetics of Archaea is still in its infancy, progress is being made in developing gene transfer systems for these prokaryotes. A major complication is that many Archaea are extremophiles, and this presents problems for establishing a genetic system. For example, hyperthermophiles cannot be plated on standard agar media; alternative gelling agents must be used for media to remain solid at extremely high temperatures. In addition, most antibiotics are not effective against Archaea, thereby complicating mutant selection. In spite of the many obstacles, the effective use of several genetic tools, including transformation, viral transduction, transposon mutagenesis, and conjugation, has been documented in some Archaea, primarily methanogens, extreme halophiles, and the hyperthermophile Sulfolobus (Figure 10.23).

10.11 | Mobile DNA: Transposable Elements Discuss maps of the transposable elements IS2 and Tn5 and the general mechanisms of transposition (Figures 10.24 and 10.25). Point out to students that the two common features of insertion sequences and transposons are a gene-encoding transposase (tnp) and short inverted repeats at both ends. Clearly distinguish the difference between conservative transposition (as in Tn5), wherein the element is moved from one chromosomal location to another, and replicative transposition (as in Mu), in which the original copy remains at its

chromosomal locus and another copy is inserted elsewhere (Figure 10.26). Familiarity with transposons and transposon mutagenesis (Figure 10.27) is necessary to understand contemporary genetic engineering concepts and biotechnological applications.

10.12 | Preserving Genome Integrity: CRISPR Interference The clustered regularly interspaced short palindromic repeats, or CRISPR, system is a sort of RNA-based, prokaryotic “immune system” that protects cells from invading viral or otherwise foreign DNA. The system consists of foreign nucleic acid spacer sequences that have previously entered the cell and are stored within a specific region on the chromosome. The spacers are separated by identical repeats that provide cleavage sites for CRISPR-associated (Cas) proteins, which bind and cut the primary transcript into short CRISPR RNA transcripts (crRNAs; Figure 10.28). The crRNAs then bind homologous foreign sequences, including viral genomes, to label them for destruction by other Cas proteins.

Although not universal, CRISPR systems are widespread in prokaryotes. However, phages having higher mutation rates contain regularly changing genomes that are more of a “moving target” and are therefore less vulnerable to this defense system.

Answers to Review Questions

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1. A wild-type strain is a bacterial strain isolated from nature or one used as a parent in a genetics investigation The term “wild-type” may be used to refer to a whole organism or just to the status of a particular gene that is under investigation. Selection is an especially powerful genetic tool because it allows the isolation of a single mutant from a population containing millions or even billions of parental cells. A good example of a selectable mutation is drug resistance: An antibiotic-resistant mutant can grow in the presence of an antibiotic that inhibits or kills the parent and is thus selected under these conditions.

In replica plating, a colony from a master plate can be transferred onto an agar plate lacking the nutrient by using a sterile loop. Parental colonies will grow normally, whereas those of the mutant will not. Thus, the inability of a colony to grow on medium lacking the nutrient signals that it is a mutant or auxotroph, and the auxotroph can be isolated.

2. The metabolic roles that histidine and lactose play in Escherichia coli are very different.

Histidine is an amino acid that must be synthesized or obtained from the environment for cells to synthesize proteins and grow. A His– strain would be auxotrophic because it must acquire all necessary histidine from its environment. Lactose is a substrate that may serve as a source of carbon and energy, but it is not required for growth when other usable substrates are present. A Lac– strain might be auxotrophic if it were grown on a medium in which lactose was the only carbon and energy source available.

3. Silent mutations are heritable changes in the genome that have no apparent effect on the phenotype. In other words, they are not detectable by nonsequencing observations.

Because the genetic code is degenerate, a change in the third position of the codon triplet often encodes the same amino acid, as described in question 1 above. Such a mutation results in no structural change to the protein.

4. The average mutation rate for most microorganisms is between 10–6 and 10–7 per kilobase pair during each round of replication. This rate can significantly increase when cells are exposed to radiation or chemical or biological mutagens.

5. A transposon, such as Tn5, would be a biological mutagen. Transposons induce mutations by inserting themselves into genes, resulting in an interruption of the normal gene sequence. Ultraviolet radiation is an example of a physical mutagen; UV light induces the formation of pyrimidine dimers that inhibit the proper function of DNA polymerase.

Nucleotide base analogs are examples of chemical mutagens that impede proper base pairing, resulting in the insertion of an incorrect base into the new strand of DNA during replication.

6. A heteroduplex occurs when a DNA double helix is formed from single strands of DNA from two separate molecules. These structures form as a result of homologous recombination between DNA molecules having highly similar sequences, and this process is key for successful horizontal transfer of genetic material.

7. Cellular uptake of naked DNA from the environment is most efficient for small, linear fragments of DNA. In addition, one strand of the DNA is often degraded by nucleases before uptake, and only a single-stranded fragment enters the cell. Because they must remain double-stranded and circular to be replicated, successful natural transformation of plasmids is rare. .

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8. Transduction is the transfer of host cell genes from one cell to another by a virus. In generalized transduction, DNA derived from virtually any portion of the host genome is packaged inside the mature virion in place of the virus genome. In specialized transduction, DNA from a specific region of the host chromosome is integrated directly into the virus genome, usually replacing some of the virus genes.

9. The F plasmid contains genes that regulate DNA replication as well as a number of transposable elements that allow the plasmid to integrate into the host chromosome. In addition, the F plasmid has a large region of DNA, the tra region, containing genes that encode transfer functions. Many genes in the tra region participate in mating pair formation, and most of these have to do with the synthesis of a surface structure, the sex pilus. Only donor cells produce these pili. Different conjugative plasmids may have slightly different tra regions, and the pili may vary somewhat in structure. The F plasmid and its relatives encode F pili.

10. Cells possessing a nonintegrated F plasmid are called F+. Those with an F plasmid integrated into the chromosome are called Hfr strains. This term refers to the high rates of genetic recombination between genes on the donor and recipient chromosomes.

The F plasmid and the chromosome of E. coli both carry several copies of mobile elements called insertion sequences. These provide regions of sequence homology between chromosomal and F plasmid DNA. Consequently, homologous recombination between an IS on the F plasmid and a corresponding IS on the chromosome results in integration of the F plasmid into the host chromosome. Once integrated, the plasmid no longer replicates independently, but the tra operon still functions normally and the strain synthesizes pili. When a recipient is encountered, conjugation is triggered just as in an F+ cell, and DNA transfer is initiated at the oriT (origin of transfer) site. However, because the plasmid is now part of the chromosome, after part of the plasmid DNA is transferred chromosomal genes begin to be transferred.

11. Genetic selection in Archaea is difficult for several reasons. Antibiotics are generally ineffective in Archaea, and the extreme growth conditions many Archaea require are problematic for genetics experiments. Examples of selective agents that are effective include novobiocin (a DNA gyrase inhibitor) and mevinolin (an inhibitor of isoprenoid biosynthesis). Puromycin and neomycin have been shown to inhibit protein synthesis in methanogens.

12. Transposons and insertion sequences (ISs) are both genetic elements that can move from one location on a host chromosome to another. However, the only gene ISs contain is a transposase for excision and integration, whereas transposons may contain additional genes in addition to transposase, such as those encoding proteins for antibiotic resistance.

13. DNA in the CRISPR region of a prokaryotic chromosome is essentially an amassed memory bank of viral and other hostile sequences that provide a template for an RNAbased defense system. For crRNAs to bind in a homologous manner to foreign nucleic acids and tag them for destruction, the cell must have been pre-exposed to similar invading sequences. .

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Answers to Application Questions 1. A constitutive mutant may be formed if an altered repressor protein fails to recognize the operator (regulator constitutive) or if the operator locus fails to interact with a normal repressor (operator constitutive). In either case, the phenotype would be constitutive expression of the gene. The two constitutive mutants may be distinguished genetically using the cis-trans test in merodiploid cells.

2. Because chemical mutagens tend to alter the structure of specific groups on nucleotide bases in the DNA, they are not directed toward a specific gene. Site-specific mutations using synthetic DNA and current recombinant DNA technology allow one to construct a modified form of a DNA sequence, insert it into a recipient cell, and select for mutants at a specific region. Mutations of this type will be site specific but not gene specific. Sitedirected mutagenesis requires that the gene to be mutated already be cloned for specific targeting of the base pairs to be changed. The mutant gene is then inserted into the host for production of the mutant protein.

3. Transformation of several genes in a single experiment is greatly restricted by the size of the DNA fragments liberated by the transforming cells. Because the average size of DNA fragments to be transformed contains about 10,000 nucleotides (approximately10 genes) and there will be thousands of these present during a transformation event, there is competition between these fragments for access to the cell surface for uptake. Consequently, only a few of the genes of a cell may be transferred since a recipient cell will pick up only a few of the DNA fragments. In addition, only about one out of several hundred fragments will contain the marker being studied. Similar to the size limitation of DNA fragments in transformation, a finite number of genes can be transferred into a recipient via a transducing phage since each gene is about 1000 nucleotides in length.

4. The non encapsulated mutants on an agar plate would appear as small, rough colonies instead of larger, smooth colonies. For isolation of an arginine auxotroph that is resistant to penicillin the isolation media should comprise of arginine as the sole source of carbon.

M9 minimal media supplemented with arginine instead of glucose could be used Once arginine auxotrophs are screened, they could be plated on the same medium supplemented with penicillin. Colonies that appear on it are the required mutants, which are penicillin resistant arginine auxotrophs.

The Ames test is one of the most useful screens for determining the potential mutagenicity of a compound. The Ames test assays for back mutations instead of forward mutations (generating auxotrophs from the wild type) because revertants can be more easily selected. It is important that the auxotrophic strain used in the Ames test carry a point mutation so that the reversion rate is measurable. Cells of such an auxotroph do not grow on a medium lacking the required nutrient (for example, an amino acid), and even very large populations of cells can be spread on the plate without the formation of visible colonies. However, if revertants (back mutants) are present, those cells form colonies.

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CHAPTER

11

Genetic Engineering and Biotechnology

Summary Chapter 10 presented information on how scientists have exploited the genetic transfer abilities of microorganisms (transformation, transduction, and conjugation) to move genes and study genetic processes. However, there are limitations in using these in vivo techniques.

Chapter 11 therefore describes a variety of in vitro approaches that are routinely used by geneticists to manipulate genes and DNA molecules.

11.1–11.2 | Restriction Enzymes and Nucleic Acid Separation; Nucleic Acid Hybridization These sections describe some of the basic tools employed for manipulating DNA. Discuss restriction enzymes and their recognition sequences in some detail. Point out that microorganisms (mostly prokaryotic) produce restriction enzymes naturally for protection against viral genomic invasion. The exploitation of these enzymes gave rise to what we now refer to as genetic engineering. Use the EcoRI and EcoRV examples shown in Figure 11.1 to show specific restriction enzyme recognition sequences that generate “sticky end” and “blunt end” fragment products, respectively (Figure 11.1a). Also shown are the same recognition sequences following modification by the EcoRI and EcoRV methylases, which prevent cutting of the cell’s own DNA by the corresponding restriction enzymes (Figure 11.1b). Note that restriction enzymes are classified into three classes. Types I and III bind to their recognition sequence but cut DNA some distance away from that binding site. Type II enzymes cut within their recognition sequence and thus are most useful in molecular biology and genetic engineering. Table 11.1 lists several restriction endonucleases from various species of bacteria and their corresponding recognition sequences.

Section 11.1 also describes agarose gel electrophoresis (Figure 11.2). This widely used technique allows for the separation of DNA fragments based on size. In this technique, an electrical current is applied to direct the movement of negatively charged DNA molecules through an agarose gel matrix toward a positive electrode. Smaller and more compact molecules move more quickly through the gel, whereas larger or less compact molecules migrate slowly. Visualization of DNA fragments is usually aided by the application of DNA-binding stains, such as ethidium bromide, which fluoresces under ultraviolet light. A DNA ladder containing known sizes of DNA fragments usually serves as a standard from which to com.

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pare DNA fragments in the sample (Figure 11.2b). The separated fragments can then be extracted from the gel and purified for DNA sequencing or a variety of other purposes.

Next, discuss nucleic acid hybridization, and show how this mechanism is central to the functioning of Southern and Northern blots (Figure 11.3), as well as fluorescence in situ hybridization (FISH) techniques (Figure 11.4). The concepts of DNA denaturation, hybridization kinetics, and nucleic acid probes may be initially difficult for students to comprehend.

However, stress to students that the basis for any nucleic acid hybridization technique is complementary base pairing of DNA (or RNA) molecules. You may find it beneficial to use as many visual sources as possible, including any of several video tutorials available as ancillary materials to the text and those found free online.

11.3 | Polymerase Chain Reaction (PCR) Spend considerable time and detail discussing the polymerase chain reaction (PCR) (Figure 11.5). Carefully discuss the steps (and principles of each) in a PCR cycle. This technique uses all of the principles of DNA replication and is one of the most widely used techniques in molecular biology. In addition to Taq polymerase, derived from the thermophile Thermus aquaticus, the isolation and identification of novel DNA polymerases from hyperthermophiles (e.g., Pfu from Pyrococcus furiosus) have provided researchers with an even more thermostable polymerase for highly accurate amplification of DNA. Give examples of the various applications of PCR, from phylogenetics to forensics.

The power of PCR has been expanded with the development of reverse transcription PCR (RT-PCR), which uses the enzyme reverse transcriptase to create double-stranded cDNA from an mRNA template (Figure 11.6), and quantitative (real-time) PCR (qPCR), which can be used to quantify the amount of target DNA or RNA in the initial sample.

These techniques have been used for the detection and diagnosis of pathogens from clinical samples and for environmental sample analysis.

11.4 | Essentials of Molecular Cloning This section discusses molecular cloning methods used as a means to investigate biological questions. Inform students that the methods described were developed from knowledge gained from investigating topics such as DNA replication and protein synthesis in microbial systems. For example, cloning (Figure 11.7) requires the use of restriction enzymes, plasmids, DNA ligase, and genetic transfer systems, all of which arose from the study of microbial systems and can now be purchased in rather expensive kits. The students may perform many of the techniques described here as laboratory activities in more advanced courses.

However, it is important for them to understand the principles behind these techniques if they are to be truly useful as tools in their future scientific work.

If the gene of interest is cloned using PCR (Section 11.3), then it has already been identified (this technique requires that primers be synthesized from previously identified sequence information), and thus, a single clone is the objective. Most of the time, however, the scientist is trying to identify and clone a single gene from the chromosome by creating a gene library .

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of the chromosomal DNA. This then creates the problem of finding the desired clone within the library. Multiple approaches are available for finding the clone of interest: • Identifying a gene that is not expressed in the host requires the use of a labeled (either radioactive or fluorescent) nucleic acid probe that hybridizes to the target sequence (i.e., the cloned gene) on the DNA of lysed host cells (Figure 11.8a). Explain to students that by varying the hybridization conditions, the stringency of hybridization can be controlled. For example, strict conditions require nearly exact complementary base pairing for hybridization to occur, which allows for increased specificity and prevents false positives.

• Identifying a cloning host that is expressing the gene (and thus producing the protein) requires that the cloning host must not naturally produce the protein or that a mutant host strain defective in the ability to produce the protein be available. Thus, it may be possible to identify the proper clone via complementation if the protein has a function that can be detected by an enzyme assay, for example. The library could also be screened with a labeled antibody that was generated against the protein of interest and will detect its presence in a particular host cell (Figure 11.8b). This does not require that the expressed protein have a detectable function. Though highly specific, this method requires the availability of an antibody that binds the protein antigen in question.

11.5–11.6 | Molecular Methods for Mutagenesis; Gene Fusions and Reporter Genes The artificial synthesis of DNA has become a central component of genetic engineering, and a number of commercial systems are available for synthesizing strands of DNA of 12 to over 100 nucleotides in length (called oligonucleotides). Longer DNA molecules are synthesized by joining individual oligonucleotides using DNA ligase. Interestingly, artificial DNA synthesis proceeds in the 3 → 5 direction, which is opposite that of DNA replication. Synthetic DNA molecules are used for a variety of purposes in molecular biology, such as primers for PCR (see Figure 11.5), probes for nucleic acid hybridization (Figure 11.6a), and as oligonucleotide primers for site-directed mutagenesis (Figure 11.9).

Remind students that in the previous discussion of mutagenesis (Section 10.4), the mutations introduced into the DNA were random, and thus usually required clever, and sometimes tedious, selection protocols. Site-directed mutagenesis, in contrast, can precisely target mutations to a given site on the chromosome using either synthetic DNA in conjunction with DNA cloning techniques (Figure 11.9) or PCR with primers that contain the desired nucleotide sequence changes. Point out to students that a useful application of this technique is the ability to change the amino acid sequence of a protein (a result of nucleotide sequence changes to its corresponding gene) to study its effect on that protein’s activity. Continue with a discussion of cassette mutagenesis as a tool for targeted gene disruption (Figure 11.10). Using this method, the insertion of a cassette at a particular site on the DNA performs two functions: (1) It deactivates the target gene, thereby creating a gene knockout, and (2) it confers resistance to a particular antibiotic, which allows for selection of cells containing the cassette. .

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Perhaps one of the most important outcomes of the development of in vitro gene manipulation tools was its effect on the study of gene regulation. The most common approach is to place the coding sequence from one source next to the regulatory region from another source.

This construct is then fused to a reporter gene to monitor expression under a variety of conditions. One of the most commonly used reporter genes has been the lacZ gene, which encodes -galactosidase (see Section 7.3). The activity of -galactosidase can be easily assayed and/or detected on indicator plates (see Figures 11.14 and 11.20). Many other reporter genes are now available, many of which provide more versatility and sensitivity than lacZ as a reporter system. Green fluorescent protein (GFP) is an excellent example of such a reporter (Figure 11.11), and since its initial use, other variations of GFP have been developed.

Reporter genes are commonly used when constructing gene fusions, where the regulatory region from a particular gene is fused to the coding sequence of a reporter gene to evaluate expression of the gene of interest (Figure 11.12). Gene fusion methods can be used to determine whether regulation occurs at the level of transcription (via an operon fusion) or translation (via a protein fusion).

11.7–11.8 | Plasmids as Cloning Vectors and Hosts for Cloning Vectors Some of the major concepts of genetic engineering, such as molecular cloning, were discussed earlier in the chapter. In these sections, the authors expand on this topic by describing the variations and advances in the tools and strategies employed in cloning and genetic engineering.

Figure 11.13 provides a nice overview of the essential features of plasmid pUC19, a commonly used cloning vector. Discuss the 10 characteristics described in the text that make pUC19 a useful cloning vector. The procedure for cloning using plasmid pUC19 is shown in Figure 11.14. Point out the key components of this technique, including the use of ampicillin to select for cells containing the plasmid and the incorporation of the lacZ gene as a reporter.

The disruption of lacZ by foreign DNA results in insertional inactivation; thus, functional -galactosidase can no longer be produced. Students should understand that the lack of -galactosidase activity results in white colonies, thereby indicating which cells have been transformed. Some plasmid vectors have been developed specifically to clone DNA products from PCR reactions. Such commercially available vectors are usually linearized plasmids with overhanging thymidine residues that pair with adenine extensions on the PCR product (Figure 11.15).

One of the most important components in any successful genetic engineering system is the choice of a suitable host strain for uptake, replication, and expression of the cloned DNA. Discuss the desired characteristics of a host organism, and highlight those specific for gene expression in prokaryotic versus eukaryotic hosts (Figure 11.16).

• Prokaryotic host cells: Point out to students the fact that laboratory strains of E. coli have been and still are the “workhorses” for routine molecular biology manipulations. However, these strains were derived from human intestinal isolates, some of which are potential pathogens and thus are not as desirable for large-scale production of pharmaceuticals and .

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other products. By contrast, the gram-positive, nonpathogenic bacterium Bacillus subtilis naturally secretes proteins into the medium (an advantage for large-scale drug production) and is amenable to genetic manipulation. Unfortunately, exogenous DNA is not always stable in B. subtilis (Figure 11.16).

The take-home message for students is that there is a real need for the development of alternative host organisms for expression of foreign genes by the biotechnology industry.

This is especially important for genes isolated from eukaryotic sources (plants, animals, fungi, protists) and genes from extremophiles—many of which will not be transcribed, translated, and/or produce a functionally active protein in a nonextremophilic E. coli-type host. For example, one may expect that although a cloned gene for a membrane-bound enzyme from a hyperthermophilic bacterium may be able to be translated into its protein product in E. coli, its enzymatic activity may be undetectable due to the growth temperature requirements of the host and its protein(s). One solution to this problem is to first identify a “compatible” hyperthermophilic host organism, and then develop techniques for uptake and expression of cloned genes in that organism.

• Eukaryotic host cells: Most eukaryotic recombinant DNA work has been done in the yeast Saccharomyces cerevisiae due to the extensive genetic tools developed in this organism (Figure 11.16). Ask students why it would be desirable to use a eukaryotic host cell for expression of eukaryotic DNA. Recently, another yeast expression system employing Pichia pastoris has been shown to possess advantages over S. cerevisiae for expression of particular proteins. Hopefully, the students’ understanding of transcription and translation differences between prokaryotes and eukaryotes will elicit a quick and confident response regarding the need for primary RNA transcript splicing and possible posttranslational processing mechanisms, both of which are difficult to genetically engineer into a cloning vector for use in a prokaryotic host. Also mention the availability of mammalian cell lines for use as hosts for studies in cancer, human genetics, and infectious diseases.

However, these systems are more expensive and difficult to maintain and, thus, are generally used only when a suitable alternative is not available. Insect cell lines and compatible vectors are also useful for many eukaryotic genetic studies, and sophisticated systems are also available in plants and plant tissue culture lines, the latter of which has spawned the huge and rapidly growing agricultural biotechnology industry.

• As students learned in Chapter 10, the term transformation refers to a mechanism of DNA uptake in prokaryotes. However, in mammalian cells it is used to describe the conversion of a normal cell to a malignant cell. The mechanism used for uptake of exogenous DNA in eukaryotes is referred to as transfection. There are several techniques used to transfect mammalian cells, including microinjection, electroporation, and microparticle gun bombardment (Figure 11.14). The particle gun method has been successfully used to transfect yeasts, algae, plant cells, mitochondria, and chloroplasts.

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11.9 | Shuttle Vectors and Expression Vectors Discuss the use and properties of specialized vectors (i.e., shuttle vectors and expression vectors) available for use in both prokaryotic and eukaryotic hosts. Shuttle vectors can be stably replicated in two unrelated organisms (e.g., E. coli and B. subtilis, or E. coli and yeasts), thus allowing for gene transfer between them. Emphasize again the components required of a useful vector: origin(s) of replication, promoters, terminators, selectable markers, reporter genes, a polylinker, and the ability to regulate the expression of the cloned gene (ribosome-binding site, operator/repressor sequences). Figure 11.17 shows the key features of a shuttle vector that can be maintained and is used in both yeast and E. coli.

Expression vectors are designed to allow the researcher to control the expression of a cloned gene (Figure 11.18). In the biotechnology industry, the goal is usually to obtain the maximum yield of its protein product. The design of expression vectors has to allow for precise control of transcription of the cloned gene and must ensure the efficient translation of mRNA. Discuss the bacteriophage T7 control elements (promoter and terminator sequences) and their use in expression vectors (Figure 11.19). Point out to students how the lac system is used in conjunction with T7 RNA polymerase to provide an easily regulated expression system that yields large amounts of the cloned gene product. For maximum translational efficiency, site-directed mutagenesis may be necessary to align the codon usage of the cloned gene with that of the host organism.

11.10 | Other Cloning Vectors Lambda is a useful cloning vector due to its long history in molecular genetics and the fact that it can hold about twice as much DNA (~20 kbp) as most plasmids. This larger capacity allows for the cloning of large genomic regions, such as operons and eukaryotic genes. Discuss the steps involved in cloning with lambda, a process that requires restriction enzymes, DNA ligase, and lambda-lysed cell extracts, which serve as a source of head and tail proteins for phage maturation (Figure 11.20).

Cosmid vectors are plasmid vectors that are ligated to lambda cos sites and can be packaged into stable phage particles. The main advantages of using cosmid vectors are that they allow for the cloning of large DNA inserts (up to 50 kbp), and they provide for long-term storage of recombinant DNA molecules.

Vectors are routinely employed for the cloning and sequencing of a particular gene or set of genes. However, some vectors are more specialized and provide advantages for genomic analysis. Discuss the advantages and disadvantages of using artificial chromosomes in genome sequencing projects. Note that bacterial artificial chromosomes (BACs; Figure 11.21) can accept foreign DNA inserts of about 300 kbp, whereas up to 800 kbp of DNA can be cloned into yeast artificial chromosomes (YACs; Figure 11.22).

11.11 | Expressing Mammalian Genes in Bacteria

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Review some of the major problems and challenges of expressing eukaryotic genes in bacteria, as discussed in the text. Because of these problems, several eukaryotic organisms have been genetically modified to serve as host cells for animal protein expression, including transgenic plants and animals (see Section 11.13).

The focus of Section 11.11 is the expression of mammalian genes in bacteria to obtain useful products. Dramatic advances have been made in the identification and cloning of mammalian genes; however, expression of these genes in bacteria requires synthesis of cDNA as a strategy to remove introns (Figure 11.23). Eukaryotic mRNA can be isolated using its unique poly(A) tail by hybridization to cellulose-supported poly(T) in a chromatographic column. The purified mRNA can then be reverse-transcribed into double-stranded cDNA, which now lacks introns and can be inserted into an appropriate expression vector for cloning. However, because cDNA also lacks promoters and regulatory sequences, bacterial promoters and ribosome-binding sites are engineered separately into the vector. Keep in mind that because a cDNA gene library is generated from mRNA, only those genes expressed under a specific set of growth conditions are represented.

If the sequence of the gene of interest is known, a cDNA probe can be synthesized and used to screen the gene library. Alternatively, if amino acid sequence information of the protein is available, one can use reverse translation to construct a DNA probe for gene identification (Figure 11.24). Impress upon the students that due to the degeneracy of the genetic code, many possible DNA probe combinations will be available for a given stretch of amino acid sequence, and thus only one probe in the pool of oligonucleotides will be an exact match. This has implications for designing hybridization stringency conditions for screening for the gene of interest. An exact sequence match is not necessary for hybridization if lower stringency conditions are used, especially with longer probes.

Expressing a protein of interest in a foreign host can also be problematic for other reasons, including improper folding and degradation by host proteases. Point out to students that after the successful expression of the protein of interest, there must be efficient and cost-effective methods for purifying the protein. Consequently, most expression vectors encode N-terminal or C-terminal amino acid sequences that result in a recombinant fusion protein that facilitates the subsequent purification of the recombinant protein (Figure 11.25).

11.12 | Somatotropin and Other Mammalian Proteins Perhaps the most intense area of research in biotechnology today involves the production of human proteins, especially hormones, because they are present in such small amounts in human cells and are thus prime candidates for microbial production approaches. Discuss the genetic engineering and expression of the growth hormone somatotropin, which, unlike insulin, is a single polypeptide encoded by a single gene (Figure 11.26). Note that the administration of somatotropin does not work in all cases of stunted growth, as some dwarf individuals lack growth hormone receptors but produce normal levels of somatotropin. In the dairy industry, recombinant bovine somatotropin is used to stimulate milk production and increase milk yield (Figure 11.26).

Discuss other genetically engineered mammalian proteins and their uses (Table 11.2).

The practical examples of tissue plasminogen activator (TPA) and blood clotting factors .

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VII, VIII, and IX should be especially interesting to students planning careers in medicine.

Point out to students that in addition to hormones, enzymes are genetically engineered and produced in significant quantities for human use. For example, human DNase I thins mucus in the bronchial tubes of individuals with cystic fibrosis by digesting DNA released by lysed cells of Pseudomonas aeruginosa. This pathogen commonly infects and forms biofilms in the lung tissue of cystic fibrosis patients.

11.13 | Transgenic Organisms in Agriculture and Aquaculture The terms transgenic organisms and genetically modified organisms (GMOs) are often used interchangeably, but this is inaccurate. Transgenic organisms contain a transgene (a gene from another organism), whereas a GMO is an organism that has been genetically engineered and may or may not contain a transgene.

In this section, the genetic engineering of plants is discussed, along with public concerns and protests over the use of genetically modified plants (especially in some European Union countries). A detailed discussion of the structure, function, and use of the Ti plasmid from the plant pathogen Agrobacterium tumefaciens is warranted considering the significant impact this bacterium has had on the agricultural industry (Figure 11.27). Although the Ti plasmid system works well with broadleaf dicots, it is not suitable for grassy monocots, including corn. However, other methods are available for inserting DNA into these plants, including the DNA gun system shown in Figure 11.28.

Note that a major focus of transgenic plants is on herbicide resistance (e.g., “Roundup ready” soybeans; Figure 11.29) and insect resistance via Bt toxin, a crystalline insecticidal protein derived from Bacillus thuringiensis (Figure 11.30). However, some transgenic plants are engineered for other purposes, including a higher nutritional content, a longer shelf life, and the production of pharmaceuticals. Point out the many successes in the construction of transgenic plants that have improved insect/disease resistance and product quality and that have made these plants factories for the production of recombinant proteins and even vaccines.

Finally, discuss the application of genetic engineering to the aquaculture industry using the development of transgenic “fast-growing salmon” as an example (Figure 11.31). Your students will be interested to know that the two salmon in the figure are the same age.

Transgenic salmon (top) reach a mature size much faster than non-genetically modified salmon (bottom). This is because, unlike the more seasonal production of growth hormone in wild-type salmon, the genetically engineered salmon contain a growth hormone gene with a promoter that is active year-round, resulting in fish that produce growth hormone constantly.

11.14–11.17 | Genetically Engineered Vaccines, Mining Genomes, Engineering Metabolic Pathways, and Synthetic Biology .

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The design and production of genetically engineered vaccines (Section 11.14) is an intense research focus in the pharmaceutical industry. Explain to students that typical vaccines are suspensions of killed or modified pathogens, with the antigenic component usually being a surface protein (e.g., a virus coat protein). A number of genetically engineered vaccines are currently in use, and several others are under development. These include several types of recombinant vaccines (Figure 11.32). Vector vaccines are recombinant vaccines that promote immunity through an innocuous carrier virus that contains genes from a pathogenic virus.

Polyvalent vaccines are engineered vaccines that confer immunity against two different diseases. Subunit vaccines contain only certain immunogenic proteins (or portions of proteins) from a pathogen, such as a viral coat protein.

Remind students of the term metagenomics (Section 6.10) and discuss the metagenome, the total genomic content of an environment. The vast majority of microorganisms present in a given environment cannot be sustained in laboratory culture using current technologies.

Given the fact that most of the genetic information on Earth exists in microorganisms, the genomes present in a particular environment will most certainly contain many novel genes that encode proteins with potential biotechnological applications. Differentiate between the techniques of environmental (Figure 11.33) and targeted gene mining. Note that there are advantages and disadvantages of isolating environmental RNA versus DNA, and discuss these differences with the students. Also point out the use of bacterial artificial chromosomes (BACs; see Section 11.10) in screening for entire metabolic pathways or in screening samples expected to be of higher biodiversity.

The expression of a single protein encoded by a single gene is relatively straightforward using modern genetic techniques. However, some of the most important molecules with potential medical or biotechnological applications are metabolites that are often transient and produced in biochemical pathways that require several enzymatic steps. Consequently, metabolic pathway engineering (Section 11.16) is an active research area in which an existing metabolic pathway is improved by using genes from more than one organism. In some cases, an entire metabolic pathway is introduced and expressed in a host organism. An example of pathway engineering is the production of indigo in E. coli (Figure 11.34).

The methodologies of genetic engineering have been taken to a new level in the burgeoning field of synthetic biology (Section 11.17). Recently, the genome of Mycoplasma capricolum was replaced with a synthetic chromosome based on the Mycoplasma mycoides genome, resulting in a viable cell with all of the usual properties of the latter species. Figure 11.35 shows an interesting example of small-scale synthetic biology in the form of “bacterial photographs.” By combining a photoreceptor from the cyanobacterium Synechocystis with a sensor protein from Escherichia coli, a light-sensitive fusion protein was synthesized that regulates production of β-galactosidase. When light is transmitted through a mask over a lawn of the engineered bacteria, shaded portions of the Petri plate become pigmented from the production of a black dye as β-galactosidase cleaves Xgal, a lactose analog, in the growth medium, thus reproducing the image on the plate culture. The point of this technology, of course, is not to pursue a novel form of photography using microorganisms, but rather it is to develop tools and techniques that may someday be useful in solving practical problems that threaten human welfare and the environment.

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Answers to Review Questions 1. Restriction enzymes are enzymes that cut double-stranded DNA molecules at specific sites based on the nucleotide sequence. The primary function of these enzymes is likely to protect the cell from invasion by foreign DNA from viral infection. The bacterium protects its own DNA from degradation by the action of modification enzymes, a major example of which involves methylation of the host DNA so that the restriction enzyme cannot recognize its cognate sequence.

2. Nucleic acid hybridization is a process where DNA is denatured (that is, the two strands are separated) and the single strands form hybrid double-stranded molecules with other single-stranded DNA (or RNA) molecules by complementary (or almost complementary) base pairing.

In southern blotting, probes of known sequence are hybridized to target DNA fragments that have been separated by gel electrophoresis. The hybridization procedure in which DNA is the target sequence in the gel, and RNA or DNA is the probe, is called a Southern blot. By contrast, a Northern blot uses RNA as the target sequence and DNA or RNA as the probe to detect gene expression. In fluorescence in situ hybridization (FISH), a range of different fluorescent signals can be covalently linked to oligonucleotide (short single-stranded DNA or RNA molecules) probes to target specific DNA sequences.

3. PCR gene amplification relies on the basic principle of DNA polymerases, which recognize double-stranded DNA containing a free 3–OH. The addition of primers complementary to each strand, along with nucleoside triphosphates, allows the reaction to proceed by the following steps: (1) heating to denature the double-stranded DNA, (2) lowering the temperature to allow annealing of the primers, and (3) raising the reaction mixture to the optimum temperature for a thermophilic DNA polymerase to allow for chain elongation. These cycles are repeated many times to amplify the target sequence of interest.

The use of thermostable polymerases has reduced the problem of nonspecific binding of primers that occurs at lower temperatures, and it has eliminated the need to add new enzymes following each heat denaturation step.

4. The essential characteristics of a cloning vector are (1) small size, making DNA isolation and manipulation easier; (2) independent origin of replication; (3) multiple copy number, allowing for amplification of cloned DNA; and (4) selectable markers for identification of the cells containing the cloning vector or recombinant clone. Characteristics that make plasmids, in particular, useful for molecular cloning (in addition to those just stated) include (1) a polylinker, or multiple cloning site; (2) color selection for immediate identification of recombinant plasmids; and (3) promoters for regulating expression of the cloned gene (e.g., M13 phage promoters for generation of single-stranded DNA). The F plasmid is much too large to be useful as a cloning vector and does not contain any selectable markers.

5. The steps in gene cloning include isolation and fragmentation of the source DNA, insertion of the DNA fragment into a cloning vector, and introduction of the cloned DNA into a host organism. Identification of the correct clone can be done by DNA sequencing or by restriction digests performed on plasmids extracted from a large number of colonies.

Hybridization is often used to detect the presence of specific genes in genomes that have .

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not yet been sequenced. Alternatively, antibodies can be used to detect a protein of interest; here, the antigen is the protein encoded by the cloned gene. Because the antibody combines specifically with the antigen, observing the binding of the antibody can identify colonies that contain the antigen. Because very little of the antigen is present in each colony, only a small amount of antibody is bound, and so a highly sensitive procedure for detecting bound antibody must be used. In practice, radioisotopes, fluorescent chemicals, or enzymes are used.

6. Major uses for artificially synthesized DNA include (1) primers for DNA sequencing and PCR, (2) nucleic acid hybridization probes for detection of specific DNA or RNA fragments, such as those in a clinical or environmental sample, and (3) primers for sitedirected mutagenesis.

7. In vitro mutagenesis or site-directed mutagenesis uses synthetic DNA plus DNA cloning techniques to introduce mutations into genes at precisely determined sites. By altering gene sequences to produce amino acid sequence changes, site-directed mutagenesis is used to manipulate protein characteristics such as enzyme activity or protein-binding affinity. Site-directed mutagenesis can be used to investigate the activity of proteins with known amino acid substitutions. Using site-directed mutagenesis, enzymologists can link virtually any aspect of an enzyme’s activity, such as catalysis, resistance, or susceptibility to chemical or physical agents, or interactions with other proteins, to specific amino acids in the protein.

8. A reporter gene is a gene that allows one to identify a cell or colony containing a cloned gene. Two examples include the lacZ and GFP genes, which allow visible identification of the expression of a cloned gene via either pigmented colonies or fluorescence, respectively.

9. Gene fusions are single genetic constructs that contain components of two different genes. The most common use of gene fusion constructs is to study gene regulation. For example, a promoter for a particular gene could be fused to the coding region of a reporter gene to study transcriptional regulation of that gene. Alternatively, the promoter for a gene could be removed and the gene fused to a different regulatory element to study its regulation from a different promoter.

10. Insertional inactivation of the lacZ gene by a cloned insert results in white colonies on a selection plate containing Xgal, a colorless analog of -galactose that turns blue when cleaved by -galactosidase. Thus, blue colonies represent cells that contain plasmid without insert (i.e., those cells producing functional -galactosidase), and white colonies consist of cells that contain plasmid with cloned inserts (i.e., those cells having a disrupted lacZ gene).

11. E. coli is an excellent choice for initial cloning work, but it is problematic as an expression host because it is found in the human intestine and some wild-type strains are potentially harmful. A major problem with using any bacterial host, including E. coli, is the lack of systems to correctly modify eukaryotic proteins. Another problem with using E. coli is that, like all gram-negative bacteria, it has an outer membrane that hinders protein secretion. The main disadvantage of using B. subtilis as a cloning host is plasmid instability. It is often difficult to maintain plasmid replication over many subcultures of the .

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organism. Also, foreign DNA is not as well maintained in B. subtilis as in E. coli; thus the cloned DNA is often unexpectedly lost.

12. A shuttle vector is a cloning vector that can stably replicate in two or more different organisms, allowing for the transfer of genes between unrelated species. An expression vector is a vector that can be used to clone the desired gene and contains the proper regulatory sequences to control the desired level of expression of that gene for further study and manipulation.

13. If the cloned gene contains introns, as eukaryotic genes typically do, the correct protein product will not be made if the expression host is a prokaryote. This problem can also be corrected by using synthetic DNA. However, the usual method to create an intron-free gene is to obtain the mRNA (from which the introns have already been removed) and use reverse transcriptase to generate a complementary DNA (cDNA) copy.

14. Viral cloning vectors (such as lambda) have several advantages over plasmids, including the ability to hold much larger amounts of DNA than most plasmids. In addition, cloned DNA can be efficiently packaged into phage particles in vitro, which can then be used to infect a suitable E. coli host. Lambda is especially useful because its biology is well understood, and because the central third of its genome is not essential for infectivity, it can be removed to allow for cloning and packaging of large segments of DNA. 15. Artificial chromosomes are single copy cloning vectors that allow for the insertion of extremely long DNA segments (nearly 1 Mb), which is especially useful for genomic sequencing projects. Necessary components of artificial chromosomes, such as an origin of replication and a selectable marker gene, are shown in Figures 11.21 and 11.22. BACs (bacterial artificial chromosomes) can carry >300 kbp of foreign DNA and are derived from the F plasmid. YACs (yeast artificial chromosomes) are similar in their ability to carry large fragments of DNA (200 kbp–800 kbp). However, they are designed to replicate in yeasts in that they contain a yeast origin of replication, telomeres (for replication of linear chromosomes), and centromeres (for segregation during mitosis).

16. Reverse transcriptase is useful for cloning animal genes into bacteria because this enzyme makes a cDNA copy of the mRNA, which effectively removes introns normally found in animal genes.

17. Genetic engineering has been used to transform microorganisms into tiny factories for the production of valuable products such as fuels, chemicals, drugs, and human hormones, such as insulin. Genetic engineering can also be applied in many different ways to the production of vaccines.

18. The Ti plasmid is associated with the tumorogenicity of Agrobacterium tumefaciens.

Because all of the plasmid DNA is not required for plant cell transformation, foreign DNA may be added in place of nonessential DNA. Introduction of genes cloned into the Ti plasmid are used to transfer genes conferring improved phenotypes to plants.

19. A subunit vaccine is one in which only a portion of the proteins from a pathogen are used to elicit an immunogenic response. The safety of these vaccines is due to their marked lack of pathogenicity since the vaccines contain only some of the proteins from the pathogen. .

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20. In gene mining potentially useful novel genes are isolated from the environment without first culturing the organisms that carry them. DNA (or RNA) is isolated directly from environmental samples and cloned into suitable vectors to construct a metagenomic library.

21. Pathway engineering is the use of cloning to assemble a new or improved metabolic pathway using genes from multiple organisms to optimally produce a desired end product. Antibiotic synthesis is generally dependent upon the action of several enzymes in a complex pathway, thereby complicating the optimization of production. The engineering of a single enzyme is much more straightforward, and the effects are readily apparent.

22. Genetic engineering methodologies focus on the modification of a single, existing biochemical pathway. Synthetic biologists seek to engineer novel biological systems by linking together known biological components in various combinations.

Answers to Application Questions 1. Genetic improvement of plants and animals by traditional selection and breeding has a long history, but recombinant DNA technology has led to revolutionary changes. Genetically engineered plants or animals contain a gene from another organism—called a transgene—they are transgenic organisms. The public knows these as genetically modified organisms (GMOs). They are of high economic importance especially in agriculture and aquaculture. Major areas targeted for genetic improvement in plants include herbicide, insect, and microbial disease resistance, as well as improved product quality. The main genetically modified (GM) crops today are soybeans, corn, cotton, and canola. Almost all the GM soybeans and canola planted were herbicide resistant, whereas the corn and cotton were herbicide resistant or insect resistant, or both. Another example is the insect resistant Bt-cotton developed from the Ti plasmid of the bacterium Agrobacterium tumefaciens, which can transfer DNA encoding the Bt-toxin into plant cells. The fast growing transgenic salmon is a practical example of commercial success from the aquaculture products.

Herbicide resistance is genetically engineered into a crop plant to protect it from herbicides applied to kill weeds. Many herbicides inhibit a key plant enzyme or protein necessary for growth. For example, the herbicide glyphosate (RoundupTM) kills plants by inhibiting an enzyme necessary for making aromatic amino acids. Some bacteria contain an equivalent enzyme and are also killed by glyphosate. However, mutant bacteria were selected that were resistant to glyphosate and contained a resistant form of the enzyme.

The gene encoding this resistant enzyme from A. tumefaciens was cloned, modified for expression in plants, and transferred into important crop plants such as soybeans. When sprayed with glyphosate, plants containing the bacterial gene are not killed. Thus, glyphosate can be used to kill weeds that compete for water and nutrients with the growing crop plants. Herbicide-resistant soybeans are now widely planted in the United States.

2. The sample for isolation of microbes for cold-active lipases has to be collected from an appropriate location, e.g., tanneries and dairy or milk product processing units from places where cold climate prevails. Metagenomic library construction for the target gene .

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must then be done. This should be followed by the screening of lipase activity with 1% tributyrin/tricaprylin/triolein and the fosmid clone is to be selected. The insert could be sequenced by shotgun sequencing. This would help in performing the phylogenetic analysis of the gene. The gene could then be expressed in Escherichia coli and the enzyme could be purified by metalchelating chromatography and characterized by standard protocols like SDS-PAGE, Zymogram, amino acid sequencing, etc.

3. The use of luciferase as a reporter gene makes cells expressing it luminescent against a large background of other colonies. However, luciferase expression requires several other factors and more than one gene. GFP, on the other hand, requires no accessory factors and is expressed in most cells. It is stable and does little harm to the cell membrane. Its use can be problematic in cells that autofluoresce in that wavelength range, which is a factor for some photosynthetic organisms. There are now alternative versions, including blue (BFP) and yellow (YFP) fluorescent genes, that can help overcome this problem.

4. The gene encoding the protein that may be effective against a cancer would be subcloned into an expression vector. Expression in a bacterial host may allow for rapid, large-scale production of the protein as long as (1) the protein is not modified or improperly folded in this host and (2) the recombinant protein is not degraded by endogenous proteases.

Genetically modified strains of E. coli exist that reduce the occurrence of either of these situations. Cloning the gene into an E. coli strain would allow you to obtain (and maintain) the gene. However, it may be best to express the protein in a eukaryotic host, especially if it requires some posttranslational modifications.

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CHAPTER

12

Microbial Evolution and Systematics

Summary The evolution of living organisms is the unifying theme that underlies all of biology. This chapter explores microbial evolution from the origin of Earth and the first hypothesized life forms that eventually diversified to shape the biosphere. The authors discuss the analytical tools used in microbial phylogenetic studies, including rRNA gene sequencing that led to the reclassification of life into three domains: Bacteria, Archaea, and Eukarya. The final sections of the chapter present the various approaches and analytical tools employed in the field of microbial systematics.

12.1 | Formation and Early History of Earth Scientists generally agree that Earth’s early atmosphere lacked significant quantities of O2 but contained a variety of other gases, including H2O (vapor), CH4, CO2, N2, H2S, and some H2. The environment at that time was highly reducing and had an elevated surface temperature. Energy was available from solar irradiation, volcanic activity, electrical discharges, and radionuclide decay. It is hypothesized that the reorganization of these key chemicals under such conditions lead to the abiotic synthesis of organic compounds necessary for life such as amino acids, fatty acids, and other important biological “building blocks.” A timeline may be used as a reference for the discussion of biological evolution (Figure 12.1). Note the appearance of molecular oxygen from oxygenic photosynthesis by cyanobacteria. The steady increase in O2 concentration must have been a critical parameter for the diversification of life.

This type of narrative can lead to a lively discussion about what traits the earliest microorganisms would possess. Most students should already be familiar with background information concerning this topic from general biology courses and from Chapter 1 of this text.

The idea that the early prebiological synthesis of organic molecules occurred in an aqueous environment is problematic because the elevated temperatures of an early Earth would prevent accumulation of liquid water. Sedimentary rock, which forms under liquid, appears to have been present about 3.9 billion years ago, as evidenced from samples discovered in southwestern Greenland. It is now largely accepted that Earth had cooled enough to be compatible with life within only a few hundred million years after it formed. Fossilized evidence for microbial life in the form of rod- and coccus-shaped cells was found in 3.45-billion-yearold rock from South Africa (Figure 12.2).

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Although details regarding the origin of life on Earth remain a great mystery, one popular hypothesis proposes that life originated in the subsurface at hydrothermal vents on the ocean floor. Here, conditions were less hostile and presumably more stable, and high-energy electron donors (e.g., H2 and H2S) and other inorganic nutrients were in plentiful supply from geothermal (abiotic) activity (Figure 12.3). Organic compounds produced during this time by abiotic mechanisms would accumulate in the environment since chemoorganotrophic metabolisms that would promote their decomposition would not have been present.

One requirement for life is the ability to self-replicate. The discovery of catalytic RNA molecules, or ribozymes (see Section 7.15), that can synthesize nucleotides and catalyze several other reactions makes the idea of RNA-based life an attractive hypothesis. The steady flow of reduced inorganic compounds would have provided the energy to drive prebiotic chemistry. The appearance of lipid membranes containing protein molecules to allow for semipermeability would also have to occur. Such lipoprotein vesicles could trap RNA and DNA, leading to the first replicating cells (Figure 12.4). This early population of cells is now referred to as the last universal common ancestor (LUCA) that diverged into Bacteria and Archaea (Figure 12.4).

Next, discuss the topic of early metabolism by considering Earth’s environmental conditions at the time life arose. Accepting the presence of reducing conditions during early biochemical evolution, one must consider what type of simple redox system could have served as a source of energy for a primitive microorganism. The abundance of H2 and CO2 and lack of O2 on early Earth suggest that the first cells may have been anaerobic autotrophs. Ferrous iron (Fe2+) reacting with hydrogen sulfide (H2S; both thought to be present on early Earth) yields a favorable free energy (∆G0 = –42 kJ) and forms H2 as an end product, which could then be used to reduce elemental sulfur (S0), as seen in Figure 12.5. There are at least three other reasons that favor this scenario: (1) The metabolism is very simple, requiring chemical substrates that were likely present in plentiful supply; (2) energy coupling for the reaction H2 + S0 → H2S requires only a cytoplasmic membrane and a few enzymes; and (3) some extant hyperthermophiles carry out these reactions.

12.2 | Photosynthesis and the Oxidation of Earth In rocks under ~3.5 billion years of age, one can see large accumulations of fossilized stromatolites⎯layers of filamentous prokaryotes sandwiched between sediment (Figure 12.6).

Compare Figure 12.6b with Figure 12.6d to note the remarkable similarity of the microorganisms in modern stromatolites to those in ancient fossilized stromatolites. Figure 12.7 shows a one billion-year-old rock formation that contains fossilized filamentous bacteria and spherical eukaryotic cells that are similar in appearance to modern cyanobacteria and green algae, both oxygenic phototrophs.

Competition for resources between early cells would have resulted in a long period of microbial diversification. The LUCA would most likely have existed nearly 4 billion years ago (Figure 12.4), and molecular evidence suggests that Bacteria and Archaea would have diverged soon after. This divergence was accompanied by the evolution of distinct metabolisms. It is estimated that anoxygenic photosynthesis arose ~3.4 billion years ago in Bacteria only. By ~2.8 billion years ago, the cyanobacteria developed the ability to carry out oxygenic photosynthesis, using H2O as an electron donor and releasing O2 as a waste product. The .

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accumulation of O2 in the atmosphere to significant levels took approximately 300 million years, as the O2 produced by cyanobacteria spontaneously oxidized Fe2+ to insoluble Fe3+, the outcome of which generated banded iron formations (Figure 12.8). The consequences of oxygenic photosynthesis for Earth were enormous, as the atmosphere changed from anoxic to oxic, leading to the development of a protective ozone (O3) shield around the planet, the evolution of new metabolic pathways, and the eventual evolution of multicellular organisms.

12.3 | Endosymbiotic Origin of Eukaryotes Scientists had two opposing ideas about the origin of the eukaryotic cell. It was debated whether compartmentalization with concomitant localization of function in discrete organelles was autogenous or if compartmentalization was due to serial endosymbiosis. Recent information strongly supports endosymbiosis of ancestral bacteria as the origin of mitochondria and chloroplasts. These organelles possess several prominent features of the bacteria from which they are putatively derived, including prokaryotic ribosomes and circular DNA containing prokaryotic-like genes. Figure 12.9 illustrates two popular models for endosymbiosis and the origin of the eukaryotic cell. The first of these describes the endosymbiosis of the mitochondrial ancestor after the nucleation of the cell (Figure 12.9a), while the second model, the hydrogen hypothesis, shows a H2-producing mitochondrial ancestor being taken up endosymbiotically by a H2-consuming archaeal cell before formation of the nucleus (Figure 12.9b). In either case, endosymbiosis of the cyanobacterial ancestor is believed to have occurred after mitochondrial incorporation. This is an excellent topic to promote class discussion, as it usually generates good student participation. Figure 12.10 can be used to show molecular features shared by cells of all three domains, as well as those that are unique to each.

12.4 | Molecular Phylogeny and the Tree of Life Early attempts to show evolutionary relationships of living organisms were based on phenotypic and metabolic properties and were unable to resolve the various lineages of microorganisms (Figure 12.11). The development and use of phylogenetic analyses for organismal classification, especially comparisons of small subunit ribosomal RNA gene sequences (Figure 12.12), provided a new and more accurate perspective of evolutionary relationships.

Point out to your students the skewed view of the prominence and diversity of prokaryotic organisms (“Monera”) implied by the Whittaker Tree in Figure 12.11b versus that apparent in the modern three-domain concept of living organisms first realized by Carl Woese in the 1970s (Figure 12.13).

Molecular and phylogenetic evidence indicates that horizontal gene transfer played a large role in the early evolution of cells but perhaps became less extensive as distinct lineages appeared. Two prominent bifurcations in the evolutionary descent of early microorganisms are proposed: the separation of the Bacteria and Archaea followed by the further separation of the Archaea and Eukarya (Figure 12.13). Consisting of at least 84 phyla containing organisms found in nearly every environment on Earth, by far the greatest physiological and phylogenetic diversity of living organisms occurs within the domain Bacteria (Table 12.1). By contrast, only five phyla of Archaea have been described based on cultivated strains. Howev.

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er, species of Archaea occupy some of the harshest environments on Earth, including those that are extremely hot (above 100°C) or saline, where Bacteria are not found. Many bacterial and some archaeal phyla are known only from the description of phylotypes, SSU rRNA gene sequences obtained from environmental samples and subjected to phylogenetic analysis (see Section 18.5). Eukaryotic diversity likely developed much more recently and is believed to coincide with the accumulation of molecular oxygen in Earth’s atmosphere approximately 600 million years ago. The diversity of the Bacteria is discussed in detail in Chapters 14 and 15, the Archaea in Chapter 16, and the microbial Eukarya in Chapter 17.

12.5 | Molecular Phylogeny: Making Sense of Molecular Sequences The major workhorse of phylogenetic analysis using DNA sequences is the polymerase chain reaction (PCR; see Section 6.11), which provides an ample number of DNA copies for sequencing. The PCR products are separated by agarose gel electrophoresis, and the bands (~1,465 bp long) are cut from the gel, purified, and subjected to DNA sequencing. Use Figure 12.14 to provide students with an overview of 16S rRNA gene amplification, sequencing, and analysis. Note that domain-specific primers are available for amplifying the 16S rRNA gene of any organism within a specific domain. In addition, universal primers can be used to amplify small-subunit (SSU) rRNA genes from any organism, regardless of domain. Following gene amplification and sequencing, phylogenetic analysis continues with a process called sequence alignment. This method is used to compare the relatedness of one organism to one or more other organisms based on the degree of sequence identity in the genes being aligned (Figure 12.15). If you have adequate time, you may wish to show your students the function of the web-based BLAST (Basic Local Alignment Search Tool) searching algorithm incorporated into both the National Institutes of Health (http://www.ncbi.nlm.nih.gov/BLAST) and Ribosomal Database Project (http://rdp.cme.msu.edu) websites. This exercise is especially relevant for microbiology courses designed for science majors.

Discuss the principles of using sequence alignments to create either unrooted or rooted forms of phylogenetic trees, or cladograms (Figure 12.16a). Discuss the meaning of the nodes and branches, and note that the branches define (1) the order of descent and (2) the ancestry of the nodes (Figure 12.16b). Explain to students that computers use either algorithms or sets of optimality criteria for tree construction in which nucleotide sequence differences in multiple organisms at certain positions are used to build a distance matrix, which can then be used to produce a phylogenetic tree (Figure 12.17). Tree-building algorithms include the Neighbor Joining methods and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Optimality criteria methods include parsimony, maximum likelihood, and Bayesian analysis, with each differing slightly in terms of the assumed model of evolution (e.g., by accounting for the fact that certain nucleotide mutations occur more often than others).

Because phylogenetic trees can be built only from extant sequences, evolutionary distances may be underestimated in cases where homoplasy, or convergent evolution, has occurred. In these instances, recurrent mutations can return mutated DNA to its original, unaltered sequence, thereby masking the true divergence of organisms over time (Figure 12.18).

A second limitation of phylogenetic trees is the assumption that genes used for phylogenetic analyses were inherited vertically from one generation to another. However, recall from .

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Chapter 10 that horizontal gene transfer may be extensive in an organism’s evolutionary history, and this may be especially the case for microorganisms (Figure 12.19). Therefore, it is important to point out to your students the difference between a gene phylogeny and an organismal phylogeny. Despite this distinction, however, make sure students are aware that because of their low rates of horizontal transfer and highly conserved nature, SSU rRNA gene phylogenies have been shown to align closely with whole genome phylogenetic trees and are therefore considered to accurately represent organismal phylogeny.

12.6 | The Evolutionary Process Some of the processes driving evolution were presented in Chapters 6 and 10 (e.g., mutations, genetic exchange, gene duplications, and horizontal gene transfer). Mutations and selection are revisited here in the context of microbial evolution. In addition, genetic drift is discussed as a means of altering gene frequencies in populations of organisms over time independently of natural selection (Figure 12.20). Summarize these topics as they relate to the evolution of microbial diversity and metabolism. Remind students that because of their tremendous biotic potential, microorganisms have the ability to quickly adapt to changing conditions, and in some cases, spontaneous mutants having a selective advantage can quickly outcompete other cells (Figures 12.21 and 12.22).

Although differing traits within a species can develop quickly, speciation itself appears to be a slow process. Changes in gene sequences can be used as molecular clocks to estimate the amount of time passed since major lineage divergences in the evolutionary history of organisms. These divergences can range from relatively recent speciation events to the bifurcation of the Archaea from the Eukarya, an event estimated to have occurred about 2.8 billion years ago. Such estimates of evolutionary divergence are made more robust when the molecular clock approach is combined with evidence from the geological record (e.g., data obtained from stable isotope analyses).

12.7 | The Evolution of Microbial Genomes The idea of a core genome versus a pan genome was discussed in Section 6.13, and the concept is further elaborated upon here. As more and more genomes are sequenced and analyzed, it has become clear that separate prokaryotic strains within a species vary widely in their genetic content. This property is exemplified by the various sequenced strains of both pathogenic and nonpathogenic Escherichia coli, which have genomes ranging in size from 4068 to 5379 total genes and a core genome consisting of only 1976 genes. Use Figure 12.23 to illustrate the dynamic nature of microbial genomes, which may quickly change size over time, largely as a result of horizontal gene transfer and gene deletions. Deletions occur more frequently than insertions in microbial genomes, and they presumably account for the extremely small genomes of certain obligate endosymbiotic and pathogenic species. See the feature box Explore the Microbial World: The Black Queen Hypothesis for more detail on this topic.

12.8 | The Species Concept in Microbiology .

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Explain to your students that although the definition of a species in a multicellular organism (i.e., the biological species concept) may be somewhat obvious based upon observable characteristics, such as interbreeding behaviors between populations, this is not the case with prokaryotes. Even considering genetic recombination, one cannot define prokaryotic species based upon sexual reproduction and reproductive isolation. Therefore, prokaryotes are subject to a phylogenetic species concept, an idea that requires the utilization of genetic taxonomic tools, such as genomic DNA:DNA hybridization (Figure 12.24). The currently accepted definition holds that prokaryotic strains having ≥70% genomic DNA:DNA hybridization and ≥97% 16S rRNA gene sequence identity can reliably be considered to be of the same species (Figure 12.25). Such parameters should dictate that strains composing a single species are monophyletic and therefore exclusively share a recent common ancestor.

Discuss the limitations or questions concerning the current definition of bacterial species with your students, and then lead your discussion into the question of how many extant microbial species exist. Although only about 10,000 prokaryotic species have been validly named, the expected diversity of all Bacteria and Archaea on Earth is considered to be several orders of magnitude higher than this number and many times higher than all plant and animal species combined.

12.9 | Taxonomic Methods in Systematics Inform your students that systematics combines phylogeny and taxonomy to develop a cohesive picture of the diversity and relationships between organisms. The concept of a prokaryotic species uses a polyphasic approach that combines three characterization methods—phylogenetic, genotypic, and phenotypic—for the identification and description of prokaryotes.

Although small subunit rRNA gene sequence analyses provide a useful phylogenetic comparison (Section 12.5), closely related species, or strains within a species, may not be reliably distinguished by such methods alone. Figure 12.26 shows the use of multigene phylogenetic analysis to resolve individual strains within species of Photobacterium that cannot be differentiated solely by 16S rRNA gene sequence analysis. To obtain unequivocal discrimination between bacterial strains within species, multilocus sequence typing (MLST) is often employed. MLST looks at several different chromosomal “housekeeping” genes to determine an allelic profile, which is used to generate a phenogram (Figure 12.27). While too sensitive to resolve higher order taxa, this technique has proven useful in clinical microbiology in distinguishing and tracking the distribution of pathogenic versus nonpathogenic strains of bacterial species.

A sequencing-independent method, called ribotyping, can also be used for bacterial identification. Ribotyping is a genome fingerprinting technique in which DNA is first digested with various restriction enzymes. Gel electrophoresis is then used to produce a DNA banding pattern that is transferred to a nylon membrane and treated with a labeled rRNA gene probe (Figure 12.28). The specific band pattern that results is compared to a reference database for identification of organisms. This technique is often used for bacterial identification in clinical diagnostics and for microbial analysis in the food and beverage industries. Another common genome fingerprinting method is DNA profiling, which is performed using techniques such as repetitive extragenic palindromic PCR (rep-PCR) and amplified fragment length polymor.

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phism (AFLP), both of which provide a type of strain-specific DNA fingerprint (Figure 12.29). New techniques gaining popularity in microbial systematics are multigene and whole genome analyses. These methods reduce or eliminate limitations associated with single gene phylogenetic analyses, such as erroneous taxonomic conclusions that can result from instances of horizontal gene transfer.

In combination with phylogenetic and genotypic methods, phenotypic analyses provide an important third component to microbial systematics. Table 12.2 lists some phenotypic traits used in the identification and description of species. Provide examples for the students as presented in this text section. The FAME (fatty acid methyl ester) technique is used to determine the types and proportions of plasma membrane fatty acids, and it is widely used in clinical, public health, and food and water inspection laboratories (Figure 12.30). Discuss the advantages and drawbacks of this technique.

12.10 | Classification and Nomenclature Conclude this chapter presentation with the topics of classification and nomenclature. Table 12.3 shows the taxonomic ranks of bacterial classification from domain to species using the purple sulfur bacterium Allochromatium warmingii as an example. Review with your students the binomial system of classification (genus name followed by specific epithet) devised by Carl Linnaeus, and explain to them that new names must follow the rules defined by the International Code of Nomenclature of Bacteria. In addition, a detailed characterization of the organism with its proposed name must appear as a peer-reviewed publication. New validly published taxa are announced in the International Journal for Systematic and Evolutionary Microbiology, the principal scientific journal for the taxonomy and classification of prokaryotes.

Identify the two primary resources available to microbiologists for the characterization of newly isolated prokaryotes: Bergey’s Manual of Systematic Bacteriology and The Prokaryotes. Point out to students that just as museums store and maintain plant and animal specimens for future study, national culture collections preserve type strains (strains that formally represent a particular species) of microorganisms for research purposes (Table 12.4). However, a major difference is that the type strains are preserved as viable cultures that can be obtained as actively growing cells or in a lyophilized (freeze-dried) state. For a new species to be validly published, the type strain of the new species must be deposited into at least two national culture collections so that it is publicly available to the scientific community.

Answers to Review Questions 1. From measurements made using a variety of techniques, Earth is estimated to be about 4.6 billion years old. Subsequent cooling and water condensation into oceans is widely believed to have occurred ~4.3 billion years ago. The oldest known microfossils came from rocks of the Barberton Greenstone Belt, South Africa, and were dated at ~3.45 billion years old.

2. Stromatolites are laminated microbial mats, typically composed of layers of filamentous Bacteria and other microorganisms, which may become fossilized. Some fossilized stromatolites are found in rocks that are 3.5 billion years old, providing some of our earliest evidence of life on Earth. .

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3. The metabolism of cyanobacteria yielded O2 that oxidized reduced minerals containing Fe2+ to iron oxides containing Fe3+. Only after the abundant Fe2+ on Earth was consumed could O2 accumulate in the atmosphere, and not until 600–900 million years ago did atmospheric O2 reach present-day levels (~21%). As O2 accumulated on Earth, the atmosphere gradually changed from anoxic to oxic, which created conditions for the evolution of various new metabolic schemes, such as sulphide oxidation, nitrification, and the various other aerobic chemolithotrophic processes.

4. One hypothesis is that eukaryotes originally arose as a nucleus-bearing cell line that later acquired mitochondria and chloroplasts by endosymbiosis. In this hypothesis, the nucleus-bearing cell line arose in a lineage of cells that split from the Archaea; the nucleus is thought to have arisen in this cell line during evolutionary experimentation with increasing cell and genome size, probably in response to oxic events that were transforming the geochemistry of Earth. The hydrogen hypothesis proposes that the eukaryotic cell arose from an association between a H2-producing species of Bacteria, the symbiont, which eventually gave rise to the mitochondrion, and a species of H2-consuming Archaea, the host. In this hypothesis, the nucleus arose after genes for lipid synthesis were transferred from the symbiont to the host. This transfer led to the synthesis of lipids containing fatty acids by the host, lipids that may have been more conducive to the formation of internal membranes, such as the nuclear membrane system. The simultaneous increase in size of the host genome led to sequestering DNA within a membrane, which better organized it and made replication and gene expression more efficient.

5. In addition to evidence provided by structural and morphological considerations, phylogenetic analyses of small subunit rRNA genes provide strong evidence for the threedomain classification of living organisms. DNA sequences of other conserved genes and even whole genomes also support this view.

6. Properties common to Archaea and Eukarya include the following: a resistance to many antibiotics, the lack of a peptidoglycan cell wall, an initiator tRNA that carries methionine, the presence of histone proteins, a TATA box-containing promoter, sensitivity to diphtheria toxin, the requirement of transcription factors, and introns in the tRNA. Some properties common to Archaea and Bacteria include common cell size and general morphological forms, prokaryotic cell plan, 70S ribosomes, anaerobic metabolic potential, circular DNA, simultaneous transcription and translation within the same cell compartment, unicellularity, binary cell division, and chemolithotrophic growth.

7. Small subunit ribosomal RNAs are good choices for phylogenetic studies because they are (1) universally distributed; (2) functionally constant; (3) well-conserved over broad phylogenetic distances; and (4) of adequate length to provide an evolutionary view that encompasses all organisms. A weakness is that the essential function of SSU ribosomal RNAs has limited the number of changes that can occur over time and makes it difficult to discriminate between prokaryotic species.

8. Sequence alignments are necessary for proper phylogenetic tree construction because some mutations result in the insertion or deletion of one or more nucleotide bases from a DNA sequence. This causes gene sequences to differ in length, making the addition of gaps necessary to ensure a proper homological comparison of each nucleotide position

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9. Algorithms used to build phylogenetic trees include the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and Neighbor Joining methods. Alternatively, phylogenetic methods that employ optimality criteria include parsimony, maximum likelihood, and Bayesian analyses.

10. A gene tree depicts phylogenetic associations from the evolutionary history of a single gene, whereas an organismal tree depicts the evolutionary history of a cell by taking horizontal gene transfers into account (see Figure 12.19). Because SSU rRNA genes are transferred horizontally at very low frequency, trees built from such sequences are considered to provide an accurate view of organismal phylogeny.

11. Genomic DNA is isolated from pure cultures of the three bacterial isolates, and PCR is used to amplify SSU rRNA genes from each isolate in separate reactions. The amplified DNA is subjected to gel electrophoresis, and bands are removed from the gel and purified for gene sequencing. The sequenced SSU rRNA genes of the three isolates can then be compared to each other and to rRNA gene sequences from other bacteria using computer programs that search vast databases of stored sequence information. DNA sequences of the three isolated strains are aligned with SSU rRNA sequences from related bacteria, and the resulting sequence alignment is used to generate a phylogenetic tree, which provides useful information for systematics applications.

12. Mutations are random changes in DNA sequence that accumulate in all DNA sequences over time; they are a fundamental source of the natural variation that drives the evolutionary process. Mutations take several forms, including substitutions, deletions, insertions, and duplications. Genetic drift is a random process that can cause gene frequencies to change over time, resulting in evolution in the absence of natural selection. Genetic drift occurs because some members of a population will have more offspring than others simply as a result of chance; over time these chance events can result in evolutionary change in the absence of selection. Genetic drift is most powerful in small populations and in populations that experience frequent “bottleneck” events. The latter occur when a population experiences a severe reduction in population size followed by regrowth from the cells that remain.

13. Fitness is the ability of an organism to produce progeny and contribute to the genetic composition of future generations. The fitness of an organism can change based on whether random mutations are deleterious or beneficial, and this is determined by the selective pressures present in the organism’s environment.

14. Gene deletions play an important role in microbial genome dynamics. Deletions occur with far greater frequency than insertions in microbial genomes, and this bias toward deletions is the force that maintains the small size of microbial genomes.

15. Most horizontal gene transfer events take place between close relatives and occur through homologous recombination of DNA segments of 50 to 500 base pairs. However, insertions due to nonhomologous recombination are also common in microbial genomes.

Insertions typically contain about 4 genes, but segments of up to 10 genes or more occasionally occur. Deletions play an even larger role in shaping the core and pan genomes of a species. Genetic material gained through horizontal transfer is frequently lost from the genome over time if it provides no advantage to the fitness of the organism. Thus, inser-

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tions tend to add genes to the pan genome of a species, whereas deletions tend to define the core genome since any essential genes that are lost would constitute a lethal mutation.

16. The “species problem” in microbiology is that there is currently no universally accepted concept of what constitutes a microbial species. The definition of a microbial species is difficult to articulate primarily because the morphological and reproductive characteristics that provide the foundation for many species determinations of higher taxa are not feasible for the classification of microorganisms, especially those that are prokaryotic.

In addition, the high degree of horizontal gene transfer among prokaryotes obscures phylogenetic analyses that provide important insights but cannot in themselves allow for firm taxonomic conclusions.

17. An accurate estimation of the number of bacterial species is not possible based upon our current understanding and technology, and this is exacerbated by uncertainties as to what exactly defines a bacterial species. It is probable that the total number of bacterial species exceeds by several orders of magnitude the approximately 10,000 species that have been formally characterized to date. Thus, it is likely that at least 1,000,000 different species of bacteria exist.

18. Major phenotypic characteristics used in classical bacterial taxonomy include the Gram stain, fatty acid content, motility, endospore formation, cell shape and arrangement, the types of metabolisms employed to sustain cell growth, nutritional requirements, and cell wall analysis. The Gram stain and the cell wall have been particularly useful in determining phylogenetic relatedness, as well as identification. Genotypic characteristics used for taxonomic purposes include GC ratios, genomic DNA:DNA hybridization, the sequence of SSU rRNA genes and other genes of phylogenetic usefulness, DNA profiling, multilocus sequence typing, and multigene or whole genome phylogenetic analyses.

19. FAME (fatty acid methyl ester) analyses are used for bacterial identification by characterizing the types and proportions of fatty acids present in the cell membranes of isolated strains. FAME analyses require rigid standardization since fatty acid profiles in a single organism can vary as a function of temperature and growth medium.

20. Gene sequence analyses, multilocus sequence typing, genome fingerprinting (ribotyping), multigene and whole genome analyses, and phenotypic analysis (FAME analysis).

21. When a new microorganism is isolated from nature and thought to be unique, a decision must be made as to whether it is sufficiently different from other prokaryotes to be described as a new taxon. To achieve formal validation of taxonomic standing as a new genus or species, a detailed description of the organism’s characteristics and distinguishing traits, along with its proposed name, must be published, and viable cultures of the organism must be deposited in at least two international culture collections. The manuscript describing and naming a new taxon undergoes peer review before publication. A major vehicle for the description of new taxa is the International Journal of Systematic and Evolutionary Microbiology (IJSEM), the official publication of record for the taxonomy and classification of Bacteria, Archaea, and microbial eukaryotes. In each issue, the IJSEM also publishes an approved list of newly validated names. By providing validation of newly proposed names, publication in IJSEM paves the way for their inclusion in taxonomic reference sources.

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Answers to Application Questions 1. The early atmosphere was devoid of significant amounts of O2; the most abundant gases were CH4, CO2, N2, H2S, and H2. It is also likely that a considerable amount of HCN was present. Moreover, the temperature of Earth was hotter, H2O existed only as water vapor, and there was probably much more UV light from the sun. These conditions obviously do not exist today since the cooling of Earth formed liquid water, and oxygenic photosynthesis by cyanobacteria produced an abundance of O2 in Earth’s atmosphere. Two reasons why animals could not have existed on early Earth include the following: (1) There was no atmospheric O2, which serves as terminal electron acceptor in aerobic respiration; and (2) there was a lack of reduced organic compounds for chemoorganotrophic metabolism, which is used by all animals for ATP production and cellular biosynthesis.

The biosphere has been dramatically altered by microbial metabolism. The diversification of early microbial life altered the chemical environment by the production of waste products and cellular material, with the evolution of molecular oxygen as a waste product having the most dramatic effect on the biosphere. Without oxygenic photosynthesis, the accumulation of O2 in the atmosphere would not have occurred, and the enormous diversity of extant life dependent upon aerobic metabolisms would presumably not exist.

2. CLUSTAL 2.1 multiple sequence alignment Taxon_2

-----GTTCGGTAA- 9

Taxon_6

----AAATCGTCAAA 11

Taxon_5

GTAAAACACGAT--- 12

Taxon_1

---GTTCCCTTT--- 9

Taxon_4

---CTTCCCTTT--- 9

Taxon_3

GAAAAACCCTTT--- 12

The phylogenetic tree could be built by multiple sequence alignment. In the sequence alignment we can count the number of differences between each pair of sequences to build a distance matrix. This distance matrix can be used to build a tree where the cumulative lengths of the horizontal branches between any two species in the tree are proportional to the number of nucleotide differences between these species.

3. To determine whether the four different strains are actually members of the same species, one could use multilocus sequence typing, a genome fingerprinting technique, or DNA:DNA hybridization.

4. To determine if a new form of microbial life represented a fourth domain, one would have to look at a number of characteristics, including SSU rRNA sequences, membrane lipids, and so on, as listed in Table 12.1. Answers here may vary.

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CHAPTER

13

Metabolic Diversity of Microorganisms

Summary This chapter focuses on the tremendous diversity in metabolic strategies employed by microorganisms. The material has been extensively reorganized and now includes information from Chapters 13 (Phototrophy, Chemolithotrophy, and Major Biosyntheses) and 14 (Catabolism of Organic Compounds) from the previous edition. The major metabolisms addressed in this chapter include phototrophy, chemolithotrophy, fermentations, syntrophy, a variety of anaerobic respirations (e.g., those dependent upon the reduction of nitrogen and sulfur compounds, acetogenesis, and methanogenesis), and hydrocarbon metabolism. Emphasize the essential roles that microbial metabolisms play in maintaining and generating the nutrient cycles critical to life on Earth. Although students may consider this an overstatement of the facts, nothing could be further from the truth. Introductory biology courses typically discuss the role of photosynthetic primary production as the ultimate source of organic carbon for all living organisms. The roles played by consumers (e.g., animals) and decomposers (e.g., fungi) in recycling organic carbon back to CO2 are also mentioned, but the immense role of microorganisms in driving nutrient cycling is not discussed in detail. Students in an introductory microbiology course should, at the very least, be exposed to general metabolic schemes in the context of microbial ecology and diversity.

As you discuss specific groups of microorganisms presented in Chapter 13, you may want to refer students to or include information from complementary sections in Chapters 14 and 15, which expound upon the immense diversity of the Bacteria. The material discussed in these later chapters will provide students with useful examples to enhance their understanding and appreciation of the fascinating topics discussed in Chapter 13.

13.1–13.4 | Photosynthesis and Chlorophylls; Carotenoids and Phycobilins; Anoxygenic Photosynthesis; Oxygenic Photosynthesis The material presented in these sections provides the biochemical background required to understand the activities of photosynthetic organisms in nature. Although photosynthesis is usually considered in some detail in general biology courses, students at the freshman level often do not have adequate background to fully appreciate or understand the process. Discuss the two general concepts that constitute the hallmarks of photosynthesis: (1) the use of lightabsorbing pigments to initiate a series of electron transport reactions that are coupled to .

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ATP synthesis and the generation of reducing power (NAD[P]H) and (2) the use of ATP energy and reducing power for the reduction of CO2 to cell material ([CH2O]n). The former concept is referred to as the light reactions of photosynthesis, whereas the latter compose the dark reactions.

To comprehend photosynthesis at a biochemical level, students must first understand oxidation and reduction, redox potential, and the general thermodynamic principles of metabolism (see Chapter 3 and Appendix 1). Inform the students that nearly all phototrophs are also autotrophs (they use CO2 as their sole carbon source); however, some photosynthetic microorganisms, called photoheterotrophs, use organic carbon as a carbon source during phototrophic growth. Figure 13.1 describes photosynthesis in terms of energy source and reducing power. Students should understand that the only significant difference between oxygenic and anoxygenic photosynthesis is the ultimate source of electrons for the reduction of CO2.

Molecular oxygen (O2) is evolved in oxygenic photosynthesis because water (H2O) is the electron donor—oxygen evolution is not required for photosynthesis, as some students assume. However, the evolution of a photosystem that could be reduced using H2O as electron donor was a fortuitous event for aerobic-respiring organisms! In anoxygenic photosynthesis, the electron donor typically comes from an environmental source, such as reduced sulfur compounds (e.g., H2S) or hydrogen (H2).

Contrast the relatively minor differences in the chemical structures of chlorophyll (Chl) a and bacteriochlorophyll (Bchl) a, and note how these small differences result in very different absorption spectra (Figure 13.2). Point out that many plants contain more than one type of chlorophyll molecule, with chlorophyll a and b being the most common. Anoxygenic phototrophs possess one or more of a wide range of bacteriochlorophylls, each with different absorption maxima (Figure 13.3). Challenge students to hypothesize why different organisms possess different light-absorbing pigments if a single type functions satisfactorily in light absorption and electron transport. Discuss the arrangement of light-harvesting antenna pigments, chlorophylls/bacteriochlorophylls that funnel light energy to the reaction center (RC) in a photosynthetic membrane (Figure 13.4). Note that chlorophyll pigments are integrated into membranes: thylakoids in chloroplasts of eukaryotes (Figure 13.5), and an internal membrane system in prokaryotes (Figures 13.6 and 13.7). The chlorosomes of green sulfur and green nonsulfur bacteria (the latter are also called filamentous anoxygenic phototrophs [FAPs]) are interesting and efficient light-harvesting structures that contain Bchl c, d, or e and allow for growth of these bacteria at very low light intensities (Figure 13.7). The light energy absorbed by chlorosomes is funneled to the RC through the Bchl a-containing FennaMatthews-Olson (FMO) pigment-protein complex.

Discuss the types and functions of accessory pigments (e.g., carotenoids, and phycobiliproteins) found in phototrophs (Figures 13.8–13.10). Point out the primary roles of these pigments. Carotenoids confer photoprotective properties to cells by absorbing high-energy wavelengths of light that could otherwise catalyze photooxidation reactions that produce toxic forms of oxygen. Phycobiliproteins aggregate into structures called phycobilisomes and function as the main light-harvesting pigments in cyanobacteria and red algal chloroplasts (Figure 13.10).

The details of anoxygenic photosynthesis (Section 13.3) are described in the text using purple phototrophic bacteria as the model, primarily because much of the biochemical and molecular genetic knowledge of the process has come from using these organisms as the research tool. Discuss the reaction center of purple bacteria (Figures 13.11 and 13.12). Figure .

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13.12a shows a general scheme of electron flow in a purple bacterium. Focus student attention on the reduction potential scale. Note that this scale dramatically demonstrates the change in reduction potential for the special pair Bchl a molecules following the absorption of a photon of light. The ground state reduction potential of the RC, called P870, is highly positive (E0 = +0.5V), making it a poor electron donor, whereas its “excited” reduction potential (E0 = –1.0V) converts it into a strong electron donor with respect to the other carriers shown in the electron transport chain.

• Point out to your students that during autotrophic growth, electrons donated by P870 are drawn from the transport cycle to create reducing power for CO2 fixation. This is achieved by the reduction of NAD(P)+ to NAD(P)H, which occurs via reverse electron flow from the quinone pool (an energy-requiring process). Electrons must then be replaced from some external electron donor, such as H2S, S2O32–, S0, or Fe2+. Both purple and green sulfur bacteria are capable of oxidizing H2S as electron donor, producing globules of S0 in the process. However, while sulfur globules are stored intracellularly in purple bacteria, they accumulate outside the cells of green sulfur bacteria. Some cyanobacteria, such as Oscillatoria limnetica, are also capable of anoxygenic photosynthesis using sulfide as electron donor, and similar to green sulfur bacteria, globules of elemental sulfur accumulate outside the cells (see Figure 13.15).

• Use Figure 13.12b to show students that the electron transport complex also allows the establishment of a proton motive force (PMF) for ATP synthesis. This is a good opportunity to relate the similarities of the processes of photosynthesis and respiration, as discussed in Chapter 3. Rather than being complete opposites, as is often suggested, the mechanisms of photosynthesis and respiration have much in common, though their overall purposes differ. Traditional textbook presentations often summarize the biochemistry of these processes by stating that “the reactants of one are the products of the other,” which is an oversimplification that can be misleading.

• Figure 13.13 compares the evolutionarily derived photosynthetic complex arrangements present in different anoxygenic phototrophs with respect to electron flow and generation of reducing power. Note that the different RC bacteriochlorophylls helped to create ecological niches for the evolutionary coexistence of a diversity of bacterial phototrophs.

• The progress made in understanding anoxygenic photosynthesis in purple bacteria has come about due to the wide variety of techniques available for genetic manipulation of these organisms.

Next, contrast electron flow in oxygenic photosynthesis (Section 13.4), which involves two photosystems as opposed to the one-photosystem arrangement of anoxygenic phototrophs.

The “Z” scheme shown in Figure 13.14 may be somewhat familiar to students from introductory biology courses. Compare this figure to Figures 13.12 and 13.13. Include the following key points in your discussion: • The key to using H2O as the electron donor for photosystem II (PS II) is due to the reduction potential of the ground state P680 Chl a molecule being more electropositive than the O2/H2O couple (Figure 13.14). P680 is therefore an extremely poor electron donor but an excellent electron acceptor.

• The movement of electrons from P680* of PS II to the RC (P700) of PS I generates a PMF for the synthesis of ATP. These reactions are referred to as noncyclic photophosphoryla.

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tion based upon the fact that the electron transferred to PS I does not return to reduce P680.

• PS I electron transport results in production of NAD(P)H for reduction of CO2 to (CH2O)n.

If sufficient NAD(P)H is present in the cell, then cyclic photophosphorylation can occur in which the electron is shuttled from ferredoxin back to the cytochrome bf complex (Figure 13.14).

13.5 | Autotrophic Pathways The most widely distributed pathway for CO2 fixation is the Calvin cycle (Figures 13.16 and 13.17). One of the key enzymes of the pathway, ribulose 1,5-bisphosphate carboxylase (RubisCO), catalyzes the incorporation of a molecule of CO2 into one molecule of ribulose 1,5-bisphosphate to yield two molecules of phosphoglycerate (PGA)—a glycolytic intermediate (see Section 3.8). Point out to students that the CO2 incorporated into PGA is at the same oxidation state as atmospheric CO2, and thus is not truly “fixed” (or reduced) to the level of cell material. The actual fixation occurs via the reduction of PGA to glyceraldehyde-3phosphate (Figure 13.26b). Several autotrophic prokaryotes that employ the Calvin cycle produce polyhedral inclusions called carboxysomes, which contain crystalline arrays of RubisCO molecules that allow for more rapid incorporation of CO2 into the Calvin cycle (Figure 13.18). Point out that autotrophy via the Calvin Cycle is an energetically expensive process, requiring a total of 12 NAD(P)H and 18 ATP to produce a single hexose sugar from 6 molecules of CO2.

Also described in Section 13.5 are two alternative methods employed by some bacteria for CO2 fixation: the reverse citric acid cycle (Figure 13.19a) and the hydroxypropionate pathway (Figure 13.19b). Two important points from this section include the following: • Use of the reverse citric acid cycle for CO2 fixation has been documented in phototrophic green sulfur bacteria; in the early-branching autotroph, Aquifex; and in certain nonphototrophic, hyperthermophilic Archaea. The cycle incorporates 3 molecules of CO2 into a molecule of glyceraldehyde 3-P, a glycolytic intermediate (Figure 13.19a). A unique enzyme of the pathway is citrate lyase, which uses ATP to cleave citrate into acetyl-CoA and oxaloacetate.

• The hydroxypropionate pathway incorporates two CO2 molecules into a molecule of glyoxylate through a hydroxypropionate intermediate (Figure 13.19b). Among phototrophs, the hydroxypropionate pathway has been confirmed only in the green nonsulfur bacterium Chloroflexus. However, the pathway is also present in several nonphototrophic genera of early-branching, hyperthermophilic Archaea, suggesting that the hydroxypropionate pathway may be the oldest mechanism of autotrophy.

13.6–13.10 | Chemolithotrophy Chemolithotrophic metabolism was introduced in Chapter 3 and was described in general terms as a method for the generation of energy via oxidation of inorganic electron donors.

In the next five sections, the strategies, problems, and advantages of a chemolithotrophic lifestyle are discussed. .

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Section 13.6 describes the energetics of chemolithotrophy, including an abbreviated list of potential inorganic electron donors and the energy yielded from their oxidation (Table 13.1). Remind students that by looking at reduction potentials, such as those shown in the table, one should be able to predict the different types of chemolithotrophic metabolisms present in nature (i.e., which reduction-oxidation couples can generate enough free energy to drive ATP synthesis). This will again reinforce to students that only thermodynamically favorable reactions supply the potential energy necessary for ATP synthesis; Table 13.1 provides a summary of the chemolithotrophic reactions carried out by microorganisms. Summarize the important electron donor oxidations involving hydrogen (H2), sulfide/sulfur (HS–/S0), ferrous iron (Fe2+), ammonium (NH4+), and nitrite (NO2–) using Figures 13.20–13.28.

Note that like purple phototrophs, reducing power (NADH) necessary for autotrophic growth is produced by reverse electron flow in many chemolithotrophs, thereby limiting ATP yield.

• Point out to students that hydrogen bacteria (Section 13.7) represent an interesting group because (1) they are facultative chemolithotrophs that metabolize organic compounds when available, and (2) some species can grow in a mineral salts medium containing H2, O2, CO2, and N2—all gases! Discuss the energetics of H2 oxidation and the role of cytoplasmic hydrogenase, which produces reducing power (NADH) for CO2 fixation, versus membrane-bound hydrogenase, which is necessary for energy conservation (Figure 13.20).

• Reduced sulfur compounds, such as hydrogen sulfide (H2S), elemental sulfur (S0), and thiosulfate (S2O32–), are excellent electron donors for supporting chemolithotrophic growth. In many organisms, the oxidation of H2S occurs in stages. The initial product of the oxidation is S0, which collects as insoluble globules that are either intracellular or extracellular, depending on the organism (Figure 13.21). If the supply of sulfide becomes depleted, the S0 globules can then serve as the energy source by being oxidized completely to SO42– (sulfate). Some bacteria, such as Paracoccus pantotrophus, utilize the sulfur oxidation (Sox) system for growth, whereby sulfide is oxidized directly to sulfate without producing intermediate sulfur compounds (Figure 13.22a). While sulfur-chemolithotrophs are primarily aerobic, some species can respire anaerobically with nitrate as the terminal electron acceptor. Discuss the energetics for the oxidation of each sulfur species (Figure 13.22).

• Iron-oxidizing, acidophilic bacteria (Section 13.9), such as Acidithiobacillus ferrooxidans, present an interesting energetic problem for students because the “fall” of electrons from the oxidation of Fe2+ to Fe3+ (E0 +0.77V at pH 2) is barely enough free energy (–32.9 kJ) to synthesize a single ATP! The key energetic solution in this case is the natural proton gradient provided from an acidic environment. Protons that enter the cytoplasm through ATP synthase are combined with O2 and electrons from the oxidation of Fe2+ to produce H2O (Figures 13.23–13.25). However, because excess protons would acidify the cytoplasm of the cell, only enough protons enter the cell as there are electrons to combine with them.

Therefore, ATP synthesis is ultimately controlled by the availability of Fe2+.

• Ammonia (NH3) and nitrite (NO2–) are employed by nitrifying bacteria (Section 13.10) as electron donors for chemolithotrophic growth under strictly aerobic conditions. Discuss the bioenergetics and interesting enzymology of nitrification. Key enzymes include ammo.

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nia monooxygenase and hydroxylamine oxidoreductase in ammonia-oxidizing bacteria and nitrite oxidoreductase in nitrite-oxidizing bacteria (Figures 13.26 and 13.27, respectively).

Note, however, that ammonia can also be oxidized under anoxic conditions, a process referred to as anammox (Figure 13.28). The anammox reaction is interesting in that ammonium (NH4+) serves as the electron donor with nitrite (NO2–) as the electron acceptor, yielding dinitrogen (N2) and water. Several genera of anammox bacteria are known, a few of which carry out this reaction in a membrane bilayer-enclosed structure called the anammoxosome (one of the few examples of what could technically be considered an organelle in prokaryotic cells). Anammox is of practical and ecological significance because the anaerobic removal of ammonia from wastewater helps to prevent eutrophication of rivers and streams.

13.11–13.15 | Fermentations Begin your discussion of anaerobic metabolisms by pointing out to students that in many anoxic habitats, typical inorganic electron acceptors used in anaerobic respirations (e.g., SO42–, NO3–, Fe3+; see Section 13.16) are not available, requiring that organisms ferment organic compounds to obtain energy (Figure 13.29). Discuss with students the major problems organisms have when catabolizing organic compounds: conserving energy for ATP synthesis and maintaining redox balance (Section 13.11). Note that redox balance in fermentation is internal, wherein a fermentable substrate becomes both oxidized and reduced.

Fermentations use energy inherent in a variety of energy-rich compounds to generate ATP via substrate-level phosphorylation (SLP). The number of different fermentation strategies used by microorganisms is impressive, but remind students that fermentations have two things in common: (1) With rare exceptions, ATP synthesis occurs via substrate-level phosphorylation, and (2) redox balance is maintained by the production of one or more fermentation products that are generated from the original substrate (Figure 13.29). Point out the variety of energy-rich compounds available for substrate-level phosphorylation, as shown in Table 13.3. Use Table 13.4 to show students the major types of fermentations, most of which are classified based upon the products they form (e.g., alcohol, mixed acid, and propionic acid). Unusual fermentations are noted in Table 13.5. Also note that redox balance in fermentations is often maintained by the production of H2, which is either catalyzed by the enzyme hydrogenase or associated with the production of formate (Figure 13.30). Depending upon the type and level of the course (or the biochemistry background of the students), you can decide how much detail to include in these sections.

Note that lactic acid bacteria are either homofermenters, producing lactic acid as the sole fermentation product, or heterofermenters, producing other products in addition to lactic acid (e.g., ethanol and CO2; Table 13.4 and Figure 13.31). The difference in these two fermentations is largely due to the activity of aldolase, a glycolytic enzyme that is present in homofermenters but absent in heterofermenters (Figure 13.31).

The Entner-Doudoroff pathway is a variation of the glycolytic pathway with the interesting difference being that one of the two molecules of pyruvate produced from glucose is formed directly, without concomitant substrate-level phosphorylation. The pathway therefore nets only one-half of the ATP produced from glycolysis. Key genera employing this pathway of glucose catabolism are Zymomonas and Pseudomonas. .

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In mixed-acid fermentations, either a mixture of acids (acetic, lactic, or succinic) or acids plus neutral products are formed (Figure 13.32). These fermentations provide the basis for the Methyl Red/Voges-Proskauer (MR-VP) test, which is often performed as a laboratory exercise to distinguish enteric bacteria, such as the mixed acid fermenter Escherichia coli from the 2,3-butanediol-producing Enterobacter aerogenes.

Section 13.13 provides two examples demonstrating fermentative diversity: clostridial fermentations and propionic acid fermentation. The major product from sugar fermentation by saccharolytic clostridia is butyric acid (Figure 13.33). Your students may find it interesting that fermentation by Clostridium acetobutylicum was the primary method of producing the solvents butanol and acetone during World War II. The production of butyric acid lowers the pH of these cultures, derepressing genes for neutral solvent production. Acetone production by C. acetobutylicum requires the subsequent reduction of butyrate to butanol so that the two NADH produced during glycolysis can be recycled back to NAD+ (Figure 13.33). This reaction maintains redox balance in the cell and allows glycolysis to continue. Note that some species of Clostridium are proteolytic in that they ferment either individual amino acids or an amino acid pair, where one amino acid is the electron donor and the other is the electron acceptor (a process known as the Stickland reaction; Figure 13.34). Similar to this, Clostridium kluyveri is capable of fermenting ethanol with acetate as the electron acceptor (Table 13.4). Because ethanol is itself a fermentation product, this process is a type of secondary fermentation. Also discuss the key components of the propionic acid fermentation, another type of secondary fermentation in which lactate is the major substrate (Figure 13.35). Propionic acid bacteria are necessary for the production of Swiss (Emmentaler) cheese, where acetic and propionic acids provide the distinctive flavor, and CO2 gas bubbles produce the eyes (holes) in the cheese.

Present students with the following problem: “If some bacteria carry out fermentations that do not produce enough free energy to synthesize ATP via substrate-level phosphorylation (i.e., less than –32 kJ), what mechanism might such bacteria employ to allow for ATP synthesis?” Lead the discussion of Section 13.14 back to the generation of an electrochemical gradient for ATP synthesis (either a proton or sodium motive force). Point out that such fermentations are either linked to ion pumps that generate an electrochemical potential, as in the fermentation of succinate (Figure 13.36a), or a PMF is formed indirectly through the consumption of cytoplasmic protons, as in the fermentation of oxalate (Figure 13.36b).

The process of syntrophy is presented in Section 13.15. Introduce to your students the concept of co-culture metabolisms. In these cooperative arrangements, pairs of organisms carry out metabolic strategies that are not possible by either organism alone (Table 13.6). A key example is interspecies H2 transfer, wherein H2 produced by one member is directly consumed by the other (Figure 13.37). Students need to understand the energetics of syntrophic reactions⎯specifically, the mechanism by which a reaction that was unfavorable (endergonic) under standard conditions (i.e., 1 atm, pH 7, 25C, and 1 molar concentration of all reactants and products) becomes favorable (exergonic) when considering the concentrations of the substrates in the natural environment. In the example shown in Figure 13.37, the immediate consumption of H2 by the methanogen provides an overall free energy change of –111.3 kJ, thus allowing the syntroph to conserve energy via ethanol fermentation (ordinarily an endergonic process). Figure 13.38 shows a similar example in which the normally endergonic (G0 = +48.2 kJ) oxidation of the fatty acid butyrate to acetate and H2 is made energet.

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ically favorable (G = –18 kJ) in Syntrophomonas when the reaction is coupled to the immediate removal of H2 by another organism, such as a methanogen. Because H2 concentrations are constantly kept extremely low, the reaction becomes one that releases enough free energy to support growth of both organisms.

13.16–13.21 | Anaerobic Respirations In natural systems, microorganisms employ an amazing diversity of anaerobic metabolisms in addition to the fermentations discussed in Sections 13.11–13.15. The major types of anaerobic respirations are discussed in Sections 13.16–13.21. Adjust to an appropriate level the amount of detail presented to your students from these sections.

The general principles of anaerobic respirations are covered in Section 13.16. Point out to students that the more electropositive the reduction potential of the electron acceptor in the redox couple, the more energy is released during the oxidation of the electron donor (Figure 13.39; also see Figure 3.9). The reduction of electron acceptors for the purpose of energy conservation is referred to as dissimilative metabolism, whereas assimilative metabolism is the reduction of inorganic compounds for use in biosynthesis (e.g., amino groups [–NH2] and sulfhydryl groups [–SH] can be derived from the reduction of NO3– and SO42–, respectively).

Nitrate reduction and denitrification are covered in Section 13.17. Ammonia and nitrate are the most common forms of reactive inorganic nitrogen in nature. Point out that the dissimilative reduction of nitrate yields gaseous products, which can therefore be lost from the immediate environment—a process referred to as denitrification (Table 13.7 and Figures 13.40 and 13.41). Discuss the biochemistry of dissimilative nitrate reduction as shown in Figure 13.41. Compare the electron transport chains of E. coli cells growing aerobically versus by nitrate respiration (Figure 13.41a and b). Discuss how dissimilative nitrate reduction by E .coli differs from denitrification by Pseudomonas stutzeri, in which NO3– is reduced all the way to N2 (Figure 13.41c).

Sulfate and sulfur reduction are discussed in Section 13.18. Show students the different oxidation states of relevant sulfur compounds, along with the electron donors commonly used for sulfate reduction (Table 13.8). Mention to them that the differences in reduction potentials between many of the available electron donors and acceptors are not large, and consequently these organisms might be expected to grow somewhat more slowly than organisms respiring O2 or NO3–. Sulfate-reducing bacteria (SRB) provide a good energetic example of this concept (Figures 13.42 and 13.43). Note that many SRB oxidize acetate to CO2 via the acetylCoA pathway (see Figure 13.45). SRB that can grow autotrophically using H2 as an energy source/electron donor use this same pathway with the reactions operating in the reverse direction for CO2 fixation. Some SRB can also disproportionate sulfur compounds, in which a substrate is both oxidized and reduced during the same metabolism to form two separate products (the disproportionation of thiosulfate to sulfate and sulfide is a good example).

Also note the unusual chemolithotrophic metabolism of phosphite oxidation linked to sulfate reduction by Desulfotignum phosphitoxidans. Finally, point out that some organisms that cannot use sulfate as an electron acceptor can instead respire elemental sulfur. The inability to reduce sulfate is presumably a result of being unable to activate sulfate to adenosine 5phosphosulfate (APS), a necessary ATP-consuming step in the conversion of sulfate to sulfite

.

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(Figure 13.42). Regardless of whether SO42– or S0 is used as the electron acceptor, H2S is produced, thereby linking the two processes ecologically.

Section 13.19 covers acetogenesis, which is a type of anaerobic respiration in which CO2 functions as an electron acceptor to produce acetate. Like the methanogens (discussed in the next section), acetogens are strict anaerobes that use H2 as a major electron donor (Figure 13.44), although acetogens can also metabolize a variety of organic compounds, including sugars, alcohols, and organic and amino acids. Note that all homoacetogens incorporate CO2 into acetate using the reductive acetyl-CoA pathway, which requires the key enzyme carbon monoxide dehydrogenase, the coenzyme tetrahydrofolate, and vitamin B12 as a cofactor (Table 13. 9 and Figure 13.45). Many acetogens can also grow chemoorganotrophically by sugar fermentation, and acetate is the sole fermentation product.

Section 13.20 discusses the process of methanogenesis in detail. Point out that methanogenesis is carried out exclusively by species of Archaea. These organisms are commonly found in freshwater sediments (Figure 13.46), sewage sludge digesters, and mammalian intestinal tracts. Figures 13.47 and 13.49 provide an excellent summary of the two classes of key coenzymes employed in the methanogenesis pathway: (1) C1 carriers and (2) electron carriers for reduction of CO2 to CH4. Coenzyme F420, for example, is a key electron donor in multiple steps of the reduction of CO2 in methanogens, and its presence in living cells is readily detected using fluorescence microscopy (Figure 13.48). Note that in addition to H2 + CO2, methylated compounds (e.g., methanol and acetate) can be used as substrates for methanogenesis (Figures 13.50 and 13.51). Depending on the growth substrate, energy conservation in methanogens occurs through oxidative phosphorylation at the expense of either a proton or sodium motive force; substrate-level phosphorylation does not occur.

Other inorganic and organic electron acceptors used in anaerobic respirations (Section 13.21) should be briefly discussed with the students, including ferric ion, manganese, chlorate, fumarate, and arsenate reductions (Figures 13.52 and 13.53). Point out to students that the microbiological reduction of a variety of alternative electron acceptors, such as arsenic trisulfide and any of several chlorinated compounds (e.g., polychlorinated biphenyls, chlorobenzoate, and tetrachloroethylene), can benefit the environment by converting persistent toxins to less harmful or nontoxic forms (Table 13.10). Metabolisms of this type are therefore potentially useful for bioremediation of contaminated sites (see Chapter 21).

Proton reduction is perhaps the simplest form of anaerobic respiration. Evidence for the evolution of primitive metabolism (Section 12.1) may be presented by discussing the proton reduction pathway in the hyperthermophilic archaeon, Pyrococcus furiosus. Originally thought to use the glycolytic pathway to oxidize sugars, P. furiosus was found to lack a key step in glycolysis⎯the production of 1,3-bisphosphoglyceric acid from glyceraldehyde 3-phosphate. Because the synthesis of 1,3-bisphosphoglyceric acid is bypassed, the organism cannot make ATP by substrate-level phosphorylation at the first of two possible steps for energy conservation in the pathway (Figure 13.54). However, additional substrate-level phosphorylation occurs in this organism with the oxidation of pyruvate to acetate (Figure 13.54). A transmembrane hydrogenase functions as the sole proton pump to establish a proton motive force in P. furiosus. Despite an incomplete glycolytic pathway, the combination of substrate-level phosphorylation and oxidative phosphorylation in P. furiosus actually produces more ATP per glucose molecule metabolized than most glucose fermenters are capable of producing. .

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13.22–13.24 | Hydrocarbon Metabolism The aerobic oxidation of hydrocarbons is a topic that should be familiar to most students in the biological sciences. It is relevant to issues that include the cleanup of toxic waste disposal sites, bioremediation of pesticide and fertilizer accumulation, and the biological degradation of oil/fuel and solvent spills that affect plant and animal life. Students in microbiology courses should be aware of the chemical and enzymatic requirements microorganisms must meet to oxidize these organic carbon compounds. Discuss the key role of oxygenase enzymes in the aerobic catabolism of aliphatic and aromatic hydrocarbons (Figures 13.55 and 13.56), both of which are typically oxidized completely to CO2. Although aromatic hydrocarbons can also be degraded anaerobically (see Figure 13.59b), growth by aromatic hydrocarbon oxidation is much faster in microorganisms growing aerobically.

In contrast to methanogenesis, in which CO2 is reduced to CH4, methylotrophs and methanotrophs are aerobic organisms that oxidize C1 compounds for energy and biosynthesis (Section 13.23). Discuss the biochemistry and bioenergetics of methane oxidation, noting that methanotrophs are a type of methylotroph that can use CH4 as the electron donor. The initial critical step of the process is the oxidation of methane to methanol using the enzyme methane monooxygenase (Figure 13.57). Differentiate between the two pathways for incorporation of C1 compounds into cell material. In the serine pathway (Figure 13.58a), each molecule of acetyl-CoA is synthesized from one molecule each of formaldehyde (CH2O) and CO2 at the expense of two molecules each of NADH and ATP. This is accomplished using serine transhydroxymethylase (an enzyme unique to the pathway) and several citric acid cycle enzymes. The ribulose monophosphate pathway (Figure 13.58b) is an alternative and more efficient mechanism of C1 assimilation because all cell carbon is derived from formaldehyde (CH2O), and therefore no reducing equivalents are required. Formaldehyde incorporation is catalyzed by the unique enzyme hexulosephosphate synthase.

Anoxic hydrocarbon catabolism is covered in Section 13.24. This metabolism is carried out by anaerobically respiring bacteria and may include either aliphatic or aromatic hydrocarbons as substrates. The most investigated route for oxidation of aliphatic compounds is hexane (C6H14) catabolism in denitrifying bacteria (Figure 13.59a). The anaerobic degradation of the aromatic hydrocarbon toluene is primarily carried out by denitrifying or sulfatereducing bacteria and results in the formation of benzoyl-CoA (Figure 13.59b). The latter compound can be further degraded by ring reduction in anoxic environments using the benzoyl-CoA pathway (Figure 13.60). This is in contrast to the ring oxidation method of aromatic degradation carried out aerobically (Figure 13.56). Use Figure 13.60 to show the anaerobic degradation of benzoate by the purple phototrophic bacterium Rhodopseudomonas palustris, an organism with an extraordinary ability for catabolizing a wide variety of aromatic compounds.

Methane produced in anoxic freshwater ecosystems by methanogens is typically oxidized to CO2 by methanotrophs once it reaches oxic zones. However, the oxidation of methane can also occur under anoxic conditions. This was originally observed in marine sediments by consortia of sulfate-reducing bacteria and methanotrophic Archaea (Figure 13.61). It is now known that this phenomenon is not limited to these two groups of organisms, as anoxicmethane oxidation (AMO) has also been linked to denitrification and Fe3+ and Mn4+ reduction. The denitrifying bacterium Methylomirabilis oxyfera is capable of AMO using a unique mechanism in which NO3– is reduced to NO2– and then nitric oxide (NO), which is then split .

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into N2 and O2 using the enzyme NO dismutase. The O2 can then accept electrons during the oxidation of CH4 to CO2, meaning that this organism produces its own O2 as a terminal electron acceptor in an anoxic environment!

Answers to Review Questions 1. The major differences between oxygenic and anoxygenic phototrophs lie in their reaction center photosystems and electron donors used for photosynthesis. Anoxygenic phototrophs use electron donors other than water (e.g., sulfide and other reduced substances from their environment) and contain a single photosystem for generation of ATP and reducing power. Oxygenic phototrophs use water as an electron donor and therefore evolve O2 as a byproduct of photosynthesis. They also have two photosystems: PS II for ATP synthesis and PS I for reducing power.

2. The two most indispensable light-sensitive pigments are chlorophylls, found in plants, algae, and the cyanobacteria, and bacteriochlorophylls, found in the purple and green bacteria. Other pigments are carotenoids and phycobilins. Chlorophyll and bacteriochlorophyll are related to tetrapyrroles that are the parent structure of the cytochromes. Unlike cytochromes, chlorophylls contain magnesium instead of iron at the center of the ring. The absorption spectrum of Chlorophyll a shows strong absorbance near 680 nm and 430 nm whereas Bacteriochlorophyll a absorbs maximally between 800 and 925 nm.

3. β-carotene is a common carotenoid. Carotenoids function primarily as photoprotective agents. Bright light can be harmful to cells because it can catalyze photooxidation reactions that can produce toxic forms of oxygen. Like superoxide and other forms of toxic oxygen, singlet oxygen can spontaneously oxidize photocomplexes, rendering them nonfunctional. Carotenoids quench toxic oxygen species by absorbing much of this harmful light and in this way prevent these dangerous photooxidations.

4. Just as in respiratory metabolisms, ATP production in an anoxygenic phototroph comes from the proton motive force generated during electron transport. However, in phototrophs, electron transport is initiated by the light-induced change in the reduction potential of RC pigments, allowing the RC to reduce a series of low-potential electron carriers while pumping protons outside the membrane. The return of these protons into the cytoplasm through ATP synthase provides the energy for ATP synthesis. This process is referred to as cyclic photophosphorylation. Electron transport in phototrophs uses some of the same electron carriers as those found in nonphototrophs. Unlike in respiration, however, there is no net input or consumption of electrons in cyclic photophosphorylation—the electrons simply travel in a closed loop.

5. Because the E0 of the quinone is 0 V and that of the NAD+/NADH couple is –0.32 V, reduction of NAD+ requires reverse electron flow and consumption of ATP. Reducing power during phototrophic growth in purple bacteria comes from reduced substances in their environment (e.g., H2S, S2O32–, S0), which are oxidized by c-type cytochromes and ultimately reduce NAD+ to NADH via the quinone pool. In cyanobacteria, NAD(P)+ is reduced to NAD(P)H directly from electron transport reactions from photosystem I. 6. PS II (P680) must have a highly electropositive reduction potential to allow H2O to serve as the electron donor (1/2 O2/H2O = E0 +0.82 V), a byproduct of which is O2. PS I (P700) is at a lower reduction potential than P680 and is reduced by the final electron .

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carrier from PS II, plastocyanin. Therefore, the highly electropositive reduction potential of PS II is essential for establishing the chain of electron carriers that can result in the eventual reduction of PS I. 7. The unique enzymes of the Calvin cycle (enzymes that may be used as markers for possessing the cycle) are (1) ribulose bisphosphate carboxylase (RubisCO) and (2) phosphoribulokinase. The reactions catalyzed by these enzymes are 1. CO2 + ribulose 1,5-bisphosphate + H2O → 2 phosphoglyceric acid 2. ribulose 5-phosphate + ATP → ribulose bisphosphate + ADP The absence of either of these enzyme activities would prevent autotrophic growth via the Calvin cycle.

8. Among phototrophs, Chloroflexus uses the hydroxypropionate pathway for autotrophic growth. Chlorobium, Sulfolobus, Thermoproteus, and Aquifex are known to employ the reverse citric acid cycle.

9. Chemolithotrophs oxidize inorganic electron donors to conserve energy and obtain reducing power. Energy conservation occurs from respiratory processes that generate a proton motive force. Most chemolithotrophs can grow autotrophically. It has been found that for a chemolithotroph to grow as an autotroph, the formation of ATP is not enough.

Reducing power (NADH) is also necessary to reduce CO2 to cell material. Reducing power for purple bacteria can come from many sources, in particular reduced sulfur compounds such as H2S. ATP generation in chemolithotrophs is similar to that in respiring chemoorganotrophs except that the electron donor is inorganic rather than organic.

Reducing power in chemolithotrophs is obtained in either of two ways: directly from the inorganic compound (if it has a sufficiently negative reduction potential, such as H2), or from reverse electron transport reactions, if the inorganic electron donor is more electropositive than NADH. The chemolithotrophic hydrogen bacteria use H2 as an electron donor, reducing O2 to H2O. The enzyme hydrogenase is required to oxidize H2, and H2 also supplies reducing power for the fixation of CO2 in these autotrophs. Hydrogenase catalyzes the synthesis of ATP during H2 oxidation by O2. Electrons from H2 are initially transferred to a quinone acceptor and through a series of cytochromes to generate a proton motive force that eventually reduces O2 to water.

10. Reduced sulfur compounds such as H2S, S2O32−, and S0 are electron donors for energy conservation in sulphur chemolithotrophs. Electrons from these substances enter electron transport chains, yielding a proton motive force. Sulfur chemolithotrophs are also autotrophs and fix CO2 by the Calvin cycle. Chemolithotrophic iron bacteria oxidize Fe2+ as an electron donor. Most iron bacteria grow at acidic pH and are often associated with acidic pollution from mineral and coal mining. A few chemolithotrophic and phototrophic bacteria can oxidize Fe2+ to Fe3+ anaerobically.

11. Hydroxylamine oxidoreductase is a key enzyme that participates in the oxidation of reduced nitrogen compounds. It oxidizes NH2OH to NO2−, removing four electrons in the process. The anoxic removal of NH3 and amines by the formation of N2 helps reduce the input of fixed nitrogen from wastewater treatment facilities into rivers and streams, thereby maintaining higher water quality than would otherwise be possible.

12. In substrate-level phosphorylation, the high-energy phosphate bonds from energy-rich organic compounds are transferred directly to ADP to make ATP. Oxidative phosphory.

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lation involves the oxidation of reduced electron carriers (e.g., NADH and FADH2) by membrane-bound electron transport complexes, resulting in the generation of a proton (or sodium) motive force for the synthesis of ATP via a membrane-bound ATP synthase.

13. Lactobacillus: lactic acid; ethanol Clostridium: propionic acid; ethanol; butyric acid; acetate; butanol Propionibacterium: propionic acid Escherichia: mixed acid; ethanol; 2,3-butanediol; succinic acid; lactic acid; acetate; formate Some clostridia ferment only an amino acid pair. In this situation one amino acid functions as the electron donor and is oxidized, whereas the other amino acid is the electron acceptor and is reduced. This coupled amino acid fermentation is called a Stickland reaction.

14. Fermentative organisms have an additional option for energy conservation when the energy released in the reaction is insufficient for substrate-level phosphorylation. This option is to generate either a proton or sodium motive force for oxidative phosphorylation. A proton motive force can be produced indirectly using a formate-oxalate antiporter; the influx of oxalate and efflux of formate consumes cytoplasmic protons. A sodium motive force is produced by coupling a membrane-bound Na+ pump to the decarboxylation of an organic acid, such as succinate. In either case, the electrochemical gradient produced is used to generate chemical energy using ATP synthases.

15. Syntrophy is often referred to as interspecies H2 transfer because the H2 released by one organism is consumed as the energy source (electron donor) by another organism. In most cases, the free energy of the metabolic reaction carried out by the first organism (the syntroph) is highly unfavorable unless the second organism immediately consumes the H2 produced. Thus, the activity of the second organism lowers the partial pressure of H2 for the initial reaction and changes the energetics of the reaction to yield an overall –∆G. 16. The reduction potential of the NO3–/NO2– couple (+0.43 V) is much more electropositive than that of the SO42–/HS– couple (–0.217 V). Therefore, the use of NO3– as an electron acceptor results in a greater release of free energy from a reaction. Thus, NO3– is a better electron acceptor than SO42–.

17. Dissimilative nitrate reduction in Escherichia coli is characterized by the reduction of NO3– only to NO2–, whereas in Pseudomonas, denitrification to N2 occurs.

18. Hydrogenase is constitutively expressed in Desulfovibrio because during sulfate reduction, H2 is produced from the catabolism of organic compounds. For example, lactate, an excellent electron donor and energy source for species of Desulfovibrio, is oxidized to pyruvate by lactate dehydrogenase, and this produces H2 as a byproduct. More H2 is produced from the subsequent oxidation of pyruvate to acetate. The membrane-bound hydrogenase splits the H2 into electrons, which enter a transport chain and eventually reduce SO42– to H2S, and protons, which establish a proton motive force for ATP production.

19. A key enzyme of the acetyl-CoA pathway is carbon monoxide (CO) dehydrogenase. CO dehydrogenase contains Ni, Zn, and Fe as cofactors and catalyzes the formation of carbon monoxide and water from carbon dioxide and hydrogen. The CO produced by CO .

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dehydrogenase ends up as the carbonyl carbon of acetate in acetogenesis. When acetate is the substrate for methanogenesis, it is first activated to acetyl-CoA, which interacts with CO dehydrogenase from the acetyl-CoA pathway.

The methyl group of acetate originates from the reduction of CO2 by a series of reactions in which the coenzyme tetrahydrofolate plays a major role during acetogenesis.

Methanopterin is a methanogenic coenzyme that resembles the vitamin folic acid and plays a role analogous to that of tetrahydrofolate in methanogenesis.

The coenzyme methanofuran is required for the first step of methanogenesis. It has a five membered furan ring and an amino nitrogen atom that binds CO2. CO2 is activated by a methanofuran-containing enzyme and reduced to the formyl level. The coenzymes F420 and 7-mercaptoheptanoylthreonine phosphate (also called coenzyme B, CoB) are electron donors in methanogenesis.

In acetate and methanol-grown cells, energy conservation is linked to the terminal step in methanogenesis, the methyl reductase step. In this reaction, the interaction of CoB with CH3–CoM and methyl reductase forms CH4 and a heterodisulfide product, CoM-S— S-CoB. The latter is reduced by H2 to regenerate CoM-SH and CoB-SH. This reduction, carried out by the enzyme heterodisulfide reductase, is exergonic and is coupled to the pumping of H+ across the membrane by using the membrane-associated electron carrier called methanophenazine.

20. Ferric iron (Fe3+) is an excellent electron acceptor (E0 = +0.77 V at pH 2) that is widespread in nature and used by a variety of chemoorganotrophic and chemolithotrophic bacteria in anaerobic respirations. For example, the model organism for the study of this process, Geobacter metallireducens, can degrade environmental pollutants such as toluene using Fe3+ as the electron acceptor. In a similar manner, some sulfate-reducing bacteria, such as Desulfomonile, can grow anaerobically using H2 or organic compounds as electron donors and chlorobenzoate (E0 = +0.297 V) as the electron acceptor, reducing it to benzoate and HCl. Many reduced chlorinated compounds are toxic, and thus reductive dechlorination serves as an environmentally significant bioremediation process.

21. Monooxygenases catalyze the incorporation of one oxygen atom from O2 into the molecule being oxidized, whereas both oxygen atoms are incorporated into dioxygenase-catalyzed reactions. Oxygen incorporation into hydrocarbons by oxygenases is necessary because oxygen serves as a direct reactant in the catabolic process.

22. Methanotrophs (largely aerobic Bacteria) oxidize methane as an energy source, whereas methanogens are anaerobic Archaea that produce methane in their energy metabolism. Unlike the serine pathway, which requires both NADH and ATP to synthesize acetyl-CoA from one molecule each of formaldehyde (CH2O) and CO2 (Figure 13.58a), the ribulose monophosphate pathway (Figure 13.58b) is a more efficient mechanism of C1 assimilation because all cell carbon is derived from formaldehyde (CH2O), and therefore no reducing equivalents are consumed.

23. Denitrifying and sulfate-reducing bacteria can degrade aliphatic or aromatic hydrocarbons without the use of oxygenase enzymes under anoxic conditions by incorporating oxygen atoms from other .

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molecules, such as fumarate, into the hydrocarbon. In doing so, fatty acid derivatives can be formed, and these can be catabolized by beta-oxidation.

Answers to Application Questions 1. The photosynthetic pigments of purple bacteria are located in intracellular membrane systems composed of lamellar stacks or vesicular chromatophores. In cyanobacteria, pigments occur in thylakoid membranes with attached phycobilisomes. In green algae, chlorophylls are found in the lamellae of chloroplasts. Pigment-containing RCs must be in close proximity to a membrane-associated electron transport system to facilitate the conversion of light energy to chemical energy. Therefore, the generation of ATP and NAD(P)H requires that the RC also be membrane bound.

2. The chlorophyll pigments and all the other components of the light-gathering apparatus exist within membranes in the cell. The location of these photosynthetic membranes differs between prokaryotic and eukaryotic microorganisms. In eukaryotic phototrophs, photosynthesis takes place in intracellular organelles, the chloroplasts, where the chlorophylls are attached to sheetlike membranes. These photosynthetic membrane systems are called thylakoids, and stacks of thylakoids form grana. The thylakoids are arranged so that the chloroplast is divided into two regions, the matrix space that surrounds the thylakoids, called the stroma, and the inner space within the thylakoid array. This arrangement makes possible the generation of a light-driven proton motive force that is used to synthesize ATP. Chloroplasts are absent from prokaryotic phototrophs. In purple bacteria, the photosynthetic pigments are integrated into internal membrane systems that arise from invagination of the cytoplasmic membrane. Membrane vesicles called chromatophores or membrane stacks called lamellae are common membrane arrangements in purple bacteria. The ultimate structure for capturing low light intensities is the chlorosome. Chlorosomes are present in the anoxygenic green sulfur bacteria (Chlorobium) and green nonsulfur bacteria (Chloroflexus). Chlorosomes function as giant antenna systems, but unlike the antennae of purple bacteria or cyanobacteria, bacteriochlorophyll molecules in the chlorosome are not attached to proteins. Chlorosomes contain bacteriochlorophyll c, d, or e, arranged in dense arrays running along the long axis of the structure. Light energy absorbed by these antenna pigments is transferred to bacteriochlorophyll a in the reaction center in the cytoplasmic membrane through a small protein called the FMO protein. Bchl c, d, and e are important photosynthetic pigments present in green sulfur bacteria. Bacteriochlorophylls c, d, and e consist of isomeric mixtures with the different substituents at R3 position.

Absorption properties can be determined by suspending intact cells of a phototroph in a viscous liquid such as 60% sucrose (this reduces light scattering and smooths out spectra) and running absorption spectra. In vivo absorption maxima are the physiologically relevant absorption peaks.

3. Chemolithotrophs use an inorganic compound as an electron donor and energy source, whereas chemoorganotrophs use organic compounds. However, in both types of organisms, ATP can be produced using membrane-bound electron transport chains that generate a proton motive force for oxidative phosphorylation. A major reason why chemoor-ganotrophs respiring glucose have a dramatically higher growth yield than a chemolithotroph respiring sulfur is a function of the reduction potential of the electron donor/acceptor couples employed in each metabolism. For glucose-respiring prokaryotic chemoorganotrophs, the net free-energy differential yields about 38 ATP molecules per .

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mole of glucose. Moreover, glucose can also serve as a reduced carbon source for biosynthesis. Most sulfur-chemolithotrophs obtain their cellular carbon autotrophically from CO2 fixation via the Calvin cycle. This is energetically expensive and consumes about 3 ATP molecules per mole of CO2 reduced, which drops the net ATP yield compared with chemoorganotrophs, resulting in an overall lower growth yield.

4. The energetics of butyrate fermentation (butyrate → acetate) are highly unfavorable (+48.2 kJ) and, thus, cannot be carried out in pure culture. However, butyrate can be catabolized under anoxic conditions in pure culture via anaerobic respiration. Because of interspecies H2 transfer, butyrate fermentation is possible in a syntrophic mixed culture if an H2-consuming organism is present (Section 13.15).

5. Both acetogens and methanogens use carbon dioxide (CO2) as an electron acceptor for energy conservation, and CO2 is typically abundant in anoxic habitats because it is a major product of the energy metabolisms of anaerobic chemoorganotrophs. When acetate is the substrate for methanogenesis, it is first activated to acetyl-CoA, which interacts with CO dehydrogenase from the acetyl-CoA pathway. The methyl group of acetate is then transferred to the corrinoid enzyme to yield CH3–corrinoid, and from there it follows the CoM-mediated terminal step of methanogenesis. Simultaneously, the CO group is oxidized to yield CO2 and electrons.

For reason that acetogens and methanogens use carbon dioxide in the procress of methanogenesis, these would definitely help in reducing its concentration from the atmosphere, which otherwise causes global warming.

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CHAPTER

14

Functional Diversity of Bacteria

Summary In Chapter 13, the authors covered the major examples that illustrate the enormous metabolic diversity of microorganisms. Chapter 14 focuses on the functional diversity of Bacteria, a concept that complements the phylogenetic and metabolic diversity of these organisms. After defining the principles of functional diversity in microorganisms, the authors consider a variety of microorganisms that serve to illustrate these concepts, including the various phyla of phototrophic Bacteria; sulfur and nitrogen cycling prokaryotes; bacteria that reduce or oxidize iron compounds; hydrogen, methane, and acetate-metabolizing bacteria; predatory Bacteria; and those Bacteria that are morphologically or otherwise phenotypically distinct..

14.1 | Making Sense of Microbial Diversity This section compares and contrasts phylogenetic diversity with functional diversity. Phylogenetic analyses draw important comparisons between microorganisms that allow for the study of lineages and the assignment of taxonomic classifications. These conclusions are most often based on sequence analysis of ribosomal RNA genes or other universal genes that are highly conserved. By contrast, functional diversity pertains to descriptions of microorganisms based on form and function in relation to physiology and ecology.

Although functional diversity often aligns well with phylogenetic diversity, this is not always the case. Figure 14.1 illustrates this point by comparing phylogenetic relatedness to the major functional traits of microorganisms. Point out to your students that many major ecological processes, such as nitrogen fixation and dissimilative sulfur metabolism, are represented across widely divergent lineages, even spanning both domains of prokaryotes (the Bacteria and Archaea) in multiple cases. At least three mechanisms account for shared functional traits between divergent organisms: (1) gene loss by some, but not all, descendants of a common ancestor; (2) convergent evolution of a trait conferred by genes that are not homologous; and (3) horizontal gene transfer that produces homologous genes in distantly related lineages.

14.2–14.8 | Diversity of Phototrophic Bacteria Photosynthesis is widespread among the Bacteria, with representatives from six phyla capable of (bacterio)chlorophyll-based phototrophic growth. Five of the six phyla (Proteobacteria, Chlorobi, Chloroflexi, Firmicutes, and Acidobacteria) consist of only anoxygenic .

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phototrophs that contain either type I or type II photosynthetic reaction centers (RCs). Type I RCs are most similar in structure to photosystem (PS) I of oxygenic phototrophs, whereas type II RCs are most similar to PS II of oxygenic phototrophs. The sixth bacterial phylum containing phototrophs is the Cyanobacteria, which, like algae and green plants, are oxygenic phototrophs that contain both types of photosystems (PS I and PS II). Pigments in phototrophs are always embedded within a membrane—either the cytoplasmic membrane or within intracytoplasmic membrane systems that can be quite extensive. While most phototrophs can grow autotrophically, it may surprise some students to know that many phototrophs grow preferentially using certain organic substrates as carbon sources, and some have an absolute requirement for organic carbon. Present a brief overview of the key features of genera and representative species from each of these phyla as suitable for your course content.

Briefly discuss the evolutionary importance of cyanobacteria and the prochlorophytes (Section 14.3), as they are oxygenic phototrophs that were responsible for the oxygenation of Earth’s atmosphere over two billion years ago. Cyanobacteria are distantly related to grampositive bacteria and are a morphologically diverse group (Table 14.1 and Figures 14.2 and 14.3). Cyanobacteria contain only chlorophyll a and phycobilin pigments, such as phycocyanin (see Figure 13.10 and 14.5), whereas prochlorophytes contain chlorophyll a and b but lack phycobilins. Pigments of both groups are contained within internal thylakoid membranes (Figure 14.4). Filamentous cyanobacteria can fragment into short sections of cells called hormogonia, which can disseminate from the parent filament by gliding motility (Figure 14.6a and b). Some species also form resting structures called akinetes, which are dormant for a period of time before germinating into vegetative cells upon encountering favorable conditions (Figure 14.6c). In this regard, they are similar to endospores; however, endospores have a much greater degree of heat resistance than do akinetes. Other notable features of cyanobacteria include the following: (1) widespread capacity to carry out N2 fixation (Figure 14.7), often via the formation of heterocysts, differentiated cells that segregate oxygensensitive nitrogenase from O2-evolving photosynthesis (Figure 14.8), (2) production of gas vesicles, which regulate buoyancy in the water column, and (3) the formation of cyanophycin, a copolymer of aspartic acid and arginine that serves as a nitrogen storage product and reserve energy source.

Discuss key concepts of the ecology of cyanobacteria with the students. Summarize the interesting characteristics of Synechococcus and Prochlorococcus. The marine prochlorophyte Prochlorococcus has the distinction of being perhaps the most abundant (up to 105 cells/ml seawater) and the smallest (0.5–0.8 µm) oxygenic phototroph on Earth. Considering their vast numbers, small unicellular cyanobacteria are believed to be of great ecological importance as the dominant source of primary production in oligotrophic, open ocean waters.

Section 14.4 discusses the purple sulfur bacteria, a group that includes the genera Chromatium and Ectothiorhodospira and aligns within the Gammaproteobacteria. These anoxygenic phototrophs use H2S as the electron donor for photoautotrophic growth and typically inhabit anoxic waters in which both sulfide and light are available, including stratified lakes, sulfide springs, surficial marine and salt marsh sediments, and microbial mats (Figure 14.9). Some species are extremely alkaliphilic or halophilic. The oxidation of sulfide by these organisms is often accompanied by the accumulation of elemental sulfur globules either inside or outside the cells, which are oxidized fully to sulfate if sulfide levels become limiting (Figure 14.10). Purple bacteria contain type II RCs, often embedded within prominent vesicular or lamellar internal membrane systems (Figure 14.11). .

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The purple nonsulfur bacteria and aerobic anoxygenic phototrophs are addressed in Section 14.5. Purple nonsulfur bacteria, such as Rhodobacter and Rhodospirillum, are phylogenetically diverse, having members within both the Alpha- and Betaproteobacteria. They may contain a variety of carotenoid pigments in addition to bacteriochlorophyll a or b, and this assortment imparts a range of potential colors for cultures of these bacteria (Figure 14.12).

In addition, this group exhibits considerable morphological diversity (Figure 14.13). Purple nonsulfur bacteria are well known for their remarkable metabolic versatility, being able to grow photoheterotrophically using a wide range of organic carbon sources or photoautotrophically using H2, Fe2+, or (despite their name) low levels of sulfide as the electron donor. Most species can also grow chemotrophically by aerobic or anaerobic respiration or fermentation.

The aerobic anoxygenic phototrophs (AAPs), such as Roseobacter and Erythrobacter, are phylogenetically related to the purple nonsulfur bacteria but ecologically distinct. AAPs are obligate aerobic heterotrophs that produce bacteriochlorophyll a in the dark and use daylight to supplement their growth via photophosphorylation. They often account for a significant percentage of the microbial community in coastal, marine waters.

Green sulfur bacteria (Section 14.6) are obligately anaerobic anoxygenic phototrophs that contain unique light-harvesting structures called chlorosomes (Figures 14.14 and 14.15) that transfer light energy to type I RCs. Pigments found in green sulfur bacteria include bacteriochlorophyll a and either bacteriochlorophyll c, d, or e, depending on the species; green or brown carotenoids are also present (Figure 14.16). Green sulfur bacteria are distinguished by their ability to grow at very low light intensities and utilize sulfide (H2S), elemental sulfur (S0), and thiosulfate (S2O32–) as electron donors. Elemental sulfur produced from the oxidation of sulfide is precipitated outside the cell, unlike the purple sulfur bacterium Chromatium, in which the sulfur remains within the periplasm of the cells. The only cultured thermophilic species, Chlorobaculum tepidum, is notable, as its genome (2.15 Mbp) was the first of any anoxygenic phototroph to be sequenced. Point out to your students that interesting consortia of phototrophic green bacteria and a nonphototrophic central cell are common in aquatic environments (Figure 14.17; see also Section 22.2).

A phylogenetically distinct group of anoxygenic phototrophs that contains few genera is the phylum Chloroflexi (the green nonsulfur bacteria; Section 14.7). Members of this phylum are often called the filamentous anoxygenic phototrophs (FAPs) due to their morphological and physiological features. The best-known and most extensively studied genus of this phylum is Chloroflexus. Most isolated cultures of Chloroflexus are thermophilic and grow best photoheterotrophically rather than photoautotrophically (CO2 fixation occurs via the hydroxypropionate pathway). Like green sulfur bacteria, green nonsulfur bacteria contain chlorosomes and bacteriochlorophylls a and c. However, green nonsulfur bacteria have a type II RC that is structurally similar to the RCs of purple bacteria. Also similar to many purple bacteria, green nonsulfur bacteria are capable of dark chemoorganotrophic growth via aerobic respiration. Discuss the green nonsulfur representatives shown in Figure 14.18, including Chloroflexus, Oscillochloris, Chloronema, and Roseiflexus. Also point out the major characteristics of the aerobic chemotroph Thermomicrobium, including its unusual genome arrangement (two circular chromosomes), lack of peptidoglycan, and unique membrane lipids (Figure 14.19).

Section 14.8 discusses the final two bacterial phyla that contain phototrophs—Firmicutes and Acidobacteria. Heliobacteria are Firmicutes that group within the Clostridiales. They are an interesting group in that they are obligately anaerobic and heterotrophic, and they repre.

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sent the only known phototrophs capable of forming endospores (Figure 14.20). Heliobacteria also produce the unique photosynthetic pigment bacteriochlorophyll g for photoheterotrophic growth, and this is contained within a relatively simple type I RC found solely within the cytoplasmic membrane. Similar to their clostridial relatives, many heliobacteria are also capable of chemotrophic growth by pyruvate fermentation.

The phototrophic Acidobacteria include just one cultured representative, Chloracidobacterium thermophilum. As in green sulfur bacteria, C. thermophilum contains a type I PS with bacteriochlorophyll a and c, with the latter pigment organized in light-harvesting chlorosomes (Figure 14.21). In contrast to green sulfur bacteria, however, C. thermophilum can grow aerobically but is incapable of autotrophic growth.

14.9–14.11 | Bacterial Diversity in the Sulfur Cycle The dissimilative sulfate-reducing bacteria (Section 14.9) are a large group of morphologically diverse, primarily Deltaproteobacteria that carry out anaerobic respiration through dissimilative sulfate reduction (Figures 14.22 and 14.23a–f). Sulfate-reducing bacteria not included among the Proteobacteria include Desulfotomaculum, a genus of endospore-forming bacteria related to the clostridia, and Archaeoglobus, a thermophilic archaeon (Figure 14.22).

Most representatives are strictly anaerobic, but some species are tolerant of low levels of O2; at least one species, Desulfovibrio oxyclinae, is even capable of microaerophilic growth by aerobic respiration. Sulfate-reducing bacteria are categorized based on whether they have the ability to oxidize acetate completely to CO2. Species of Desulfovibrio, the best-studied genus, are unable to oxidize acetate and, therefore, are incomplete oxidizers. Growth of sulfate-reducing bacteria is accompanied by the production of hydrogen sulfide (H2S), which can react spontaneously with Fe2+ in anoxic environments to form ferrous sulfide (FeS), an insoluble black precipitate (Figure 14.23g). Sulfate-reducing bacteria primarily inhabit sulfate-containing aquatic and terrestrial habitats that are anoxic.

The dissimilative sulfur-reducing bacteria (Section 14.10) are also a diverse group. Like the sulfate-reducing bacteria, they are not actually all Bacteria. Several genera of sulfurreducing “bacteria” are in fact Archaea (e.g., the genera Acidianus, Sulfolobus, Pyrodictium, and Thermodiscus—all Crenarchaeota), and therefore it is more accurate to refer to members of these groups as sulfate- and sulfur-reducing “prokaryotes.” Most genera among the Bacteria are Proteobacteria. The sulfur-reducing bacteria respire S0 and certain other inorganic compounds for energy conservation, but they do not respire SO42−. Most species are strict anaerobes, but others are facultative, and some, such as Sulfospirillum, are able to grow autotrophically using H2 as the electron donor. Sulfur-reducing bacteria often inhabit areas where other bacteria are using H2S as an electron donor, oxidizing it to S0. Thus, the sulfur-reducing bacteria have access to a steady supply of oxidant to maintain anaerobic respiration.

To complement the organisms described in the previous two sections, the dissimilative sulfur-oxidizing bacteria are discussed in Section 14.11. These organisms, which span several phyla of Bacteria and Archaea, complete the cycling of sulfur in natural habitats by oxidizing reduced sulfur compounds for energy conservation (Figure 14.24). Both neutrophilic and acidophilic sulfur-oxidizing bacteria have been described, with many acidophilic species also capable of using ferrous iron (Fe2+) as an energy source. Most sulfur-oxidizing bacteria are strict aerobes, but some, such as Thiomicrospira, can also grow anaerobically by NO3– respi.

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ration. While some sulfur oxidizers can use organic compounds as energy sources, many are obligate chemolithotrophs and, as such, they are also autotrophic. In this regard, carboxysomes, intracellular polyhedral structures that contain Calvin cycle enzymes, are commonly observed in sulfur chemolithotrophs (Figure 14.25a).

Use Figures 14.25–14.29 to show the extensive morphological diversity of the sulfuroxidizing bacteria. Note, in particular, that their growth is often associated with the accumu lation of elemental sulfur either inside or outside the cells, and this often confers a white to yellow appearance to masses of cells. In addition, some of the largest prokaryotic cells known are sulfur oxidizers. Some species of Thiomargarita have cells with a diameter of up to 0.75 mm, a size visible to the naked eye! Take some time to discuss the fascinating ecological strategies of sulfur-oxidizing bacteria. The rosettes of Thiothrix (Figures 14.24a and 14.26b), the gliding filaments of Beggiatoa (Figures 14.24b and 14.27a), the large NO3− vacuoles of Thioploca (Figure 14.27b) and Thiomargarita (Figure 14.28), the convective veils of Thiovulum (Figure 14.29), and various symbioses with eukaryotic hosts all serve to illustrate the remarkable ecological diversity and resourcefulness of these organisms.

14.12–14.13 | Bacterial Diversity in the Nitrogen Cycle These sections address the diversity of prokaryotes that are important contributors to the cycling of nitrogen, beginning with the nitrogen-fixing bacteria (Section 14.12). Nitrogen-fixing bacteria are diazotrophs, organisms that reduce N2 to NH3 for cellular assimilation. Diazotrophy is believed to be an ancient process, as several bacterial phyla and one archaeal phylum contain representatives that share this trait. Nitrogen fixation requires the O2-sensitive enzyme complex nitrogenase. A key gene for nitrogenase biosynthesis is nifH, which encodes the dinitrogenase reductase component of the complex. Due to the incongruence between 16S rRNA and nifH gene phylogenies, it is likely that horizontal gene transfer has played an important role in the dissemination of genes that allow for nitrogen fixation (Figure 14.30).

Diazotrophs can form symbioses with higher organisms or be free-living, and both aerobic and anaerobic species are abundant. The best-studied symbiotic species are root-nodule bacteria (i.e., rhizobia) that associate with leguminous plants. Obligately aerobic free-living diazotrophs are large, gram-negative rods or cocci. While not a concern for anaerobes, aerobic N2-fixing bacteria must protect nitrogenase from O2 deactivation in their oxic environment.

Often these bacteria employ a high respiration rate to reduce O2 to H2O as it diffuses through the membrane, and many species also produce a capsular slime layer to retard O2 diffusion into the cell (Figure 14.31). Diazotrophic cyanobacteria form heterocysts to address the problem (Section 14.3).

Species of Azotobacter are capable of forming cysts, which are resting structures similar to endospores in their resistance to desiccation and radiation, but unlike endospores, cysts are not especially heat-resistant (Figure 14.32). Species of Azospirillum are useful in the agricultural industry for their propensity to form nonspecific symbiotic relationships with crop plants, in which the bacterium supplies the plant with fixed nitrogen in exchange for organic carbon substrates. Lastly, discuss the synthesis of alternative nitrogenases in molybdenumlimiting environments. While the usual and more efficient nitrogenase uses a molybdenum cofactor, alternative nitrogenases use either a vanadium or iron cofactor.

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Section 14.13 describes the nitrifying and denitrifying prokaryotes, organisms that either oxidize reduced inorganic nitrogen compounds for energy or use oxidized forms of nitrogen for anaerobic respiration, respectively. Nitrifying bacteria are subdivided into two groups: ammonia oxidizers, which oxidize ammonia (NH3) to nitrite (NO2−) (e.g., Nitrosomonas), and nitrite oxidizers, which oxidize NO2− to nitrate (NO3–) (e.g., Nitrobacter and Nitrospira; Figures 14.33 and 14.34). No single organism is known to be able to oxidize NH3 completely to NO3–. The key enzymes of nitrification are ammonia monooxygenase and nitrite oxidoreductase, which are often packaged into internal lamellar membranes reminiscent of those in purple bacteria (Figure 14.33).

The denitrifying bacteria are those that respire NO3– or NO2− to gaseous forms of nitrogen, including NO, N2O, and N2. However, most denitrifying bacteria are facultative aerobes, and thus will grow aerobically if O2 is present. With the exception of the denitrifying sulfur chemolithotrophs (described in Section 14.11), most denitrifying bacteria are chemoorganotrophs of various lineages, primarily of the Proteobacteria. Many pseudomonads carry out denitrification, but probably the best-studied denitrifier is the α-proteobacterium Paracoccus denitrificans. See Section 13.17 for details concerning the biochemistry of this organism.

14.14–14.19 | Diversity of Other Distinctive Chemotrophic Bacteria These sections explore the diversity of chemotrophic prokaryotes that are distinctive in various ways but whose functional ecological roles have not yet been discussed. Similar to the sulfur- and nitrogen-metabolizing bacteria discussed in previous sections, a number of prokaryotes use iron for energy conservation in dissimilative processes. Iron-reducing bacteria (Section 14.14) couple the oxidation of H2 or various organic compounds to the reduction of metals, such as Fe3+ or Mn6+. At least six bacterial phyla and one archaeal phylum contain iron-reducing species, with Geobacter, of the Deltaproteobacteria, among the best-studied and most metabolically versatile (Figure 14.35). Whereas most iron-reducing bacteria are anaerobes, Shewanella and Aeromonas are facultative and respire aerobically when O2 is available.

Iron-oxidizing bacteria (Section 14.15) use ferrous iron (Fe2+) as an electron donor and consist of both aerobic and anaerobic species across a broad range of bacterial and archaeal phyla. The physiological categorization of iron-oxidizing bacteria is equally wide, with defined functional groups of organisms that include acidophilic aerobes, neutrophilic aerobes, chemotrophic anaerobes, and phototrophic anaerobes. Iron-oxidizing bacteria inhabit aquatic ecosystems of all types where ferrous iron is available, as well as saturated terrestrial soils having anoxic zones that contain sufficient Fe2+. The neutrophilic iron-oxidizers Mariprofundus and Gallionella provide interesting morphological examples in that they produce unusual stalklike structures, which apparently serve as repositories for excreted Fe(OH)3 from the oxidation of Fe2+, thereby preventing the live cells from becoming encased in iron oxide (Figure 14.36).

Hydrogen-metabolizing bacteria (Section 14.16) are aerobes (often microaerophiles) that use H2 as an electron donor. All hydrogen bacteria contain hydrogenases, nickel-containing enzymes that bind H2 for ATP production or as a source of reducing power. Species of Ral.

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stonia (Figure 14.37), Pseudomonas, and Paracoccus are the best-studied examples. Some hydrogen bacteria are able to grow using carbon monoxide (CO) as an electron donor, in which case they are referred to as carboxydotrophic bacteria. These organisms use the Calvin cycle for autotrophic growth, but most can also grow chemoorganotrophically. Such versatility presumably helps counter the fluctuating levels of CO often found in natural conditions.

Methanotrophs are methylotrophs (Section 14.17) that oxidize methane (CH4) and other compounds lacking C–C bonds. By contrast, many nonmethanotrophic methylotrophs can catabolize organic acids, alcohols, and sugars. Methane oxidation by methanotrophs requires O2 as a reactant and the enzyme methane monooxygenase, which is localized inside a network of internal membranes. Table 14.2 presents a taxonomic and phenotypic overview of methanotrophs, which are categorized as type I or type II based on their internal membrane structure, phylogeny, and mechanism of carbon assimilation. Type I methanotrophs (Figure 14.38b) employ the ribulose monophosphate cycle for C1 assimilation, while type II methanotrophs (Figure 14.38a) use the serine pathway (see Section 13.23). Discuss the interesting symbiotic associations that some methanotrophic bacteria have with animals (Figure 14.39).

Acetic acid bacteria and acetogens are two groups of bacteria that produce acetate using distinct mechanisms (Section 14.18). Acetic acid bacteria, such as Acetobacter and Gluconobacter, are gram-negative, motile, obligate aerobes that oxidize alcohols and sugars to acetic and other acids, depending on the substrate (Figure 14.40). Because the acids they produce are excreted into their environment, acetic acid bacteria are naturally tolerant of acidic conditions. Acetic acid bacteria play a major role in the commercial production of vinegar and sorbose, a precursor of ascorbic acid (vitamin C). By contrast, the acetogens, such as Acetobacterium and certain species of Clostridium, are obligate anaerobes that use the reductive acetyl-CoA pathway to assimilate C1 compounds for biosynthesis. Many can also grow chemoorganotrophically on simple organic compounds that are oxidized to acetate and CO2.

Acetogens are found in anoxic waters, sediments, and saturated soils, as well as the digestive tracts of animals, where they play an important role in nutrient uptake by the host.

Section 14.19 describes the interesting morphologies and life cycles of predatory bacteria, including the tiny spirillum Bdellovibrio and the fruiting myxobacteria. Bdellovibrio attacks gram-negative bacteria by penetrating the periplasm and forming a spherical bdelloplast (Figures 14.41 and 14.42). The tiny parasite (~0.3 m in diameter) then feeds on the cytoplasmic contents of the host. Within 2.5–4 hours, the host cell is lysed, releasing the progeny Bdellovibrio cells (Figure 14.42). Myxobacteria (e.g., Myxococcus and Stigmatella) are rodshaped, swarming bacteria that aggregate to form a complex mass of cells called fruiting bodies (Figures 14.43–14.48). Gliding myxobacteria secrete lytic enzymes that kill other bacteria, thus releasing nutrients for the predatory cells. In addition to 16S rRNA phylogeny, myxobacteria are classified based on morphological characteristics of the vegetative cells, fruiting bodies, and myxospores (Figure 14.45). Discuss the complex and coordinated life cycle of Myxococcus xanthus, the model organism in this group (Figures 14.44 and 14.46).

14.20–14.24 | Morphological Diversity of Bacteria These sections describe bacteria with unusual morphological or other phenotypic features, beginning with the spirochetes and spirilla (Section 14.20). Spirochetes are tightly coiled gram-negative bacteria, and their morphology is so unusual that it can actually be used to .

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predict phylogeny in prokaryotes (Figures 14.49 and 14.50). Spirochetes are motile by means of unique endoflagella (also called axial filaments). These paired structures are similar to bipolar flagella, but they lie along the length of the cell in the periplasm, which is itself encased in an outer sheath (Figure 14.50). Rotation of the endoflagella places torsion on the cell that allows for a twisting form of motility. Spirilla are often confused with spirochetes, but they have several morphological differences. Spirilla have a thicker and more rigid cell structure than spirochetes (Figure 14.51), and they lack endoflagella, an outer sheath, and twisting motility. Spirochetes constitute a major lineage of Bacteria, with eight genera being described (Table 14.3). Point out that habitats for spirochetes include aquatic environments and inside animal hosts (e.g., Cristispira; Figure 14.52). Several genera of spirochetes contain human pathogens. For example, Treponema pallidum is the causative agent of syphilis, and Borrelia burgdorferi causes Lyme disease (Figure 14.53; see Sections 29.12 and 30.4, respectively).

The corkscrewlike motility of spirochetes is largely responsible for the invasiveness of these cells in human tissues. Discuss the highlights of other genera within this group as suits your course objectives.

The budding and prosthecate/stalked bacteria (Section 14.21) often exhibit unusual mechanisms of cell division (Figure 14.54) and complex cell cycles (Figure 14.55). They also possess a variety of intricate morphologies (Figures 14.56–14.58 and Table 14.4). Hyphomicrobium, a methylotroph, is a budding bacterium that forms hyphae and differentiates into swarmer cells that disseminate during its cell cycle (Figures 14.55 and 14.56). Caulobacter, a stalked, rosette-forming chemoorganotroph, is the model bacterium used in the study of cellular differentiation (Figures 14.57 and 14.59). Following the loss of motility in a swarmer cell, Caulobacter produces a prosthecate, a cytoplasm-filled stalk that is essentially an extension of the cell itself. Synthesis of the stalk coincides with DNA replication, and a daughter swarmer cell eventually separates from the stalked cell.

Sheathed bacteria (Section 14.22), such as Leptothrix and Sphaerotilus, are defined by the presence of a protein/polysaccharide sheath that surrounds chains of rod-shaped cells, giving them a filamentous appearance when viewed under a light microscope (Figures 14.60 and 14.61). They are found primarily in nutrient-rich, freshwater habitats, especially those with flowing waters. In unfavorable growth conditions, the cells abandon the sheath and become swarmer cells that seek out a new environment more suitable for growth. If suitable conditions are found, the swarmer cell can attach to a surface and reproduce to generate a new filament.

The magnetic bacteria are a fascinating group of primarily Proteobacteria that demonstrates magnetotaxis, movement in line with a magnetic field. Such movement is directed by the presence of magnetosomes, strands of intracellular inclusions of magnetite (Fe3O4), in microaerophilic species, or greigite (Fe3S4), in anaerobes (Figure 14.62). Magnetosome strands orient the cells along the north–south magnetic field in similar fashion to the needle of a compass. One hypothesis to explain magnetosome production is that bacteria use them to align themselves properly for flagellar rotation to propel them downward toward regions of the water column or sediments that are either microoxic or anoxic.

Finally, discuss the facultatively aerobic, bioluminescent genera Vibrio, Aliivibrio, and Photobacterium (Section 14.24). Bioluminescence is a population-dependent, light-generating system catalyzed by luciferase (Figure 14.63a and b). Most bioluminescent species are marine Gammaproteobacteria, and many of them populate the light organs of certain marine animals, which are used for a variety of purposes, including territoriality, attracting a mate, .

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avoiding predation, or attracting prey (Figure 14.63c–f). Discuss the regulation of bioluminescence in V. fischeri, as the research on this organism led to the discovery of a complex bacterial cell-cell communication system known as quorum sensing (see Section 7.9).

Answers to Review Questions 1. Convergent evolution is the occurrence in unrelated organisms of a similar trait that was acquired through independent evolutionary events. This is in contrast to horizontal gene transfer, in which traits are acquired in separate lineages through shared homologous genes.

2. Anoxygenic phototrophs are organisms that do not generate O2 as a product of photosynthesis. Instead of H2O, these early phototrophs likely used H2, ferrous iron, or H2S as the electron donor for photosynthesis. Anoxygenic photosynthesis is present in five bacterial phyla: the Proteobacteria, Chlorobi, Chloroflexi, Firmicutes, and Acidobacteria. Purple sulfur bacteria are anoxygenic phototrophs that use hydrogen sulfide (H2S) as an electron donor for photosynthesis.

3. Cyanobacteria are oxygenic phototrophs and therefore have both type I and type II photosystems. All species are able to fix CO2 by the Calvin cycle, many can fix N2, and most can synthesize their own vitamins. Cells harvest energy from light and fix CO2 during the day. During the night, cells generate energy by fermentation or aerobic respiration of carbon storage products such as glycogen. While CO2 is the predominant source of carbon for most species, some cyanobacteria can assimilate simple organic compounds such as glucose and acetate if light is present, a process called photoheterotrophy.

4. Chromatiaceae, store S0 granules inside their cells (in the periplasmic space) and have vesicular intracellular photosynthetic membrane systems. Ectothiorhodospiraceae oxidize H2S to S0 that is deposited outside the cell and have lamellar intracellular photosynthetic membrane systems. Isolation of purple nonsulfur bacteria is done using a mineral salts medium supplemented with an organic acid as carbon source.

5. Purple nonsulfur bacteria exhibit remarkable metabolic versatility. They are able to grow photoheterotrophically using a wide range of organic carbon sources or photoautotrophically using H2, Fe2+, or low levels of sulfide as an electron donor. Most species can also grow chemotrophically by aerobic or anaerobic respiration or fermentation. The aerobic anoxygenic phototrophs are obligate aerobic heterotrophs that produce bacteriochlorophyll a in the dark and use daylight to supplement their growth via photophosphorylation.

6. Chlorosomes are found in the Chlorobi, Chloroflexi, and phototrophic Acidobacteria.

7. Green nonsulfur bacteria contain chlorosomes and bacteriochlorophylls a and c like green sulfur bacteria. However, like the purple bacteria, they have a type II reaction center.

8. The five genera of Helicobacteria are Heliobacterium, Heliophilum, Heliorestis, Heliomonas, and Heliobacillus. They form rod-shaped or filamentous cells, although Heliophilum is unusual because its cells form into bundles that are motile as a unit. They produce highly resistant endospores containing elevated levels of calcium and dipicolinic acid.

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9. Sulfate-reducing bacteria that are complete oxidizers are able to oxidize acetate completely to CO2 and, thus, can grow using acetate as a carbon and energy source. Sulfatereducing bacteria that are incomplete oxidizers are unable to oxidize acetate, and therefore acetate is a common end product of carbon catabolism in these organisms.

10. Both sulfate- and sulfur-reducing bacteria carry out anaerobic respiration in which H2S is a final product, but they differ in their terminal electron acceptor (SO42− versus S0, respectively). In addition, nearly all sulfate-reducing bacteria are strictly anaerobic, but several sulfur-reducing bacteria are facultative aerobes.i 11. Carboxysomes contain high levels of Calvin cycle enzymes that increase the rate at which obligate chemolithotrophs fix CO2. Mixotrophs can obtain energy from the oxidation of inorganic sulfur compounds but lack enzymes of the Calvin cycle. They thus require organic compounds as carbon sources. Beggiatoa is an example of a mixotroph.

12. In Azotobacter chroococcum, two “alternative nitrogenases” are formed as back up nitrogenases when Mo limitation prevents the synthesis of normal MoFe nitrogenase. These are less efficient than the MoFe nitrogenase and contain either vanadium (V) or Fe in place of Mo. The three different types of nitrogenase (MoFe, VFe, and FeFe) are encoded by paralogous genes as a result of gene duplication events.

13. Nitrifying bacteria are aerobic chemolithotrophs that are subdivided into two groups: ammonia oxidizers, which oxidize ammonia (NH3) to nitrite (NO2−) (e.g., Nitrosomonas), and nitrite oxidizers, which oxidize NO2− to nitrate (NO3–) (e.g., Nitrobacter and Nitrospira). With the exception of the denitrifying sulfur chemolithotrophs (described in Section 14.11), most denitrifying bacteria are chemoorganotrophs that respire NO3– or NO2− to gaseous forms of nitrogen, including NO, N2O, and N2. Most denitrifying bacteria are facultative aerobes that will grow aerobically if O2 is present.

14. Dissimilative iron-reducers possess outer membrane cytochromes that facilitate electron transfer with insoluble minerals. They use either iron oxides or manganese oxides as electron acceptors apart from nitrate, fumarate, inorganic sulfur compounds, cobalt, chromium, uranium, selenium, arsenic, and humic compounds.

Electron donors are typically organic compounds such as fatty acids, alcohols, sugars, and in certain cases, even aromatic compounds. They are obligate anaerobes with a few being facultative anaerobic. Many species are also able to use H2 as an electron donor, but they are generally unable to grow autotrophically, requiring a source of organic carbon to support growth. The family Geobacteraceae contains four genera of dissimilative iron-reducing bacteria: Geobacter, Desulfuromonas, Desulfuromusa, and Pelobacter.

15. Gallionella is common in the freshwaters draining bogs, iron springs, and other habitats where ferrous iron is present. Geobacter inhabits anoxic freshwater and marine sediments, and is found both in shallow aquifers as well as in deep subsurface environments.

16. The neutrophilic, aerobic iron-oxidizing bacteria are the least diverse, with all described species belonging to the Proteobacteria. This lack of diversity is likely because of the paucity of environments that meet the physiological requirements of these bacteria. At neutral pH, Fe2+ spontaneously oxidizes in the presence of O2, and therefore these organisms must position themselves in microenvironments where anoxic waters containing reduced iron become exposed to O2. .

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17. Aerobic hydrogen-oxidizing bacteria tend to be microaerophilic because they contain hydrogenases, which are oxygen-sensitive enzymes.

18. Type I methanotrophs are Gammaproteobacteria that employ the ribulose monophosphate cycle for C1 assimilation. Type II methanotrophs are Alphaproteobacteria that use the serine pathway for C1 assimilation.

19. Acetic acid bacteria and acetogens both produce acetate as a metabolic product, but they do so using distinct mechanisms. Acetic acid bacteria, such as Acetobacter and Gluconobacter, are gram-negative, motile, obligate aerobes that oxidize alcohols and sugars to acetic acid. By contrast, the acetogens, such as Acetobacterium and certain species of Clostridium, are obligate anaerobes that use the reductive acetyl-CoA pathway to assimilate C1 compounds for biosynthesis. Many can also grow chemoorganotrophically on simple organic compounds that are oxidized to acetate and CO2.

20. The life cycle of Myxococcus involves fruiting body formation from stalks that produce myxospores that germinate into vegetative cells. The life cycle of Bdellovibrio is predatory upon other bacteria, attacking and penetrating the cell wall and reproducing in the periplasmic space of its gram-negative host.

21. Spirochetes use endoflagella to generate a corkscrewlike motility that allows the cells to infiltrate viscous materials or animal tissues. Spirilla produce conventional flagella that allow for typical swimming motility.

22. Both Caulobacter and Hyphomicrobium form cellular extensions and attach to surfaces via a holdfast. However, while Caulobacter undergoes an unequal binary fission to produce swarmer cells from the opposite end of the elongated stalk, Hyphomicrobium reproduces by budding from the terminus of its extended hypha.

23. Both proteins and polysaccharides, but not peptidoglycan, are found in the sheath of Leptothrix. In addition, iron oxides (from Fe2+ oxidation) commonly encrust the sheath of these bacteria.

24. Magnetosomes orient bacteria along the north–south magnetic field of Earth, making flagellar swimming motility more directional and likely allowing the cells to more easily locate microoxic or anoxic zones in the water column or sediments.

25. Bioluminescence is catalyzed by the enzyme luciferase. Its expression is controlled by a quorum-sensing mechanism that employs the use of the inducer molecule acyl homoserine lactone (AHL), which is constitutively expressed at low levels. If a high cell density is attained in a population, the activator protein LuxR binds AHL, which is now present in high concentration. This results in expression of the luxCDABE genes, and the cells become luminous.

Answers to Application Questions 1. Although Vibrio fischeri is a facultative aerobe, it is bioluminescent only when O2 is present. Luminescence in bacteria requires the genes luxCDABE and is catalyzed by the enzyme luciferase, which uses O2, a long-chain aliphatic aldehyde (RCHO) such as tetradecanal, and reduced flavin mononucleotide (FMNH2) as substrates.

2. Organism .

Metabolism

Aerobe/Anaerobe

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Thiobacillus

sulfur-oxidizing chemolithotroph

Aerobe

Nitrosomonas

ammonia oxidizer

Aerobe

Ralstonia eutropha

hydrogen (H2) oxidizer

Aerobe (microaerophilic)

Methylomonas

methanotroph

Aerobe

Pseudomonas

versatile chemotroph

Aerobe

Acetobacter

acetate-producing heterotroph

Aerobe

Gallionella

.

2+

ferrous iron (Fe ) oxidizer

Aerobe

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CHAPTER

15

Diversity of Bacteria

Summary In Chapter 14, the authors covered the functional diversity of Bacteria by describing the ecological role of the various physiologically and morphologically distinct groups. Chapter 15 focuses on the Bacteria in terms of their phylogenetic diversity (see Figure 15.1) and includes discussions of the Proteobacteria, Firmicutes, Bacteroidetes, Chlamydia, earlybranching hyperthermophilic Bacteria (e.g., Aquifex and Thermodesulfobacterium), and other evolutionarily distinct groups.

15.1–15.5 | Proteobacteria Present a brief overview of the key features of the different classes and representative species of Proteobacteria as suitable for your course content. Figure 15.2 provides a nice introductory visual to help orient your students to the diverse phylogeny of this phylum.

The Alphaproteobacteria (Section 15.1) are the second largest class of the phylum Proteobacteria, with nearly one thousand species described. The majority of genera fall within the orders shown in Figure 15.3 and Table 15.1, and many species are aerobes or facultative aerobes. The order Rhizobiales includes many genera that show significant metabolic diversity, such as phototrophy, chemolithotrophy, chemoorganotrophy, nitrogen fixation, and symbiotic and pathogenic associations. Symbiotic species of the Rhizobiales include the various root nodule bacteria that carry out N2-fixation in association with leguminous plants (Figure 15.4).

Given time, discuss the interesting properties of common genera in this group, including Rhizobium, the plant pathogen Agrobacterium, methanol-catabolizing Methylobacterium, the human pathogen Bartonella, and the abundant Pelagibacter. The order Rickettsiales consists of obligate intracellular parasitic/mutualistic bacteria. Several human diseases, such as typhus and spotted fever rickettsiosis (Rocky Mountain spotted fever), are caused by Rickettsia species that are transmitted through arthropod vectors (Figure 15.5). Species of Wolbachia are parasites that infect the eggs of insects (Figure 15.6). Discuss the metabolic deficiencies of these organisms and the mechanisms by which they obtain key metabolites from their hosts.

Major orders of the Betaproteobacteria (Section 15.2) are shown in Figure 15.7. The broad functional diversity of many members of this group was explored in Chapter 14. Similar to the Alphaproteobacteria described above, the Betaproteobacteria exhibit diverse metabolisms, and this is exemplified by species of the order Burkholderiales. Burkholderia cepacia is an important opportunistic pathogen that is a cause of nosocomial respiratory infections in immunocompromised patients (Figure 15.8). Point out the key genera and major metabolisms of other orders of this class of bacteria, some of which are human pathogens (Figure 15.9). .

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Species of Neisseria are especially notable because of the diseases they cause, including meningitis (see Section 29.5) and gonorrhea (see Sections 27.3 and 29.12), and because they are among the few gram-negative cocci known.

The Gammaproteobacteria (Section 15.3) represents the largest and most diverse class of Proteobacteria (Figure 15.10), and it is this class that contains the Enterobacteriales (the enteric bacteria), one of most important groups of bacteria in terms of human health. The enteric bacteria are a phylogenetically homogeneous group that can be differentiated by their morphology and, especially, their physiology (Figure 15.11). Discuss fermentation patterns in enteric bacteria (Figure 15.12). Because of their genetic similarity, identification employs biochemical diagnostic tests and differential media to separate them into two broad groups (Figures 15.12–15.14). Several strains of Escherichia coli are pathogenic under certain conditions, whereas species of Salmonella and Shigella are typically pathogenic, causing diseases such as typhoid fever and bacillary dysentery, respectively.

Other Gammaproteobacteria include the Pseudomonadales and Vibrionales (Section 15.4). Pseudomonas and pseudomonads include many gram-negative, nonfermentative aerobic rods (Figure 15.15), some species of which are strong denitrifiers. Table 15.2 presents distinguishing characteristics of representative species belonging to this diverse group. While several pseudomonad species are capable of causing serious disease under the proper conditions, they are free-living bacteria that do not require a host for propagation. They are therefore considered opportunistic pathogens. Complicating treatment of pseudomonad infections is the fact that many of these species carry an R plasmid or are capable of using transmembrane efflux pumps to remove antimicrobial drugs from the cell. They are also well-known for breaking down a variety of organic compounds for growth, and this may even include antimicrobial substances that would inhibit or kill other bacteria. As a result, many pseudomonads are naturally resistant to many antibiotics. Also for this section, discuss the gram-negative, facultatively aerobic genera Vibrio, Aliivibrio, and Photobacterium, some of which are waterborne pathogens (e.g., V. cholerae). Several species of this group can carry out bioluminescence, a population-dependent light-generating system catalyzed by luciferase (Section 14.24).

Section 15.5 presents the final two major classes of Proteobacteria, the Deltaproteobacteria and Epsilonproteobacteria. These classes contain fewer species and are less functionally diverse than previously discussed classes of Protebacteria (Figure 15.16). The Deltaproteobacteria contain many ecologically important species, such as a wide diversity of sulfatereducing bacteria (e.g., Desulfovibrionales), iron-reducing bacteria (e.g., Geobacter), and the tiny predatory bacteria Bdellovibrio and Myxococcus (Section 14.19). The Epsilonproteobacteria contain important human pathogens, including species of Campylobacter and Helicobacter, as well as nonpathogenic bacteria typically found in aquatic habitats (Table 15.3).

Many of the latter take part in sulfur cycling, but a large number of environmental Epsilonproteobacteria are known only from DNA sequencing efforts and have not been cultured.

15.6–15.12 | Firmicutes, Tenericutes, and Actinobacteria Major orders of the phyla presented in Sections 15.6–15.12 are shown in the phylogenetic tree in Figure 15.17. Present a brief overview of the key features of the highlighted genera and their representative organisms as suitable for your course content. .

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Section 15.6 addresses the Lactobacillales, an order that includes the genera Streptococcus and Lactobacillus. These nonsporulating Firmicutes are lactic acid bacteria. The lactic acid bacteria lack porphyrins and cytochromes and obtain energy solely by fermentative metabolism. Although lactic acid bacteria are unable to respire O2, they are not inhibited by its presence, and therefore these organisms are considered aerotolerant anaerobes. Species of Lactobacillus are generally homofermentative (produce lactic acid as the sole fermentation product), although some are heterofermentative. They are rarely pathogenic and are used in the production of yogurt, pickles, and sauerkraut (Figure 15.18). Species of Streptococcus and their relatives (e.g., Enterococcus, Lactococcus, and Leuconostoc; Figure 15.19) play important and varied roles in human life and are classified according to the presence of specific carbohydrate antigens. Some streptococci are serious pathogens, causing diseases such as dental caries, pneumococcal meningitis, and scarlet fever, whereas others are used for the production of buttermilk, silage, and other fermentation products.

Species of Listeria, Staphylococcus, and Sarcina are nonsporulating members of the orders Bacillales and Clostridiales (Section 15.7). The genus Listeria contains microaerophilic species that produce the enzyme catalase. A well-known representative is Listeria monocytogenes, a psychrotolerant pathogen that causes the potentially serious foodborne illness listeriosis. Species of Staphylococcus (Figure 15.20) are facultative aerobes that employ a respiratory metabolism when O2 is available but ferment organic compounds in its absence. Unlike Streptococcus, to which they are morphologically similar, staphylococci are catalase-positive. Staphylococci, which are commonly found on the surface of the skin, are salt-tolerant and desiccation-resistant. The genus Sarcina contains acid-tolerant obligate anaerobes that are often present and capable of growth in the human stomach, where they sometimes cause ulcerative infections. Species of this genus are catalase-negative and divide along planes that typically produce square-shaped packets of eight or more cells (Figure 15.21).

Section 15.8 summarizes characteristics of key genera of the endospore-forming Firmicutes: Bacillus, Clostridium, and Sporosarcina (Table 15.4). Note that they are distinguished based on cell morphology, endospore position (Table 15.5 and Figure 15.22), O2 relationship, and energy metabolism. Discuss representatives of these genera as applicable to your course objectives.

You may find it useful to briefly review endospore structure and function with your students (see Section 2.16). Key points you may wish to discuss concerning endospore-forming Firmicutes include the following: • Both aerobic and anaerobic representatives have been described. Aerobic genera include Bacillus (Table 15.5 and Figure 15.23), Paenibacillus, and Sporosarcina (Figure 15.24).

Most other endospore-forming Firmicutes are anaerobic, including Clostridium (Table 15.6 and Figure 15.22), Desulfotomaculum (a sulfate-reducing bacterium; Section 14.9), and the heliobacteria (an anoxygenic phototroph; Section 14.8).

• Species of the alkaline-tolerant Sporosarcina represent the only known coccus-shaped cells capable of forming endospores (Figure 15.24).

• Species of the well-studied genus Bacillus grow via aerobic respiration or are facultatively aerobic. By contrast, the clostridia, another well-studied group, are strict anaerobes that ferment primarily carbohydrates (saccharolytic) or amino acids (proteolytic) and conserve energy solely by substrate-level phosphorylation.

• Some important human pathogens are endospore-formers, including Bacillus anthracis, the causative agent of anthrax, and several species of clostridia: Clostridium botulinum .

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(produces an extremely potent neurotoxin that causes botulism), C. tetani (causes tetanus through the production of the potent toxin tetanospasmin), C. perfringens (causes gas gangrene and food poisoning), and C. difficile (causes life-threatening diarrhea, especially in immunocompromised patients).

Section 15.9 discusses Tenericutes, often informally called the Mycoplasmas (after the genus Mycoplasma). The important note for your students is that the key genera in this grouping, Mycoplasma and Spiroplasma, lack cell walls and are some of the smallest organisms capable of a free-living existence (Table 15.7 and Figures 15.25–15.27). The Tenericutes may have lost the genetic capacity to synthesize cell walls due to their highly specialized and stable habitats in or on plant and animal hosts. Support for this hypothesis lies in the observation that mycoplasmas have some of the smallest bacterial genomes, suggesting that a significant degree of genome reduction has occurred in these organisms. Note that because they lack cell walls, many species require sterols to strengthen their cell membranes (Table 15.7), and several species are human pathogens.

Sections 15.10 and 15.11 introduce the phylum Actinobacteria, including the coryneform bacteria (Corynebacterium and Arthrobacter) and propionic acid bacteria (Propionibacterium) in Section 15.10 and the genus Mycobacterium in Section 15.11. With the exception of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, and Corynebacterium diphtheriae, the causative agent of diphtheria, most of the species in these genera are harmless soil saprophytes. The coryneform bacteria are rod-shaped aerobes that can sometimes be distinguished microscopically by their V-shaped cell arrangements that result from snapping division (Figures 15.28 and 15.29). In addition, species of Arthrobacter can undergo unusual developmental cycles in which cell morphologies can alternate between rods and cocci (Figure 15.30). The propionic acid bacteria are anaerobes that ferment lactic acid, sugars, and some alcohols to propionic and acetic acids and CO2, and this fermentation is essential to the production of Swiss (Emmentaler) cheese.

Species of Mycobacterium (Section 15.11) are often identified in the laboratory using acid-fast staining, a technique that uses basic fuchsin dye to bind mycolic acids, waxy lipids found only in the cell walls of mycobacteria (Figure 15.31). The hydrophobicity of mycolic acids likely gives colonies of Mycobacterium their characteristic wrinkled appearance (Figure 15.32). Cells of virulent strains of Mycobacterium exhibit cordlike growth due to the presence of a glycolipid called cord factor (Figures 15.32b and 15.33).

Filamentous Actinobacteria include the genera Streptomyces, Actinomyces, and Nocardia (Section 15.12). Their defining characteristic is the generation of a branching network of filaments called a mycelium during growth (Figure 15.34). Most actinomycetes form spores, although the mechanism of spore formation and the morphologies of spore-forming structures vary according to subgroups (note that the reproductive spores of filamentous Actinobacteria are distinct from the heat-resistant endospores of the clostridia and bacilli, which are for survival and are not reproductive). The focus in this section is Streptomyces (Figures 15.35– 15.39), primarily because many of the most effective natural antibiotics used clinically have come from species within this genus (e.g., tetracycline, erythromycin, neomycin; Table 15.8 and see Sections 27.11–27.14).

15.13–15.14 | Bacteroidetes .

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The phylum Bacteroidetes contains four orders of bacteria (Sphingobacteriales, Cytophagales, Flavobacteriales, and Bacteroidales; Figure 15.40) and more than 700 described species. Metabolic diversity is extensive in this group of gram-negative rods, with species ranging from obligate and/or facultative aerobes to obligate anaerobes. The Bacteroidales (Section 15.13) are primarily obligate anaerobes, and species of Bacteroides are the numerically dominant bacteria of the human large intestine (measurements indicate that human feces contains more than 1010 cells per gram). Some species of Bacteroides are pathogenic and are the most important inhabitants of anaerobic infections. Unlike most bacteria but similar to eukaryotes, species of Bacteroides produce sphingolipids, which contain the esterifying amino alcohol sphingosine in place of glycerol (Figure 15.41).

Members of the Cytophagales (Section 15.14) are nearly all obligately aerobic chemoorganotrophs, most of which can digest cellulose (Figure 15.42). They are found in soils, water, and hot springs and other thermal environments. Some species of Cytophaga are fish pathogens that can be a considerable problem for the aquaculture industry. The Flavobacteriales are phylogenetically united, but metabolically and ecologically diverse. Species of Flavobacterium are obligate aerobes that inhabit primarily aquatic environments. Except for Flavobacterium meningosepticum, which causes meningitis in infants, they are rarely pathogenic.

Several flavobacteria are cold-adapted and show either psychrotolerant or psychrophilic growth (e.g., Polaribacter and Psychroflexus). The order Sphingobacteriales includes species of Flexibacter, which have rather complex nutritional requirements and do not degrade cellulose. They are generally saprophytic and are not pathogens. Rhodothermus and Salinibacter are close relatives of Flexibacter. Rhodothermus is an aerobic thermophile that produces heat-stable polysaccharolytic enzymes of commercial interest. Salinibacter is perhaps the most extremely halophilic of all prokaryotes, even rivaling the salt requirements of extremely halophilic Archaea. Like Halobacterium, an extremely halophilic archaeon, Salinibacter accumulates K+ as a compatible solute and uses primarily amino acids as a carbon and energy source.

15.15–15.17 | Chlamydiae, Planctomycetes, and Verrucomicrobia

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The three phyla considered in these sections (Chlamydiae, Planctomycetes, and Verrucomicrobia) are fairly closely related and appear to stem from a common ancestor (Figure 15.43).

The genera in the phylum Chlamydiae (Chlamydia, Chlamydophila, and Parachlamydia; Section 15.15) are all obligate intracellular parasites with some of the simplest metabolic capabilities of all known Bacteria (Figure 15.44). Point out to the students that these bacteria are the causative agents of a number of diseases. For example, psittacosis, caused by Chlamydophila psittaci, is an epidemic bird disease that can be transmitted to humans (Figure 15.45). Trachoma, a debilitating eye disease that is the leading cause of blindness from an infectious agent in humans, is caused by Chlamydia trachomatis, some strains of which can also infect the genitourinary tract. Chlamydial infections are among the most prevalent of sexually transmitted infections (see Section 29.13). Figure 15.44a shows the 48 h infection cycle of Chlamydia, which employs two different cell types: reticulate bodies are the multiplying, vegetative cell form, and elementary bodies are the infectious form.

The Planctomycetes (Section 15.16) consist of stalked, budding bacteria that are morphologically (Figures 15.46 and 15.47) and phylogenetically unique. These bacteria are commonly found associated with animals, soil, or food. They are facultatively aerobic chemoorganotrophs that are capable of growth via fermentation or respiration of sugars.

Interestingly, the Planctomycetes lack peptidoglycan and instead have an S-layer cell wall (see Figure 2.31); thus, they are immune to penicillins and other antibiotics that target peptidoglycan synthesis. The Planctomycetes are unique from all prokaryotes in that they have extensive intracellular compartmentalization, such as a nucleoid surrounded by a true nuclear membrane in Gemmata (Figure 15.46), the anammoxosome in Brocadia, in which ammonia is oxidized to N2 anaerobically, and the pirellulosome, a nonunit (phospholipid monolayer) membrane-bound compartment containing the nucleoid, ribosomes, and other cytoplasmic components.

The Verrucomicrobia (Section 15.17) carry out aerobic or fermentative metabolism and are distinguished by the multiple prosthecae produced by individual cells, which divide by binary fission (Figure 15.48). Species of Prosthecobacter contain two genes with significant sequence homology to genes that encode tubulin in eukaryotes. Although tubulin polymerizes to form microtubules in eukaryotic cells, the function of the tubulin gene homologues in Prosthecobacter is unknown.

15.18–15.19 | Hyperthermophilic Bacteria Point out to students the deep branching position of the hyperthermophilic Bacteria (Sections 15.18 and 15.19) in the phylogenetic tree (Figure 15.1). Discuss the major genera in each phylum. Thermotoga (Figure 15.49a) and Thermodesulfobacterium (Figure 15.50) are both anaerobic chemoorganotrophs, the former having a growth optimum of 80°C (maximum ~90°C) and the latter an optimum of 70°C. Thus, Thermodesulfobacterium is not, by definition, a true hyperthermophile; however, it is the most thermophilic sulfate-reducing bacterium known. It also possesses unusual membrane lipids that are ether-linked, as in Archaea, but contain unique C17 hydrocarbons and fatty acids rather than phytanyl side chains like Archaea (Figure 15.50b).

Aquifex (Figure 15.49b) and Thermocrinus (Figure 15.51) are hyperthermophilic H2-oxidizing chemolithotrophs that grow optimally at 85C and 80C, respectively. Certain .

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species can also use reduced sulfur compounds (e.g., S2O32– and S0) as electron donors, and autotrophic growth occurs via the reverse citric acid cycle. Stress to students that the early branching position on the phylogenetic tree and the ability to use H2 as an electron donor supports the hypothesis that hydrogen was a key electron donor on early Earth (see Figure 12.5) and supports the subsurface origin hypothesis (see Figure 12.3).

15.20–15.21 | Other Bacteria Phylum Deinococcus contains only a few genera, and the best studied, Deinococcus and Thermus, are discussed in Section 15.20. Point out to students that the latter genus contains the bacterium Thermus aquaticus, from which Taq DNA polymerase (used in various PCR techniques, Section 11.3) was obtained. Both genera are chemoorganotrophs, growing aerobically and catabolizing primarily sugars and amino and organic acids. Of the four species in the genus Deinococcus, the best studied is the radiation-resistant D. radiodurans. Although these cells are gram-positive, the cell wall contains an outer membrane similar to that of gram-negative bacteria with the exception that lipid A is absent (Figure 15.52). Discuss the radiation resistance of D. radiodurans, noting that it is also highly resistant to many other strong mutagenic agents. This is presumably due to a highly efficient DNA repair system and the presence of several different DNA repair enzymes.

Members of the phylum Acidobacteria are metabolically diverse chemoorganotrophs common in virtually all soils but especially those that are acidic. Some genera, Geothrix and Holophaga, are strict anaerobes capable of ferric iron (Fe3+) respiration or the degradation of methylated aromatic compounds, respectively. Chloracidobacterium is a phototrophic member of this phylum that inhabits alkaline hot springs (see Figure 14.21). Although it is a chlorosome-containing anoxygenic phototroph, the bacterium grows aerobically in close association with cyanobacteria.

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Nitrospira and Deferribacter each form their own phylum, although there is little known about them. Nitrospira (see Figure 14.34) oxidizes nitrite (NO2–) to nitrate (NO3–) and grows autotrophically, but these organisms are phylogenetically distinct from classical nitrifying Proteobacteria. Although species of Nitrospira lack the internal membranes of nitrifying Proteobacteria (see Figure 14.33), environmental surveys indicate that the former are present in significantly greater numbers in natural systems, and thus Nitrospira is likely a key contributor to nitrogen cycling in these environments. The genus Deferribacter contains obligate anaerobes that are unusually efficient and versatile at carrying out anaerobic respiration; certain species are capable of using a wide variety of electron acceptors, including NO3−, fumarate, Fe3+, and Mn4+. A related genus, Geovibrio, is also capable of reducing elemental sulfur to sulfide.

The phyla Synergistetes, Fusobacteria, and Fibrobacteres contain few representatives, but species described thus far are fermentative and inhabit the gastrointestinal tracts of animals and other anoxic environments. Some species of Synergistetes and Fusobacteria are pathogenic to humans, causing abscesses and oral infections such as periodontal disease. The Fibrobacteres have largely eluded laboratory culture but are readily detected using molecular surveys of 16S rRNA genes from animal digestive tracts. Whereas the Synergistetes and Fusobacteria readily ferment amino acids, species of Fibrobacter specialize in carbohydrate fermentation, including the polysaccharide cellulose.

Answers to Review Questions 1. More than 90% of characterized species are classified within one of the following phyla: Proteobacteria, Actinobacteria, Bacteroidetes, or Firmicutes, 2. Some Neisseria are free-living saprophytes that reside in the oral cavity and other moist areas on the animal body. Others are pathogenic, such as N. meningitides and N. gonorrhoeae, the causative agents of meningitis and gonorrhoea respectively.

3. The Enterobacteriales, commonly called enteric bacteria, comprise a relatively homogeneous phylogenetic group within the Gammaproteobacteria and consist of facultatively aerobic, gram-negative, nonsporulating rods that are either nonmotile or motile by peritrichous flagella. The oxidase test and the catalase test are common assays used to characterize bacteria and these tests can be used to discriminate enteric bacteria from many other Gammaproteobacteria. Enteric bacteria are oxidase-negative and catalase-positive.

4. The catalase test uses hydrogen peroxide (3%) to test for the presence of the enzyme catalase in cells. Catalase decomposes H2O2 to H2O and O2 by the following reaction: 2 H2O2 → 2 H2O + O2. A positive reaction is evident from the formation of O2 bubbles in cells exposed to H2O2. Obligate aerobes would be expected to contain this enzyme and therefore show a positive reaction (bubbling), whereas obligate anaerobes would not contain this enzyme since O2, which is no less toxic to an anaerobe than H2O2, is a product of the reaction. No bubbling would be expected in strictly anaerobic cells.

5. Examples of mixed acid fermenters are Escherichia, Salmonella, Shigella, Citrobacter, Proteus, and Yersinia, and of butanediol fermenters are Enterobacter, Klebsiella, Erwinia, and Serratia 6. Endospore-forming bacteria are found in the phylum Firmicutes in the genera Sporosarcina, Heliobacterium, Desulfotomaculum, Clostridium, Bacillus, and several others (see Table 15.4). .

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7. Homofermentative bacteria produce a single fermentation product, lactic acid. The heterofermentatives produce other products, like ethanol and CO2 along with lactate.

Homofermentative L. acidophilus produces acidophilus milk; L. delbrueckii produces yogurt, etc. Heterofermentative lactococci reside in the genus Leuconostoc, which produce the flavoring ingredients diacetyl and acetoin; they are also used as starter cultures in dairy fermentations. Some produce glucose or fructose polysaccharide slimes, and others produce polymers used as plasma extenders in blood transfusions.

Staphylococcus is a facultative aerobe; its gram-positive cocci often form the predominant colonies; it grows in clusters and is mostly pigmented. It can also grow fermentatively and produce acid from glucose both aerobically and anaerobically. They are catalase positive, relatively resistant to reduced water potential, and tolerate drying and high salt (NaCl) fairly well. Sarcina is an obligate anaerobe that divides in three perpendicular planes to yield packets of eight or more cells. They are catalase negative, extremely acid-tolerant, being able to ferment sugars and grow in environments at a pH as low as 2.

8. Despite that fact that mycoplasmas lack a cell wall or peptidoglycan, they are resistant to osmotic lysis owing to the presence of sterols and lipoglycans in their cell membranes.

They are pleomorphic and form embedded colonies that have a “fried-egg” like appearance. Coryneform bacteria are gram-positive, aerobic, nonmotile, rodshaped organisms that form irregular-shaped, club-shaped, or V-shaped cell arrangements by the process of snapping division. Mycobacteria are acid-fast and have mycolic acids as unique lipids in their cell wall.

The characteristic earthy odor of soil is caused by the production by streptomycetes of a series of complex metabolites all called geosmin. The production of sphingolipids can be found in a number of genera in the phylum Bacteroidetes including Flectobacillus, Prevotella, Porphyromonas, and Sphingobacterium, which is characterized by the long-chain amino alcohol sphingosine in place of glycerol in the lipid backbone. Cytophaga produces exoenzymes like cellulase, agarase and chitinase. They are fish pathogens that cause columnaris disease (Cytophaga columnaris), and cold-water disease (Cytophaga psychrophila).

9. Chlamydophila psittaci is the causative agent of the disease psittacosis, Chlamydia trachomatis causes trachoma and a variety of other human diseases, and Chlamydophila pneumoniae is the cause of some respiratory syndromes.

Psittacosis, an epidemic disease of birds, can be transmitted to humans causing pneumonia-like symptoms. Trachoma, a debilitating disease of the eye characterized by vascularization and scarring of the cornea, is the leading cause of blindness in humans.

Other strains of C. trachomatis infect the genitourinary tract, and chlamydial infections are currently one of the leading sexually transmitted diseases.

Genomes in chlamydias are approximately 1 Mbp in size and appear to be even more biosynthetically limited than those of the rickettsias, the other group of obligate intracellular parasites known among the Bacteria. The C. trachomatis genome lacks a gene encoding the protein FtsZ, a key protein in septum formation during cell division.

10. All Planctomycetes produce a structure enclosed by a nonunit membrane and called a pirellulosome; this structure contains the nucleoid, ribosomes, and other necessary cyto.

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plasmic components. Some catalyze the anaerobic oxidation of ammonia within the anammoxosome structure. The anammoxosome membrane is composed of unique lipids that form a rather tight seal, protecting cytoplasmic components from toxic intermediates produced during the anaerobic oxidation of ammonia.

11. Thermotoga, Aquifex, and Thermocrinus are all hyperthermophilic bacteria with growth temperature optima at or above 80°C. All are capable of using H2 as an electron donor.

12. Thermotoga are rod-shaped hyperthermophiles that form a sheath-like envelope, stain gram-negatively, and are nonsporulating. They are fermentative anaerobes, catabolizing sugars or starch and producing lactate, acetate, CO2, and H2 as fermentation products.

The organisms can also grow by anaerobic respiration using H2 as an electron donor and ferric iron as an electron acceptor. Thermodesulfobacterium is a strict anaerobe that uses compounds such as lactate, pyruvate, and ethanol as electron donors.

13. Thermocrinis ruber grows in the outflow of certain hot springs in Yellowstone National Park where it forms pink “streamers” consisting of a filamentous form of the cells attached to siliceous sinter. It grows optimally at 80–90°C as a chemolithotroph oxidizing H2, S2O32–, or S0 as electron donors, with O2 as an electron acceptor.

14. T. aquaticus has been isolated from geothermal systems and is the source of Taq DNA polymerase. Because it is so heat-stable, Taq polymerase allowed the polymerase chain reaction (PCR) technique for amplifying DNA to be fully automated, an advance that has revolutionized all of biology.

15. In addition to arsenate, many species of Chrysiogenetes use selenate, nitrite, nitrate, thiosulfate, and elemental sulfur in anaerobic respirations as terminal electron acceptors.

16. The genus Fibrobacter contains gram-negative fermentative strict anaerobes. However, unlike most Fusobacteria and Synergistetes, species of Fibrobacter are unable to ferment proteins or amino acids and specialize instead in the fermentation of carbohydrates, including cellulose.

Answers to Application Questions 1. Flavobacteriales are psychrophilic or psychrotolerant and can be particularly abundant in marine waters, including aquatic systems in polar environments. Flavobacterium species are primarily found in aquatic habitats, both freshwater and marine, as well as in foods and food-processing plants. Most species are obligate aerobes, though some species are able to reduce nitrate in an anaerobic respiration. Flavobacteria frequently produce yellow pigments and are generally saccharophilic; most can also degrade starch and proteins. Flavobacteria are rarely pathogenic; however, one species, Flavobacterium meningosepticum, has been implicated in cases of infant meningitis, and several fish pathogens are also known. They are aerobic and facultatively aerobic chemoorganotrophs, gram-negative rods, and are saccharolytic with many species motile by gliding.

2. In terms of their responses to O2, bacteria can be strict aerobes (require O2 for growth), facultative aerobes (grow aerobically when O2 is available or anaerobically in its absence), microaerophiles (have an O2 requirement but grow best at reduced oxygen levels), strict anaerobes (grow only in the absence of O2), or aerotolerant anaerobes .

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(do not require or use O2 for growth but can grow in its presence). Examples from this chapter of each of these classes of organisms include the following:

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Class

Organism

Strict aerobe

Listeria

Facultative aerobe

Staphylococcus

Microaerophile

Helicobacter

Strict anaerobe

Clostridium

Aerotolerant anaerobe

Streptococcus

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CHAPTER

23

Microbial Interactions with Humans

Summary The human microbiome contains a variety of different ecosystems, each with specific sets of physical, chemical, and nutrient conditions selective for particular types of resident or transient microorganisms. Microorganisms that comprise the normal microflora of the human body are introduced in the opening sections of the chapter, followed by a presentation of microbial interactions that are harmful to humans. Recall that Chapter 5 covered aspects of microbial growth and methods of controlling microbial growth—either killing or inhibiting growth of specific microorganisms. Relate this information to the nonspecific physical, anatomical, and biochemical defense mechanisms used by the human body to suppress infection by pathogenic microorganisms.

23.1–23.5 | Normal Human–Microbial Interactions These sections discuss the human microbiome—the collective normal microflora that typically colonize the human body. In your initial overview of human–microbial interactions, clearly differentiate between colonization by normal microflora and conditions that lead to pathogenic infection and disease, which are discussed in later sections of the chapter. Use Table 23.1 to provide students with a general overview of the different organisms routinely found on the following body surfaces: skin, oral cavity, respiratory tract, gastrointestinal tract, and urogenital tract. Highlight the significant points regarding the beneficial interactions of microorganisms with humans discussed in these sections. Note that most microbial colonizations begin at the surface of mucous membranes, which are typically protected by a viscous glycoprotein layer called mucus (Figure 23.1). If bacteria establish contact with epithelial cells, they may breach this barrier and penetrate to submucosal surfaces, initiating an infection (see Section 23.6).

Human skin serves as a microbiome that contains a wide diversity of mostly gram-positive bacteria (Figure 23.2), presumably due to their ability to exist in this relatively dry and sometimes salty habitat (Section 23.2). Point out that skin contains both resident and transient microflora. Microorganisms are associated primarily with the sweat ducts, sebaceous glands, and hair follicles in the skin. The degree of moisture in different areas of the skin largely determines the diversity of the microbiota that inhabit it. Students will most likely be surprised (and perhaps somewhat put off) by the fact that over 200 different genera of Bacteria and

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fungi commonly inhabit the skin, with environmental factors influencing the quantity and types of normal skin flora on an individual.

The oral cavity (Section 23.3) is one of the most complex microbial ecosystems in the body. Remind students of the microenvironment concept introduced in Section 19.3, and discuss the dynamics of the microenvironmental niches found in the oral cavity (Figures 23.3 and 23.4). Note that the oral cavity contains numerous facultative aerobes, as well as other inhabitants that are either aerobic (e.g., species of Neisseria and Moraxella) or obligately anaerobic (e.g., members of the Bacteroidetes and Firmicutes) (Table 23.2). The most abundant genus of Bacteria in the oral cavity is Streptococcus. Members of this genus are lactic acid bacteria that are aerotolerant anaerobes (see Sections 5.16 and 15.6).

The human gastrointestinal tract (Section 23.4) presents another complex system with different regions (the stomach, small intestine, and large intestine), providing distinct conditions for diverse groups of microorganisms (Figure 23.5). Helicobacter pylori is the most commonly found species in the microflora of the stomach. The colonization of the stomach wall at pH 2 by H. pylori may result in infection leading to peptic ulcers (see Section 29.10).

Point out to students that H. pylori is not invasive in the strict clinical sense, but it is found in ~80% of patients with ulcers. Discuss the biochemical and metabolic contributions of intestinal bacteria to human health (Table 23.3 and Figure 23.6), including the provision of essential vitamins (e.g., vitamins B12 and K) that human cells cannot synthesize. In some cases, the normal flora of the gastrointestinal tract may become compromised during antibiotic treatments. See the feature box Explore the Microbial World: Probiotics for a discussion of the consumption of live cultures of beneficial intestinal bacteria (probiotics) that may accelerate recolonization of desirable microflora and prevent disease by opportunistic pathogens.

Summarize the microflora of mucosal tissues, including those of the respiratory and urogenital tracts (Section 23.5). Discuss the anatomy of the respiratory tract (Figure 23.7), noting that the upper respiratory tract is constantly exposed to bacteria, whereas the lower respiratory tract is usually sterile in healthy individuals. Note that although the kidneys and bladder are sterile in healthy individuals, facultatively aerobic gram-negative rods and cocci colonize the urogenital tract by adhering to epithelial cells lining the urethra (see Table 23.1 for a list of the dominant taxa of the urogenital tract). The vagina is a complex environment that is weakly acidic in adult females and contains a large amount of glycogen. This selects for the fermentative bacterium Lactobacillus acidophilus, which maintains the acidic environment by producing lactic acid from glycogen (Figure 23.8). Abrupt changes in the normal microbiota of the vagina, as often occurs from the administration of certain antibiotics, can allow for colonization by various opportunistic pathogens, including the yeast Candida, some streptococci, and E. coli.

23.6–23.10 | Pathogenesis These sections discuss mechanisms by which particular pathogens establish an association with a host, eventually leading to disease. Make students aware of the sequence of events during microbial pathogenesis that cause tissue damage and disease: exposure → adherence → invasion → infection (Figure 23.9). The virulence of a pathogen (Section 23.6) is measured using the LD50 (lethal dose50) scale. Use Figure 23.10 to show students that highly virulent pathogens do not show any significant difference in the number of cells required to .

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kill 100% of the test population and the number of cells required to kill 50% of the test population. Explain how a decrease in virulence, called attenuation, occurs in viral and bacterial strains that are maintained in laboratory culture on artificial growth media and how this phenomenon is often used in the development of vaccines.

Impress upon the students that adherence (Section 23.7) of a pathogen to a particular host cell involves either noncovalent adherence factors, such as capsules and slime layers, or specific receptor-ligand molecular interactions using structures such as fimbriae and/or pili (Table 23.4 and Figures 23.11–23.14; see Section 2.13). Use the example of Streptococcus pneumoniae to explain that capsules also play a key role in protecting bacteria from phagocytic host defense mechanisms (Figure 23.13). Explain to students that while most strains of E. coli are nonpathogenic residents of the gastrointestinal tract, fimbriated, enterotoxic strains cause potentially serious intestinal disorders, including diarrhea (Figure 23.14). The expression of specific fimbrial proteins by enteropathogenic E. coli strains allows attachment to the small intestinal epithelium, and thus these strains are not eliminated in the feces. Table 23.4 describes the major adherence factors for attachment of microbial pathogens to host tissues.

To understand the nature of pathogenesis and disease, students must understand the dynamics of invasion, infection, and virulence (Section 23.8). Whereas some pathogens remain localized at the site of infection (e.g., boils caused by Staphylococcus skin infections; see Section 29.9), others enter the bloodstream and cause bacteremia, which can lead to a systemic bloodborne condition called septicemia. The onset of infection requires the appropriate nutrients and environmental conditions for growth of the invading pathogen. In particular, trace elements, especially iron, are not readily available to the pathogen. As discussed in Chapter 3 (see Section 3.1), iron is a key component of cytochromes and iron-sulfur proteins in electron transport chains, and therefore it is an essential element in living systems. Animal cells employ specific iron-binding proteins called transferrins and lactoferrins to sequester and recycle this important element. However, some pathogens produce iron-binding molecules called siderophores that have an extremely high affinity for iron (see Figure 23.17), enabling them to remove iron from the transferrins and lactoferrins of host cells.

Describe the ecological and metabolic events that lead to the formation of plaque and dental caries to the students (Figures 23.15 and 23.16). This interesting example of ecological succession involves the coordinated activities of numerous microorganisms, including anaerobic species. Stress to the students that plaque formation results in the generation of mixed-population biofilms, as discussed in Section 19.4 (Figure 23.16). Discuss how the presence of the lactic acid bacteria Streptococcus sobrinus and Streptococcus mutans can lead to the formation of dental caries in the presence of sucrose. Students may be interested to learn that dental caries, although nearly universal, does not occur in Tanzanian children, whose diets are essentially devoid of sucrose and whose plaque does not contain S. mutans.

Some organisms rely exclusively on toxicity or on invasiveness to promote virulence.

Discuss the mechanisms of several virulence factors produced by microorganisms (Table 23.5). Most are enzymes, such as collagenase and a variety of proteases and nucleases, which break down important host macromolecules. Note that one of the first host responses to pathogenic invasion is the formation of fibrin clots as an attempt to localize the pathogen. However, several pathogens produce fibrinolytic enzymes that dissolve clots, thus allowing tissue invasion to proceed. By contrast, some pathogens use clotting enzymes to actually induce clot formation as a strategy to elude host defense mechanisms. An example of this is .

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the coagulase produced by pathogenic Staphylococcus aureus. The activity of such enzymes leads to highly localized infections, such as pimples and boils. Many virulence determinants are encoded by Salmonella species in chromosomal regions called pathogenicity islands.

These large segments (>100 kbp) of DNA encode proteins that promote invasion and are flanked by transposable elements and insertion sequences that allow for lateral transfer from one bacterium to another (Figure 23.17; see Section 10.11).

Discuss the various classes and types of toxins produced by pathogenic microorganisms (Sections 23.9 and 23.10), including their pathogenic effects, as outlined below: • Exotoxins (Section 23.9) can be placed into three categories: (1) cytolytic toxins, which cause cell lysis by degrading the integrity of the cell membrane (Figures 23.18 and 23.19); (2) AB toxins, which can inhibit protein synthesis, as is the case for diphtheria toxin (Figure 23.20), or exist as extremely potent neurotoxins, such as the botulism and tetanus toxins (Figures 23.21 and 23.22); and (3) superantigen toxins, which intensely stimulate the immune system, causing severe inflammation and tissue damage (as will be discussed in detail in Section 24.9). Students will be interested to learn that the botulism toxins are the most potent biological toxins known⎯far more potent than any snake venom, for example. One milligram of botulism toxin is enough to kill one million guinea pigs.

Finally, discuss enterotoxins, which are a class of exotoxin that acts only on the small intestine by affecting intestinal permeability and often causing bloody diarrhea, severe dehydration, and electrolyte loss. Discuss the action of cholera enterotoxin (Figure 23.23), noting that cholera toxin is a type of AB toxin, although its mechanism of action is different from the AB exotoxins just discussed. The major properties of exotoxins and endotoxins, discussed below, are shown in Table 23.6.

• Endotoxins (Section 23.10) are cell-bound lipopolysaccharide (LPS) molecules that are part of the outer membrane of gram-negative bacteria and are released in large amounts only after cell lysis. Discuss the properties of endotoxins as shown in Table 23.6. You may wish to revisit Figures 2.28 and 2.29 to remind students of the gram-negative LPS subunits: lipid A (the endotoxic portion of the LPS), the core polysaccharide, and the O-specific polysaccharide. Although large doses of endotoxin can be lethal, they are generally much less toxic than exotoxins. For example, in mice, botulism exotoxin is about 10 million times more potent than endotoxin. Because endotoxins are pyrogenic (feverinducing), a highly sensitive assay has been developed to ensure that pharmaceuticals are completely free of endotoxin. The Limulus amoebocyte lysate (LAL) assay can detect as little as 10 picograms (pg) per ml of LPS in pharmaceutical drugs, intravenous solutions, or clinical samples (Figure 23.24).

23.11–23.12 | Host Factors in Infection and Disease The last two sections of the chapter (Sections 23.11 and 23.12) serve as an introduction to immunology, focusing the discussion on host risk factors that affect the onset of infection or disease. This is a good preface to the remaining chapters in Unit 5, which are devoted to the principles of immunology. Discuss the physical, chemical, and anatomical barriers to infection (Figure 23.25), as well as the concept of tissue specificity as it relates to adherence and

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colonization of pathogens (Table 23.7). Details of these and other tissue specificities in infectious disease are discussed in Chapters 29–32.

Host risk factors for infection are discussed in Section 23.12, and these include age, stress, and diet. Infections are more common in infants and the elderly, and they are often linked to differences in normal flora, anatomy, or immune system efficiency between these age groups and young to middle-aged adults. It is beneficial to inform students that the link between stress and illness is directly related to physiological changes in the stressed individual, some of which adversely affect the immune system. For example, excessive cortisol release in highly stressed individuals suppresses the normal inflammatory immune response and inhibits phagocyte function. Diets low in protein affect the distribution and growth of one’s normal microflora, causing the individual to be more susceptible to opportunistic pathogens. Furthermore, diets high in sucrose promote the formation of dental plaque, which contains lactic acid-producing streptococci and increases one’s propensity for the development of dental caries.

Finally, explain to your students that some individuals are prone to pathogenic infection because of certain factors that may or may not be under their control. Such compromised hosts may experience infections due to smoking, alcohol abuse, sleep deprivation, intravenous drug use, poor nutrition, or from exposure to pathogens while admitted in a health care facility (a nosocomial infection). AIDS patients and those individuals having genetic disorders that negatively affect the immune system are also considered compromised hosts. Regardless of their cause, however, all risk factors increase one’s susceptibility to infectious disease.

Answers to Review Questions 1. The skin surfaces of mammals are readily colonized by many species. The mucous membranes of the oral cavity and gastrointestinal tract acquire microorganisms through feeding and exposure to the mother’s body, which, along with other environmental sources, initiates colonization of the skin, oral cavity, upper respiratory tract, and gastrointestinal tract.

Mammalian hosts are rich in the organic nutrients and growth factors required by bacteria and provide conditions of controlled pH, osmotic pressure, and temperature that are favorable for the growth of microorganisms. The taxa prevalent in the urogenital tract include Klebsiella, Proteus, Neisseria, Lactobacillus, Corynebacterium, Staphylococcus, Candida, Prevotella, Clostridium, Peptostreptococcus, Ureaplasma, Mycoplasma, Mycobacterium, Streptococcus, and Torulopsis.

2. The most common microbial residents of the skin are corynebacteria (Actinobacteria), propionibacteria (Actinobacteria), and staphylococci (Firmicutes). These bacteria were identified using both traditional culture methods and a metagenomic molecular approach using 16S rRNA gene sequence analysis.

3. The crevices and small fissures associated with tooth and gingival surfaces in the oral cavity provide microenvironments that are oxic, microoxic, or anoxic in different areas.

Anaerobic microorganisms, such as certain members of the Firmicutes and Bacteroidetes, are able to subsist in the anoxic crevices of the mouth.

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4. The pH of the stomach is low (about pH 2) and therefore provides a barrier to microbial entry. This low pH inhibits many microorganisms, and the bacterial content of the stomach is fairly low. However, acid-tolerant organisms such as Helicobacter pylori can colonize the stomach wall, potentially causing peptic ulcers. The pH of the small intestine progresses from low to nearly neutral toward the large intestine. Oxygen levels gradually decrease while bacterial numbers increase throughout the small intestine to the large intestine, which is anoxic. The lumen of the large intestine is high in nutrient content and approximately neutral in pH. The large intestine has the highest bacterial concentration of any part of the gastrointestinal tract and contains both facultative aerobes and obligate anaerobes.

5. The vagina of adult females contains high levels of glycogen, which is fermented to lactic acid by Lactobacillus acidophilus. Therefore, the pH of the vagina remains weakly acidic. Juvenile females have a different local microflora primarily because their vaginal tracts do not produce glycogen and therefore have a higher pH than the adult female vagina.

6. Virulence is the ability of a pathogen to cause disease. Virulence is estimated using experiments that determine the LD50 of a pathogen for a particular host. Unlike pathogens having mild virulence, highly virulent pathogens show little difference between the number of cells needed to kill 50% of a test group and the number of cells that kill 100% of the test group. The LD50 is therefore more difficult to determine for highly virulent pathogens (see Figure 23.10).

7. Streptococcus pyogenes—M protein on the cell binds to receptors on respiratory mucosa Neisseria gonorrhoeae—Opa protein on the cell binds to CD66 receptors on epithelium 8. Fusobacterium species appear on dental plague, which is an oral biofilm. These embed in the matrix formed by the streptococci and extend perpendicular to the tooth surface, making an ever-thicker biofilm associating with Borrelia species, gram-positive rods, and gram-negative cocci and Actinomyces. As dental plaque accumulates, the microflora produce high concentrations of organic acids—in particular lactic acid—that cause decalcification of the tooth enamel, resulting in dental caries (tooth decay).

Several genes that direct the invasion process in Salmonella and related gramnegative pathogens are found clustered together on the chromosome as pathogenicity islands. Salmonella pathogenicity island 1 (SPI1) is a collection of virulence genes of Salmonella that encode at least 10 different proteins that promote invasion. Another Salmonella pathogenicity island, SPI2, contains genes that are responsible for causing more systemic disease and resistance to host cell defences.

9. Cytolytic toxins (e.g., -toxin of Clostridium perfringens) work by enzymatically attacking cell constituents, such as membrane phospholipids, causing lysis of host cells. The AB toxins (e.g., diphtheria toxin from Corynebacterium diphtheriae) consist of two covalently bonded subunits, A and B. The B component typically binds to a cell-surface receptor, allowing the transfer of the A subunit across the cell membrane. The A toxin then damages the cell through a variety of mechanisms, including inhibiting protein synthesis or disrupting the function of motor neurons (a neurotoxin).

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10. The typical endotoxin is a lipopolysaccharide found in the outer membrane of the gramnegative bacterial cell wall. Endotoxins cause fever because the lipid A component of the LPS stimulates host cells to release cytokines that act as endogenous pyrogens, which affect the temperature-controlling center of the brain (the hypothalamus). Endotoxins produced by enteric gram-negative bacteria most commonly affect human health.

11. There are many ways in which hosts resist infection, so answers will vary. Some include (1) physical barriers, such as the skin, mucous membranes, and ciliary action in the respiratory tract; (2) chemical barriers, such as fatty and organic acids in sebum on the skin and large variations in pH, as in the digestive tract; (3) proteinaceous antibacterials, such as lysozyme, complement proteins, and defensins; (4) mechanical flushing of the urethra during urination; (5) antimicrobial activity by phagocytic cells of the immune system; and (6) antagonistic competition for resources by nonpathogenic normal microflora.

12. A compromised host is one in whom one or more resistance mechanisms are inactive and in whom the probability of infection is therefore increased. Many hospital patients with non-infectious diseases (for example, cancer and heart disease) acquire microbial infections more readily because they are compromised hosts. Such healthcare-associated infections are called nosocomial infections. Invasive medical procedures such as catheterization, hypodermic injection, spinal puncture, biopsy, and surgery may unintentionally introduce microorganisms into the patient.

Answers to Application Questions 1. Pathogenesis is the process by which microorganisms cause disease. Microbial pathogenesis begins with exposure and adherence of microorganisms to host cells, followed by invasion, infection, and, ultimately, disease. Measles, mumps, and rubella vaccines, and rabies vaccines for animals other than humans, employ attenuated strains of each virus.

Their adherence to host tissues with the help of capsule is common to Bacillus anthracis, Escherichia coli, and Streptococcus pneumonia. Disruption of the capsule would make them vulnerable to be phagocytosed by host immune mechanisms.

The role of the normal microflora in protection against disease and the mechanism(s) by which the immune system learns to tolerate the normal microflora are important insights which help in clinical diagnosis, treatment, and prophylactic measures against any disease.

2. The most important factor in the development of dental caries is a diet high in sucrose (table sugar). A common lactic acid bacterium present in oral plaque, Streptococcus mutans, produces dextran through the activity of the enzyme dextransucrase but only in the presence of sucrose, its substrate. The sticky dextran provides a scaffold for further growth of S. mutans and allows coinfection by S. sobrinus, the organism that along with S. mutans causes dental caries. Without dietary sucrose, the highly cariogenic S. mutans is unable to synthesize the dextran layer needed to keep the bacterial cells attached to the teeth.

3. Endotoxins are lipopolysaccharides derived from the outer membrane of gram-negative bacteria such as Salmonella. Both the lipid and the polysaccharide components of endotoxin are necessary for toxicity.Enterotoxins are exotoxins that affect the small intestine. .

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Bacterial exotoxins such as botulinum and tetanus toxins are some of the most poisonous substances known. Cytotoxins such as hemolysins, and AB toxins such as diphtheria and cholera toxins are potent exotoxins. Cytotoxins, also called cytolytic toxins, are secreted, soluble, extracellular proteins produced by a variety of pathogens. Exotoxins contribute to the virulence of pathogens. Each exotoxin affects a specific host cell function. Exotoxins fall into three categories in terms of mechanisms: the cytolytic toxins, the AB toxins, and the superantigen toxins.

Cytolytic toxins damage the host cytoplasmic membrane, causing cell lysis and death. Because the lytic activity of these toxins is most easily observed in assays that use red blood cells (erythrocytes), the toxins are often called hemolysins. The production of hemolysins is demonstrated in the laboratory by streaking the pathogen on a blood agar plate (a rich medium containing 5% sterile blood). During growth of the colonies, hemolysin is released and lyses the surrounding red blood cells, releasing hemoglobin and creating a clear area, called a zone of hemolysis, around the growing colonies.

Endotoxins specifically cause lysis of amoebocytes. In the Limulus amoebocyte lysate (LAL) assay, Limulus amoebocyte extracts are mixed with the solution to be tested. If endotoxin is present, the amoebocyte extract forms a gel and precipitates, causing a change in turbidity.

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CHAPTER

24

Immunity and Host Defense

Summary In Chapter 23, students were presented with an overview of passive mechanisms of protection from pathogenic infection and disease. Chapter 24 progresses into the subject of immunity, which is the active ability of an organism to resist disease by an infecting pathogen. Following a comparison of the different types of immunity and the prevention of infectious diseases through host defense mechanisms, the final sections of the chapter describe diseases and adverse reactions that can result from mechanisms of the immune system itself.

24.1–24.4 | Immunity Introduce the concept of innate (nonspecific) immunity (Section 24.1), the noninducible, preexisting ability of specific cells (primarily pathogen-engulfing cells called phagocytes) to recognize and destroy foreign particles, including invading cells. Phagocytes recognize pathogens through shared molecular features on their cell surfaces. Adaptive immunity is an acquired ability to identify and destroy specific pathogens by the recognition of a single, unique molecular component called an antigen. Upon their identification, phagocytes present antigens to adaptive response cells called lymphocytes. Lymphocytes then produce specialized proteins that bind to specific pathogens and mark them for destruction.

Present a general overview of the cells and organs of the immune system by first describing bone marrow stem cells, which differentiate based on the influence of cytokines, and the circulation and components of blood and lymph, including leukocytes (white blood cells), plasma, and serum (Table 24.1 and Figures 24.1 and 24.2). Explain that leukocytes include both phagocytes of the innate response and lymphocytes of the adaptive response. Describe the two classes of differentiated myeloid precursor cells active in innate immunity (Figure 24.1), emphasizing their functions and mechanisms of action: (1) Monocytes develop into phagocytic antigen-presenting cells (APCs), either macrophages or dendritic cells; and (2) granulocytes include neutrophils, which are also phagocytic, and mast cells, which contain chemical mediators that can cause allergy symptoms and inflammation upon release. In contrast to monocytes and granulocytes, lymphocytes (B cells and T cells) are active in the adaptive immune response. B cells are specialized APCs that serve as the precursor to plasma cells, which produce antibodies, soluble antigen-binding proteins (see Section 24.4). T cells interact with specific antigens bound to APCs, as discussed in Section 24.3. Note that all cells involved in adaptive and innate immunity originate from bone marrow stem cells.

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Pathogens possess macromolecules on their surfaces called pathogen-associated molecular patterns (PAMPs) that are recognized by preformed receptors called pattern recognition receptors (PRRs) present on phagocytes, such as neutrophils and macrophages (Figure 24.3).

PAMPs are highly conserved and shared among closely related pathogens. Provide a few examples of PAMPs for your students, such as bacterial flagellin, lipoteichoic acids of the gram-positive cell wall, and the lipopolysaccharide (LPS) of gram-negative bacteria (e.g., E. coli, Salmonella spp., and Shigella spp.). The binding of a PRR to a PAMP initiates phagocytosis of the invading pathogen.

Adaptive immunity (Section 24.3) requires exposure to an antigen, which results in a primary immune response that elicits production of newly activated T cell and B cell clones that can confer long-term specific immunity (Figure 24.4). This specificity for a single antigen is conferred by T cell receptors (TCRs) on T cells and immunoglobulins (antibodies) on B cells. Point out that this specific immune response displays memory, meaning that subsequent exposure to the same antigen will elicit a stronger and much more rapid secondary immune response (Figure 24.4). To prevent the destruction of host cells and proteins, the adaptive immune response must also lack the ability to recognize self antigens, a concept referred to as tolerance.

Continue your discussion of adaptive immunity with the mechanistic interactions of T cells and TCRs with antigen presented by APCs (Figure 24.5). Describe the structure and role(s) of major histocompatability complex (MHC) proteins (class I and II) in this interaction. Point out that all nucleated host cells display MHC I proteins, whereas APCs (macrophages, dendritic cells, and B cells) also display MHC II proteins. Introduce the various T cell subsets, and explain their functions: T-cytotoxic (Tc) cells destroy antigen-bearing, infected target cells; and T helper (Th) cells indirectly initiate an immune response by releasing cytokines upon interaction with MHC II (Figure 24.5). An example of the latter condition is the activation of macrophages by Th1 cells (a subset of Th cell), which results in increased phagocytic activity and initiates an inflammatory response (Figure 24.6).

Discuss the role(s) of antibodies in host defense (Section 24.4). Point out that Th2 cells (an additional subset of Th cell) do not directly interact with pathogens but induce antibody production in B cells by the release of cytokines (Figure 24.7). B cells activated by cytokines differentiate into plasma cells that specialize in antibody production. Antibodies occur in five major classes (four that are soluble; Table 24.2) based on primary amino acid sequence: (1) IgM—found in blood and produced in large amounts during a primary immune response, (2) IgG—found in blood and produced in large amounts during a secondary immune response, (3) IgA—found in blood and, especially, in mucous membrane secretions and confers mucosal immunity, (4) IgE—sensitizes mast cells and is active in allergic reactions and parasite immunity, and (5) IgD—surface immunoglobulin on B lymphocytes.

Antibodies binding antigen on the surface of pathogens may have several effects (Figures 24.8 and 24.9): • They may be recognized by Fc receptors on phagocytes, initiating enhanced phagocytosis, a process referred to as opsonization.

• IgM or IgG may also activate complement proteins, which bind pathogens and cause lysis by perforating their cell membranes (primarily in gram-negative bacteria).

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• Complement proteins recruited by IgM or IgG that are attached to pathogens may also directly bind phagocytes at C3 receptors, causing opsonization (primarily in gram-positive bacteria).

• Antibodies may neutralize toxic pathogen products by binding toxins and preventing their ability to destroy host cells.

24.5–24.7 | Host Defense Inflammation (Section 24.5) is a generalized reaction to pathogens or their toxins that is characterized by erythema (redness), edema (swelling), pain, and heat (Figure 24.10). Chemical mediators called cytokines and chemokines control inflammatory reactions and are produced by phagocytes and lymphocytes. Discuss inflammation as a mechanism to rapidly activate local phagocytic cells (macrophages) and recruit others (neutrophils) to the site of infection to isolate and limit tissue damage (Figure 24.11a). If inflammation fails to localize pathogens, the inflammatory reaction can become systemic and potentially lead to life-threatening septic shock (Figure 24.11b). In this case, inflammatory cytokines functioning as endogenous pyrogens produce high fever instead of merely localized heat. In addition, the loss of fluids due to vasodilation and increased vascular permeability causes decreased blood pressure and systemic edema. For these reasons, systemic inflammation is often more life-threatening than the original infection.

Describe to your students how animals develop natural immunity, stressing the fact that natural immunity requires exposure to specific pathogens (Section 24.6). Both the innate and adaptive immune responses are essential for survival (Figure 24.12). For example, genetic defects that inhibit macrophage or neutrophil development are fatal unless individuals having these immune deficiencies are isolated from all environmental exposure. Compare and contrast natural active immunity with natural passive immunity (Table 24.3 and Figure 24.13).

Examples of natural passive immunity are the transfer of maternal IgG to the fetus across the placenta and IgA to infants through breast milk. Note that genetic problems that inhibit the natural active immune response, such as agammaglobulinemia and severe combined immune deficiency syndrome (SCID), are serious deficiencies that are often fatal. Other diseases that inhibit natural active immunity include DiGeorge syndrome, which prevents T cell maturation due to an underdeveloped thymus, and AIDS, which almost completely depletes Th cells over a period of several years (Figure 24.14).

Students will probably be interested in the topic of immunization, or artificial immunity.

Compare and contrast artificial active immunity, which is conferred by a vaccination that elicits a primary immune response and involves immune memory, from artificial passive immunity, which is conferred by an injection containing preformed antibodies (called antiserum) that disappear from the body over time. Vaccines employed in immunizations contain either killed or attenuated (weakened) pathogens or inactivated (chemically modified) forms of pathogen exotoxins called toxoids. To obtain a high enough antibody titer (quantity) to prevent disease, additional “booster” immunizations are often necessary to elicit a secondary immune response and increase the total antibody count.

Students may not be aware of the dramatic effect vaccines have had on reducing epidemic childhood diseases (e.g., measles, mumps, and rubella; Table 24.4 and Figure 24.15). .

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Infant mortality in developed countries is much lower than during the pre-vaccination era.

Moreover, herd immunity is achieved when a significant percentage of a population is resistant to a particular disease, thereby minimizing both the spread of infection and the risk of an epidemic. Point out that most children in the world are essentially still living in a “pre-vaccination era,” and the incidence of disease and infant mortality remains high in some regions.

Newer immunization strategies (Section 24.7) have emerged over the past several years that employ synthetic peptides and genetically engineered agents. Explain to students that conjugate vaccines are those in which antigenic molecules, such as a small portion of a toxin or a foreign polysaccharide, are fused to larger carrier proteins to generate an immune response without causing disease (Figure 24.16). Recombinant-vector vaccines use vaccinia virus as a cloning vector for the expression of antigenic genes from virtually any virus to induce an immune response. Recombinant-antigen vaccines are similar, but in this case, virus antigenic proteins are expressed in a microbial host (e.g., yeast cells), harvested, and used as the immunogen (see the feature box Explore the Microbial World: Vaccines and Public Health). DNA vaccines are those in which the antigenic gene of interest is cloned into a bacterial plasmid, which is injected into the host and taken up by host cells. Expression of the antigenic gene produces immunogenic proteins that trigger an immune response. Point out the important fact that the use of bioengineered molecules eliminates exposure to the pathogen and/or native antigen itself.

24.8–24.9 | Immune Diseases Discuss the various classes of hypersensitivity (Table 24.5), pointing out the differences between immediate hypersensitivity (type I, allergy) and delayed hypersensitivity (type IV) reactions. Note that type I hypersensitivities are characterized by the release of vasoactive products from the degranulation of mast cells presenting IgE within a few minutes of exposure to antigen (Figure 24.17). Chemical mediators released by mast cells, such as histamine and serotonin, cause symptoms of anaphylaxis, including vasodilation (and the corresponding drop in blood pressure), breathing distress, sneezing, itchy and/or watery eyes, and hives (Figure 24.18). If not treated quickly, such symptoms can lead to anaphylactic shock and death. Treatments include the administration of antihistamines (in mild cases) or steroids (in serious cases), and desensitization techniques in which increasing doses of the allergen are given to gradually switch antibody production from IgE to IgG and IgA. Delayed-type hypersensitivities, on the other hand, appear several hours following secondary exposure to the antigen, and peak by 24–48 hours. Examples students will be most familiar with involve contact dermatitis, such as skin reactions to poison ivy (Figure 24.19), latex, jewelry, and other chemicals and materials. These materials bind to skin and create new antigens that are presented to the immune system. The localized inflammation response to antigens of Mycobacterium tuberculosis by patients that had previously been exposed to the bacterium is a classic example of a delayed hypersensitivity reaction, and it is the basis for the tuberculin test (Figure 24.6).

Autoimmune diseases are typically the result of autoantibodies reacting against selfantigens, thus causing the destruction of host tissues. Discuss the various types of autoimmune diseases in humans (Table 24.6). In particular, describe systemic lupus erythematosis .

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(SLE) as a classic example of type III hypersensitivity that causes the formation of insoluble immune complexes that can lead to organ destruction. Note that if the autoimmune disease is organ-specific, it may be treatable by supplying the product of that organ, such as thyroxin for hypothyroidism. However, autoimmune disorders that affect multiple organs are difficult to treat and require general immunosuppression therapy that increases the likelihood of opportunistic infections. Mention the roles of heredity, gender, age, and health status in determining the incidence of specific autoimmune diseases, noting that SLE is about 20 times more likely to occur in women than in men.

Superantigens (Section 24.9) were briefly described in Chapter 23 (Section 23.9) as proteins that activate a large number of immune cells (T cells) that cause systemic inflammation.

Superantigen diseases include scarlet fever, caused by Streptococcus pyogenes, and staphylococcal food poisoning and toxic shock syndrome (Figure 24.20). Discuss the mechanisms by which superantigen reactions occur and why they result in serious host tissue damage. Make sure to point out that superantigens bind to conserved regions (but not variable regions) of MHC and TCR proteins that are positioned outside the normal antigen-binding site (Figure 24.21). Consequently, they are able to nonspecifically interact with large numbers of cells, causing massive activation of T cells and other immune responses (e.g., cytokine release and inflammation) that are similar to the symptoms of septic shock discussed earlier.

Answers to Review Questions 1. All immune cells, blood, and lymph components have a common origin in bone marrow stem cells. Maturation of B cells occurs in the bone marrow in mammals; they are then dispersed throughout the body and reside in the lymph nodes, spleen, or mucosaassociated lymphoid tissue (MALT). T cells mature in the thymus and then are dispersed throughout the body like B cells.

2. Innate immunity is the non-inducible, pre-existing ability to recognize and destroy a pathogen or its products. It does not require previous exposure to a pathogen or its products and is mediated by phagocytes. A pathogen-associated molecular pattern (PAMP) is a repeating structural component of a microorganism or virus recognized by a pattern recognition receptor. An example of a PAMP is the lipopolysaccharide (LPS) on outer membranes of all gram-negative bacteria and the lipoteichoic acids of gram-positive bacteria.

3. The cell-mediated adaptive immune response is initiated following binding of T lymphocytes to antigens presented by MHCs (i.e., infected host cells displaying antigen on MHC I proteins or APCs displaying antigen on MHC II proteins). This adaptive response requires that specific T cell receptors (TCRs) recognize and make contact with MHC I or MHC II proteins that are presenting a corresponding antigen. Antigen recognition by T lymphocytes stimulates the proliferation of T-cytotoxic cells, which secrete perforins and granzymes that kill antigen-bearing infected cells. This response is also indirectly mediated by two functionally distinct T-helper cell subsets: Th1 cells, which release cytokines to initiate the inflammatory response, and Th2 cells, which secrete cytokines to stimulate antigen-reactive B cells to differentiate into plasma cells and produce antibodies.

4. Antibody-mediated immunity usually involves both T cells and B cells. The T cell receptors have specificity for a particular antigen, and upon contact with that antigen, the T .

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cell undergoes rapid growth to produce clones of those cells. T-helper cells (specifically, the Th2 subset) stimulate B cells to produce antibodies (immunoglobulins) specific for the stimulating antigen.

5. Inflammation is initiated by the release of cytokines and chemokines from damaged cells and leukocytes, especially phagocytes and Th cells. Inflammation activates phagocytic macrophages, and their production and release of cytokines and chemokines recruits phagocytic neutrophils to the site of infection.

6.

Active immunity

Passive immunity

Exposure to antigen; immunity achieved by injecting antigen or through infection Specific immune response made by individual achieving immunity Immunity activated by antigen; immune memory in effect Immunity can be maintained via stimulation of memory cells (i.e., booster immunization) Immunity develops over a period of weeks

No exposure to antigen; immunity achieved by injecting antibodies or antigen-reactive T cells Specific immune response made by the donor of antibodies or T cells No immune system activation; no immune memory Immunity cannot be maintained and decays rapidly Immunity develops immediately

7. The antigen or antigen mixture used to induce artificial active immunity is known as a vaccine or an immunogen. Immunization with a vaccine is designed to produce artificial active immunity. A modified exotoxin that is modified chemically so it retains its antigenicity but is no longer toxic is called a toxoid. A toxoid vaccine induces long-term protective immunity against the exotoxin.

In active immunity, introduction of antigen induces changes in the host: The immune system produces antibodies in a primary immune response and produces a large number of immune memory cells. A second (“booster”) dose of the same antigen results in a faster response, yielding much higher levels of antibodies and immune T cells due to this memory, or secondary, immune response. Active immunity may remain throughout life as a result of immune memory.

Immunization with live cells or viruses is usually more effective than immunization with dead or inactivated material. It is often possible to isolate a mutant strain of a pathogen that has lost its virulence but still retains the immunizing antigens; strains of this type are called attenuated strains. Attenuated vaccines tend to provide long-lasting T cell–mediated immunity, as well as vigorous antibody immunity and a strong secondary response upon reimmunization or infection with the targeted pathogen.

8. When the whole protein to be used as a vaccine or antigen is toxic, a synthetic peptide corresponding to the antigenic part of the toxin may be produced and used as a synthetic conjugate vaccine. This strategy has great promise for creating vaccines to a number of .

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pathogens, but the entire sequence of the disease-causing protein must be known and a protective antigen recognized by lymphocytes must be identified before an effective vaccine can be engineered.

In a DNA vaccine a single pathogen gene is cloned into the plasmid and injected, so there is no chance of an infection as there might be with an attenuated vaccine. Genes for individual antigens such as tumor-specific antigens can be cloned, targeting the immune response to a particular cell component. A single bioengineered plasmid encoding an antigen can be used to infect host cells and elicit a complete immune response, inducing both immune T cells and antibodies.

Conjugate vaccines couple extracted bacterial polysaccharide to a protein toxoid, provoking a more robust immune response with better immune memory than injection of the polysaccharide antigen alone. Polysaccharide antigens are not processed efficiently

by B cells and generally provide only a primary response with little immune memory, but the conjugated protein toxoids are processed and activate Th2 cells, resulting in a primary response followed by a strong secondary response and immune memory.

9. Immediate hypersensitivity (type I) produces an immediate response to antigen exposure that is mediated by antibodies. Although usually relatively mild (e.g., allergies), some reactions can become severe so quickly that death can result from anaphylactic shock. The primary chemical mediators are histamines and serotonin released from mast cells, the latter of which have IgE receptors on their surfaces that are stimulated upon inter-action with antigen (e.g., insect venoms, pollen, certain foods, and animal dander). Delayed-type hypersensitivity is cell mediated and involves the activities of Th1 inflammatory cells. Symptoms begin to appear several hours after secondary exposure to antigen and maximum response is within 24–48 hours in most cases. The release of cyto-kines from Th1 cells attracts large numbers of macrophages to the site, and a localized inflammatory response is also observed. Examples of delayed hypersensitivity include poison ivy, mumps, and the tuberculin test.

10. Whereas conventional antigens bind to an antigen-specific site on T cell receptors (TCRs), superantigens bind to all TCRs that have common conformational structure outside of the antigen-binding site, resulting in activation of more than the normal number of T cells. Because superantigens bind outside the variable region of the TCR, 5%–25% of all T cells become activated, in contrast to less than 0.01% activation by conventional antigens.

Answers to Application Questions 1. The significance of innate immunity can be inferred from its ancient evolutionary history. For example, there are structural and evolutionary homologs of PRRs in vertebrates found in distant phylogenetic groups, such as insects. However, the nonspecific nature of innate immunity is probably no more important than the adaptive immune response to pathogens, as the inability to trigger the adaptive response is invariably fatal unless excessive measures are taken to avoid contact with the environment (Figure 24.12). In other words, in a normal environment, both innate and adaptive immunity are essential for survival.

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2. Phagocytes and lymphocytes trigger the inflammatory response upon infection. Specifically, phagocytes and Th1 cells release cytokines and chemokines (e.g., interleukins and tumor necrosis factor ) that activate macrophages and recruit other phagocytic cells, such as neutrophils, to the site of infection. The purpose of the inflammatory response is to isolate infection and rapidly dispatch invading cells. After pathogen numbers decrease, cytokine production slows, chemoattraction of phagocytes ceases, and inflammation subsides.

3. Leukocytes are nucleated white blood cells found in the blood and the lymph and participate in innate and adaptive immunity. Myeloid cells, active in innate immunity, are derived from myeloid precursor cells that can be divided into two lineages, the monocytes and the granulocytes.

The monocyte develops into specialized phagocytic cells, the antigen-presenting cells (APCs). These cells, in addition to the B cells, engulf, process, and present antigens to lymphocytes. APCs include macrophages and dendritic cells. Immature cells called monocytes are circulating precursors of macrophages and dendritic cells. Macrophages are generally the first defense cells that interact with a pathogen. They are abundant in many tissues, especially the spleen, lymph nodes, and MALT. Dendritic cells are phagocytes that specialize in presenting antigen to lymphocytes. Granulocytes are the second lineage of cells derived from myeloid precursors. Granulocytes contain cytoplasmic inclusions, or granules, that can be visualized by staining. These granules contain toxins or enzymes that are released to kill target cells. The phagocytic activity of one granulocyte, the neutrophil, also called a polymorphonuclear leukocyte (PMN), is central to innate immunity. Release of granules, a process called degranulation, from a granulocyte called a mast cell can cause allergy symptoms and inflammation. Lymphocytes (B cells and T cells) are specialized leukocytes involved exclusively in the adaptive immune response.

Type of immune response generated:

Pathogenic strains of Salmonella

Tetanus antiserum Attenuated virus

Tetanus toxoid

Phagocytes such as macrophages and neutrophils are the first line of defense against pathogens Artificial passive immunity Provide long-lasting T cell–mediated immunity, vigorous antibody immunity and a strong secondary response upon reimmunization or infection with the targeted pathogen Artificial active immunity

4. Superantigen reactions are not desirable for the host, as they override the controlled, specific, and measured amplification of adaptive immune system cells that normally results from exposure to a specific antigen. Consequently, superantigens benefit the pathogen because they promote invasion and colonization by the pathogen, and the production of superantigens by a variety of different pathogens is evidence for the evolutionary success of organisms that produce them.

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CHAPTER

25

Immune Mechanisms

Summary Chapter 25 expands upon the fundamental concepts of immunity and host defense discussed in the previous chapter by presenting the cellular and molecular mechanisms necessary for effective immunity. Overviews of innate and adaptive immune responses are provided, followed by a detailed discussion of immunogens, antigen presentation, and T lymphocyte classification and function. The chapter concludes with a consideration of antibody production and the role of antibodies and complement in destroying pathogens.

25.1–25.2 | Basic Immune Mechanisms The chapter begins with an overview of immunity that expands on the topics of innate and adaptive response mechanisms (Sections 25.1 and 25.2). Describe the main weapon of the innate immune system, phagocytic cells, whose job is to engulf pathogens and, if necessary, interact with cells that activate the adaptive immune response. Emphasize the functions and mechanisms of action of the phagocytes involved in nonspecific immunity: macrophages and dendritic cells (both of which are antigen-presenting cells, APCs), monocytes, and neutrophils (also known as polymorphonuclear leukocytes, or PMNs; Figures 25.1 and 25.2). Note that both macrophages and dendritic cells are derived from monocytes (refer to Figure 24.1). Discuss the important features of phagocytes, and be sure to address the details of the phagocytepathogen recognition system, including pattern recognition receptors (PRRs; also called toll-like receptors, TLRs; Figure 25.3) and their pathogen-associated molecular patterns (PAMPs, such as peptidoglycan; Figure 25.3). Use Figure 25.4 to discuss the production of toxic oxygen species by phagocytes for destroying invading pathogens. Remind students of earlier discussions concerning reactions that generate toxic oxygen radicals (Section 5.16).

Explain that the generation of reactive oxygen compounds does not kill the phagocyte itself, because the toxic species are sequestered in organelles called phagolysosomes (Figure 25.4).

An additional component of the innate immune response is inflammation caused by the release of cytokines and chemokines, the beneficial ramifications of which are localization of tissue damage and destruction of the invading pathogen. However, point out that some bacteria can evade destruction by phagocytes using a variety of mechanisms (e.g., carotenoids, cell wall glycolipids, leukocidins, and capsules).

Now focus on adaptive or specific immune response mechanisms (Section 25.2). Review the major cellular components of adaptive immunity, including antigen-presenting cells, T-helper cells with their T-cell receptors (TCRs), and B cells, which interact with Th2 cells

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to produce antibodies. These features of adaptive immunity are presented in more detail in Chapter 26 (Molecular Immunology). Compare and contrast cell-mediated immunity, which is dependent upon antigen presentation to T cells, and antibody-mediated immunity, which is dependent upon the action of antibodies. Finally, discuss the three important components of adaptive immunity: (1) specificity, (2) memory, and (3) tolerance (Figure 25.5). The major point to stress here is that the adaptive immune response must develop the ability to recognize self and to discriminate against foreign antigens.

25.3–25.4 | Antigens and Antigen Presentation These sections provide additional information regarding antigens and immunogens (Section 25.3) and antigen presentation to T cells (Section 25.4). Differentiate between antigens and immunogens, stressing the fact that most, but not all, antigens are also immunogenic (i.e., elicit an immune response). For example, because low-molecular-weight compounds called haptens bind antibodies, they are antigens; however, they do not induce an immune response and are therefore not immunogens. Discuss the intrinsic (size, form, complexity) and extrinsic (dose, route, foreign nature) features of immunogens that are responsible for immunogenicity (Table 25.1). Figure 25.6 shows the interaction of antibodies with antigens at specific antigenic and conformational determinants called epitopes. While antibodies recognize and bind epitopes in native conformations, TCRs bind epitopes that have been processed (partially denatured) by antigen-presenting cells (APCs) or target cells.

Section 25.4 introduces the topic of how T cells recognize and respond to encounters with activated APCs. Describe the basic structure of a T-cell receptor (Figure 25.7). Remind students of the importance of three-dimensional protein structure (tertiary and quaternary structure, Section 4.14) to the binding of substrates when describing the Vα and Vβ domains of TCRs and other molecular interactions involved in antigen presentation to T lymphocytes.

Differentiate the structure and mechanism of antigen presentation of class I major histocompatability complex proteins (MHCs), which are found on the surface of all nucleated cells, from class II MHCs, which are found only on APCs (macrophages, dendritic cells, and B cells; Figures 25.8 and 25.9). Students may be interested to know that over 200 genetic variations of MHCs (called polymorphisms; see Section 26.4) exist in humans, and this diversity results in structural and antigenic differences that often prevent successful tissue transplantation. Also, explain the function of the CD4 and CD8 coreceptors, noting that Th cells express the former, whereas Tc cells express the latter (Figure 25.9).

25.5–25.6 | T Lymphocytes and Immunity A critical component of the immune response is the lymphocyte group referred to as effector T cells. Discuss T-cytotoxic (Tc) cells and natural killer (NK) cells presented in Section 25.5.

Describe how CD8 Tc cells recognize and destroy cells presenting antigen on class I MHCs by the production and release of perforins, which are pore-forming proteins, and granzymes, which induce apoptosis (Figure 25.10). NK cells are valuable cytotoxic lymphocytes that are distinct from T lymphocytes but resemble Tc cells in that they kill compromised host cells.

Because they lack the MHC I proteins of healthy cells and instead display stress proteins, .

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NK cells are able to identify pathogen-infected or tumor cells and destroy them by releasing perforins and granzymes (Figure 25.11).

An especially important component of the immune response is T-helper (Th) cells (Section 25.6). Four subsets of Th cells have been identified (Table 25.2). Th1 and Th2 are essential activators of cell-mediated and antibody-mediated immune responses. Th1 inflammatory cells activate macrophages for increased efficiency of phagocytosis (Figure 25.12). Th2 cells stimulate antibody production by inducing B cell differentiation into plasma cells (Figure 25.13). Th17 cells are undifferentiated (naive) Th cells that function to amplify the innate immune response by releasing IL-17, a cytokine that recruits neutrophils to sites of infection (Figure 25.14a). Treg cells function to inhibit inflammation and prevent immune responses to self antigens, and therefore they are important suppressors of autoimmunity (Figure 25.14b).

25.7–25.9 | Antibodies and Immunity Discuss the structure of immunoglobulins (antibodies; Section 25.7) in some detail using IgG as the model (Figure 25.15). Emphasize to students the meaning of heavy versus light chains, which refers to the number of amino acids each chain contains. The properties of the five classes of immunoglobulins are shown in Table 25.3. The relative serum levels of each type are shown, with IgG comprising ~80% of the total. Note also that IgG is bivalent because it contains two antigen-binding sites that bind two identical epitopes. Additional key points of immunoglobulins include the following (Table 25.3): • The antigen-binding site of all antibodies involves the variable regions of the heavy and the light chains (Figures 25.16−25.19). The strength of this interaction is called the binding affinity.

• IgM is the first antibody produced in response to bacterial infection. Although most IgM antibodies have low binding affinity, they have high avidity, a measure by which the ten binding sites increase overall binding strength (Figure 25.18).

• IgA is present in most body fluids and in all mucosal surfaces of the body. Serum IgA is present as a monomer, whereas secretory IgA occurs as a dimer that is covalently linked by a J chain peptide (Figure 25.19).

• IgE and IgD are present in trace amounts in serum. Whereas IgE is also the major antibody on the surface of mast cells, IgD is found on B cells; however, the function of IgD is unknown.

Lead into your discussion of antibody production (Section 25.8) by asking students, “How is an individual able to produce antibodies to over one billion antigens with a genome containing only about 25,000 total genes?” The genetics of antibody diversity is a complex concept for most students unless they have a thorough understanding of DNA replication and genetic recombination. Introduce to students the idea of gene rearrangements (called somatic recombination) and somatic hypermutation as the major mechanisms for generating antibody diversity in B cells (Figure 25.20). Note that gene rearrangements, but not hypermutation, are responsible for TCR diversity in T cells. .

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Outline the major events of antibody production and immune memory following exposure to antigen, as discussed in the text (Figure 25.21). Point out that activated B cells can live for years as memory cells, and following re-exposure to an antigen, the memory cells can rapidly generate high titers of antibodies without the aid of T cells. During this secondary antibody response, the IgM antibodies produced during the primary response give way to mostly IgG antibodies, a phenomenon called class switching. Summarize the concept of immune memory for students by explaining that occasional reimmunization (i.e., a “booster shot”) can provide long-term protection against certain infectious diseases by maintaining a high antibody titer and sufficient numbers of memory B cells.

Details of the complement system are discussed in Section 25.9). This should provide a logical progression for students considering that this series of proteins is often activated by interactions with antigen-antibody complexes (Figure 25.22a). Discuss the classical complement pathway as a cascade of sequential protein interactions that eventually forms a membrane attack complex (MAC) that creates pores in the membrane of the pathogenic cell (Figures 25.22 and 25.23). Contrast the classical pathway with the mannose-binding lectin (MBL) and alternative pathways of complement activation. Point out that regardless of the specific complement pathway stimulated, the activity of complement proteins in host defense results in direct lysis of invading pathogenic cells and/or enhances innate immunity by marking pathogens for destruction by phagocytes, a process called opsonization (Figures 25.22 and 25.24).

Answers to Review Questions 1. Staphylococcus aureus produces pigmented compounds called carotenoids that neutralize singlet oxygen and prevent killing. Intracellular pathogens such as Mycobacterium tuberculosis (the cause of tuberculosis) grow and persist within phagocytic cells by using its cell wall glycolipids to absorb hydroxyl radicals and superoxide anions, the most lethal toxic oxygen species produced by phagocytes.

2. Phagocytes are activated by agents such as complement proteins and cytokines. Following contact with the phagocyte, the microorganism is engulfed and internalized in a phagocytic vacuole that fuses with a lysosome, forming a phagolysosome. Enzymes generate toxic oxygen compounds within the vesicle that destroy the microorganism.

These substances include peroxides, superoxide anions, hydroxyl radicals, and singlet oxygen species.

3. The defining characteristics of the antigen-specific adaptive immune response are specificity, memory, and tolerance.

4. Immunogens are molecules that induce immune responses. Antibodies are made against a variety of immunogenic substances, including proteins, lipoproteins, polysaccharides, and other substances. The intrinsic properties for effective immunogenicity of a substance are (1) a sufficiently high molecular weight (most immunogens have a molecular weight of more than 10,000); (2) sufficient complexity (nonrepeating polymers); and (3) an appropriate physical form (insoluble or aggregated complex macromolecules).

5. A linked set of genes called the major histocompatibility complex (MHC) found in all vertebrates encodes a group of proteins important in antigen presentation. The MHC proteins in humans, called human leukocyte antigens or HLAs, are the major antigens re.

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sponsible for immune-mediated organ transplant rejection. MHC proteins function primarily as antigen-presenting molecules, binding pathogen-derived peptides and displaying these peptides for interaction with TCRs. The two classes of MHC proteins are MHC class I proteins, found on the surfaces of all nucleated cells, and MHC class II proteins, found only on the surface of B lymphocytes, macrophages, and dendritic cells, all of which are APCs. 6. T-cytotoxic (Tc) cells recognize and aid in destroying cells that display foreign antigens on their surface. Tc cells require contact with infected target cells expressing class I MHC through their specific TCRs. Natural killer (NK) cells, on the other hand, do not require antigen stimulation to destroy compromised target cells, and they will not generate immunological memory. NK cells are activated by the absence or reduced expression of class I MHC proteins and the presence of stress proteins, properties that are characteristic of virus-infected or tumor cells.

7. Mycobacterium tuberculosis, Mycobacterium leprae, and Listeria monocytogenes.

Dendritic cells do not produce IL-6 to promote Th17 differentiation without interacting with the pathogen. The absence of IL-6 pushes differentiation to Treg cells that make IL10 and TGF-β, two cytokines that suppress immunity and inflammation. In the presence of self antigens and the absence of IL-6, Treg cells shut down the immune response and inhibit inflammation. This is important for controlling immune responses to self antigen and preventing autoimmunity.

8. The structural and functional differences among the five major antibody classes are described in Table 25.3 and shown in Figures 25.15–25.19.

9. The memory B cells generated by the initial exposure to antigen may live for years. A reexposure to the same antigen immediately stimulates memory B cells to differentiate to plasma cells and produce antibody; memory B cells need no T cell help. The second and each subsequent exposure to antigen causes the antibody titer to rise rapidly to a level often 10–100 times greater than the titer following the first exposure. This rise in antibody titer is the secondary antibody response. It illustrates immune memory, which is more rapid and more abundant in antibody response than the primary response. It exhibits class switching from IgM to IgG and IgA. 10. The membrane-bound C5–9 components, called the membrane attack complex (MAC), insert through the membrane to form a pore and allow the contents to leak out, lysing the cell. Both the alternative pathway and the MBL pathway nonspecifically target bacterial invaders and lead to activation of the membrane attack complex and enhanced opsonization via formation of stable C3 convertases.

All pathways make a C3 convertase. Cleavage of C3 to C3b initiates terminal reactions.

Answers to Application Questions 1. Besides the important aspects of specificity and memory, tolerance is important to the immune system and is defined as the acquired inability to make an adaptive immune response directed to self antigens. Because macromolecules in the host are also antigens, the host immune system must avoid recognizing host macromolecules as they could be .

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damaged if they were recognized as antigens by antibodies or T cells. Thus, the adaptive immune response must develop the capacity to discriminate between foreign (nonself and dangerous) antigens and host (self and not dangerous) antigens. In the presence of self antigens and the absence of IL-6, Treg cells shut down the immune response and inhibit inflammation. This is important for controlling immune responses to self antigen and preventing autoimmunity.

Polymorphisms in MHC proteins are the major antigenic barriers for tissue transplantation from one individual to another; tissue transplants not matched for MHC identity are recognized as nonself by the immune system and are rejected.

2. Memory is the ability of the immune system to recognize and respond more rapidly to a previously encountered antigen. Memory is of value because it permits the adaptive immune system to respond more vigorously to subsequent exposure to that same antigen and thus more readily fend off an infectious disease or debilitating illness. In a sense, the innate immune system has built-in memory since evolutionarily conserved PAMPs are readily recognized by equally conserved PRRs expressed on the surface of phagocytes.

3. A hereditary deficiency resulting in the inability to present antigens to Tc cells would prohibit the routine identification and destruction of virus-infected or other antigenpresenting class I MHC-expressing cells (e.g., tumor cells). This deficiency would dramatically reduce both the quality of life and the life span of an individual affected with such a disorder, because humans are constantly exposed to microbial and viral pathogenic agents. A deficiency in class II MHCs would inhibit Th cell function. This condition would be even more serious, because these cells coordinate both the humoral and cellmediated arms of the immune response (e.g., AIDS is an example of a disease that directly affects Th cell function and quantity). A hereditary deficiency that affects antigen presentation by MHCs to all T cells would most certainly be fatal early in life because these individuals would effectively lack an adaptive immune response.

4. Innate immunity is directed against features common to pathogens such as the peptidoglycan of all gram-positive bacteria or the lipopolysaccharide of all gram-negative bacteria. By contrast, adaptive immunity is directed to interactions with particular pathogen specific macromolecules such as the M-protein antigen on a single strain of Streptococcus pyogenes. The specificity of the antigen–antibody or antigen–TCR interaction is dependent on the capacity of the lymphocyte cell receptor to interact with particular antigens. Antibodies provide antigen-specific immunity that protects against extracellular pathogens and dangerous soluble proteins such as toxins. At the cellular level, complex interactions between B cells and T cells produce antibodies that provide effective antigen-specific immunity.

Electrophoresis is a technique that separates proteins and other macromolecules based on their size, charge, and conformation. From the table it is clear that IgG, being the major component of the serum with highest molecular weight amongst other antibodies, will be illustrated by the longest peak in the chromatogram. The shorter peaks in the chromatogram will be in the descending order of IgM, IgA, and others respectively.

5. Derived from myeloid stem cells, neutrophils are found predominantly in the bloodstream and bone marrow, from where they migrate to sites of active infection in tissues.

Monocytes are circulating precursors of macrophages, a major phagocytic cell type. The .

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neutrophil is an actively motile phagocyte that contains lysosomes. Interactions with APCs drive CD4 Th cells to differentiate into several subsets, each producing unique combinations of cytokines that recruit effector cells such as macrophages, antibodyproducing B cells, and neutrophils. Th17 cells are important in the first stages of the adaptive immune response; triggered by antigen interactions, Th17 cells recruit neutrophils, innate immune cells important in inflammation.

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CHAPTER

26

Molecular Immunology

Summary This chapter focuses on the complex molecular interactions between the various classes of immune system proteins and receptors discussed in Chapter 25. These interactions include MHC-antigen binding, immunoglobulin-antigen binding, and the molecular interactions controlling cell signaling and activation of the immune response. The genetics and diversity of antibodies and T-cell receptors is also presented.

26.1–26.2 | Receptors and Immunity Section 26.1 includes a more detailed discussion of pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs), topics that were briefly discussed in Chapter 25. Table 26.1 presents a summary of the receptors (PRRs) and targets (PAMPs) in the innate immune response. Inform your students that PRRs were first identified in the fruit fly Drosophila, where they were referred to as Toll receptors (see the feature box Explore the Microbial World: Drosophila Toll Receptors⎯An Ancient Response to Infections). Since that discovery, Toll-like receptors (TLRs) have been identified in many vertebrate groups, including humans. Note that a number of human TLRs can interact with more than one PAMP, and often this interaction initiates a signal transduction cascade that enhances phagocytosis and activates other effector cells (Figure 26.1).

Students were given a schematic overview of cellular receptors in Chapter 25; these molecules are examined in detail in Section 26.2, beginning with the immunoglobulin gene superfamily. Discuss the structure and evolution of the family of antigen-binding proteins: Igs, TCRs, and MHC proteins. Note their evolutionary relatedness, as evidenced from their common structural and functional domains (Figure 26.2). A key point regarding the genes encoding this family of proteins is that the amino acid sequences of their constant domains are highly homologous, and thus their evolutionary conservation supports their important structural and functional roles in the adaptive immune response. You may wish to review the concept of gene duplication and selection discussed earlier in the course to reinforce how and why these structural motifs have been conserved. This will lead to a logical conclusion regarding the evolutionary divergence of the variable domains, whose amino acid sequences could freely mutate in response to the antigenic selective pressures imposed over time.

Signal transduction in the adaptive immune system is similar to that of the innate immune system in that a cascade of ligand-receptor phosphorylation reactions triggers the expression of immune response genes. However, antigen receptors, including antibodies and TCRs,

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cannot directly interact with kinases because they lack cytoplasmic tyrosines that can be phosphorylated. Therefore, specialized tyrosine-containing adaptor molecules called immune-based tyrosine-activation motifs (ITAMs) interact with these receptors and initiate cytoplasmic signal transduction in B and T lymphocytes (Figure 26.3).

26.3–26.4 | The Major Histocompatibility Complex (MHC) The major histocompatibility complex (MHC) was introduced as an antigen-presenting component in Chapter 24, and some structural details of class I and II MHCs were described in Chapter 25. In humans, the genes encoding MHC proteins are contained in the human leukocyte antigen (HLA) complex. The human MHC region covers about 4 Mbp and is located on chromosome 6 (Figure 26.4). Discuss the structure of MHC proteins based on the computer models shown in Figure 26.5. The degree of detail in which you discuss MHC protein structure will depend upon the chemistry background of the students and/or the amount of time devoted to protein structure in Chapter 4. The most important concept, however, is the roles of class I versus class II MHC proteins in adaptive immunity. Class I proteins are expressed on all nucleated cells and function to present antigens to TCRs on T-cytotoxic cells, whereas class II MHCs are expressed only on APCs (i.e., macrophages, dendritic cells, and B cells) and function to present exogenously derived peptides to TCRs on T-helper cells.

Section 26.4 discusses the roles of polymorphism and polygeny in establishing the extensive diversity of genes that encode peptide-binding MHC class I and class II proteins (Figure 26.6). Use the example of tissue transplant rejection to explain these concepts. The majority of students may not recall the details of population genetics from their introductory biology course, so you may want to refresh their memories on the topic. Although each individual possesses only two alleles for a given trait⎯one from the mother and one from the father—the human population may contain numerous variants of each allele. The example provided in the text concerning the different class I MHC alleles at the HLA-A (2013), HLAB (2605), and HLA-C (1551) loci defines the problem inherent with transplants: the six alleles that a given individual possesses at these three loci represent only one of millions of possible combinations in the human population. A similar situation exists for class II MHC genes (HLA-DR, HLA-DP, and HLA-DQ alpha and beta chains; Figure 26.4), and thus students will understand the problem in finding a suitable match for organ transplants and tissue grafts. Finally, explain the basic mechanism of MHC binding to peptide motifs, pointing out that each MHC binds a different motif based on the position of invariant amino acids called anchor residues. Note that as long as the anchor residues are the same, a single MHC can bind and present many different peptides. This is in contrast to antibody and TCR binding, which is specific for only a single antigen.

26.5–26.7 | Antibodies and T Cell Receptors Section 26.5 presents a detailed discussion of antibody structure and the antigen-binding site of the variable domains. Spend time discussing the structural motifs shown in Figure 26.7, noting that each heavy and light chain has three highly variable complimentarity-determining regions (CDRs) that form the antigen-binding site. Point out that while most genes encode .

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only a single protein, the genes encoding the heavy and light chains of immunoglobulins can generate almost unlimited diversity. Using Figure 26.8, demonstrate the “genes in pieces” mechanism for generating antigenically distinct antibodies within a specific antibody class.

Generation of antibody diversity (Section 26.6) from limited numbers of genes specifying a given antibody molecule is a dynamic example of the power of genetic recombination and gene rearrangement. Table 26.2 summarizes the mechanisms that contribute to the remarkable antigen-binding diversity of antibodies and TCRs. Discuss allelic exclusion, the gene rearrangement mechanism B cells employ during maturation that results in the production of only one antigenically specific antibody. Also point out the contribution of somatic hypermutation to antibody diversity, typically in response to secondary exposure to antigen.

The CDRs shown in Figure 26.7 are the sites of hypermutation events that further increase the number and diversity of antigen-binding sites. Construct a frame of reference for the students on the size and scale of the interaction of antigen with the variable regions of the heavy and light chains of the antibody. For example, a 2  3 nm antigen-binding site is all that is required for the binding of an epitope consisting of 10–15 amino acids.

In a similar manner as above with antibodies, Section 26.7 discusses the structural details of T cell receptors (TCRs) as they relate to the binding of antigens embedded in MHC proteins (Figure 26.9). Point out that TCRs have a dual binding function in which the CDR3 regions of the TCR  and  chains bind the epitope, whereas the CDR1 and CDR2 regions bind to the MHC proteins. Also discuss the mechanisms for generating diversity in TCR variable regions (Figure 26.10). Point out the analogous nature of the TCR  and  chains to the light and heavy chains of immunoglobulins. The same genetic recombination and reassortment mechanisms that drive Ig diversity also function in TCR production. However, somatic hypermutation mechanisms do not contribute to diversity in TCRs as they do in antibody production.

26.8–26.10 | Molecular Switches in Immunity The final sections of the chapter describe three mechanisms essential for proper immune response following antigen recognition: (1) selection of the proper “antigen-responding” clones; (2) generation of second signals that activate T cells; and (3) production of cytokines and chemokines that activate additional immune system cells. The concept of tolerance was introduced in the last chapter in the context of autoimmune diseases, which are characterized by the initiation of the immune response to self antigens. Ask your students to think about how the immune response “learns” to discriminate between nonself antigens and self antigens. Develop the clonal selection hypothesis (Section 26.8) with students, as summarized in Figures 26.11 and 26.12.

The authors emphasize the details of T cell selection and tolerance because B cell activation ultimately depends upon selection and activation of the proper T cell population. T cell selection occurs in the thymus. The following points should be emphasized:

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• Positive selection of developing T cells requires productive interaction with MHC-self peptide complexes, which stimulate cell division; nonproductive interactions result in cessation of development and cell death (apoptosis).

• Negative selection occurs to positively selected T cells that interact very strongly with MHC proteins that are binding self antigens. These T cells die in the thymus, whereas the positively selected T cells that did not interact strongly with self antigens migrate to the lymph nodes and spleen.

• Clonal deletion is the inevitable result for more than 95% of T cell precursors following positive and negative selection (Figure 26.12). B cells also undergo clonal deletion, but this occurs in the bone marrow rather than the thymus. In addition, B cells may also display clonal anergy, a state of unresponsiveness to self antigens in the absence of a second signal from Th cells (see below).

T and B cells require two molecular signals for activation (Section 26.9). Emphasize that at this point, the T cells migrating to the lymph nodes and spleen have not yet been exposed to nonself antigens and are thus categorized as naive. Because the thymus does not contain all possible self antigens, there must be a safety mechanism for inducing anergy in T cells interacting with a self antigen for the first time after leaving the thymus. Describe the first signal (binding of TCR to MHC–foreign peptide complex) and second signal (binding of B7 protein on APC to CD28 protein on TCR) that convert naive T cells to activated effector Tc cells (Figure 26.13). Note that B cells also require two activation signals, although these are different from those that activate T cells. Antigen must first bind and cross-link surface immunoglobulins on the B cell (Figure 26.14). The second signal for B cell activation requires the interaction of antigen-presenting MHC II with the corresponding TCR of a Th cell.

An additional interaction occurs between the CD40 surface protein on the B cell and the CD40L on the Th cell. This causes the Th cell to synthesize and secrete IL-4 and other cytokines, thereby activating the B cell and stimulating antibody production (Figure 26.14).

Molecular signaling among components of the immune system is often accomplished using soluble proteins called cytokines and chemokines (Section 26.10). Cytokines (called lymphokines or interleukins when produced by lymphocytes) stimulate cell growth, differentiation, and clonal proliferation. Chemokines serve as chemoattractants, a group of small proteins that recruit phagocytes and T cells to sites of injury or infection (Table 26.3). The presence of these cells then triggers inflammatory and adaptive immune responses.

Answers to Review Questions 1. C-reactive protein, an extracellular soluble PRR, is an acute phase protein produced by the liver in response to inflammation. C-reactive protein interacts with the phosphocholine macromolecules of gram-positive bacteria cell walls. Both of these PRRs target pathogen surface PAMPs, and both bind complement proteins, leading to lysis or opsonization of the targeted cell. Structural, functional, and evolutionary homologs of the Toll receptors, called Toll-like receptors (TLRs), are widely expressed on mammalian innate immune cells. TLRs are found associated with membranes on the surface or in intracellular vesicles in macrophages, monocytes, dendritic cells, and neutrophils, all of which are phagocytes that have the ability to engulf and destroy pathogens. The NOD-like receptors (NLRs) are a family of PRRs having a nucleotide-binding oligomerization domain .

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(NOD). NLRs are soluble PRRs found in the cytoplasm. NOD1 and NOD2 interact with peptidoglycan components of gram-negative and gram-positive bacterial cell walls, respectively, stimulating production of antimicrobial peptides and inflammatory cytokines.

2. The criteria used to assign a gene and its corresponding protein to the immunoglobulin gene superfamily rely on the evolutionary relatedness of the three types of proteins in this family (TCRs, Igs, and MHC proteins). Each member of this family consists of two nonidentical polypeptides that associate to form a functional protein, and all are expressed on the surface of cells as antigen receptors. Although the specific activity and location of each of these proteins is different, they share structural, functional, and evolutionary features.

3. The class I and class II MHC proteins both consist of two noncovalently linked polypeptides. In class I molecules, a membrane-integrated  chain (MW ~42 kD) is noncovalently associated with a smaller, 2m polypeptide (MW ~12 kD) that is not membrane-bound. In class II molecules, both the  (MW ~33 kD) and  (MW 28 kD) polypeptides are integrated into the membrane, and these proteins are usually found in pairs (see Figure 25.8). The three-dimensional shape of both class I and class II molecules reveals how these proteins interact with antigens and the cells of the immune system.

4. There are at least three loci for MHC class I genes; therefore, a given individual would express six total alleles (yielding six proteins). There is a high degree of polymorphism for these alleles in the human population: HLA-A (2013), HLA-B (2605), and HLA-C (1551). Thus, the total number of MHC class I protein combinations expressed in the population would be ~8.13  109 (8,133,234,615).

5. The Ig chains required to construct a complete antigen-binding site include the variable regions of the heavy and light chains of the Ig. The antigen-binding site measures about 2 × 3 nm and is capable of binding to about 10–15 amino acids of the protein antigen.

CDRs 1, 2, and 3 of the variable region of both the heavy and light chains are involved in these interactions. CDR3 of the heavy chain is composed of V, D, and J genes, whereas CDR3 of the light chain contains V and J genes only.

6. Somatic recombination, random heavy- and light-chain reassortment, and hypermutation all contribute to the vast diversity generated from a relatively small number of Ig genes.

Somatic hypermutation occurs only in the V regions of rearranged heavy- and light-chain genes, creating B cells with mutated Ig receptors. These mutated B cells then compete for available antigen. This process selects B cells with receptors having higher antigen-binding strength than the original B cell receptor. This affinity maturation process is one of the factors responsible for a dramatically stronger secondary immune response. The affinity maturation mechanism adds additional possibilities to the generation of Ig diversity, making the potential antibody repertoire almost limitless.

7. T cell receptors (TCRs) are cell surface antigen receptors integrated into the cytoplasmic membrane of T cells. TCRs bind foreign peptides in the context of MHC proteins. The TCRs accomplish this dual binding function through a binding site composed of the V domains of the TCR α and β chains. The α-chain and β-chain V domains of TCRs contain CDR1, CDR2, and CDR3 segments that bind directly to the MHC–peptide antigen complex. Antibody diversity is expanded even more in B cells by somatic hypermu.

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tation, the mutation of Ig genes at much higher rates than the mutation rates observed in other genes. The somatic hypermutation mechanisms responsible for increased receptor diversity in Ig genes do not operate in T cells and do not generate additional TCR diversity.

8. Positive selection of developing T cells requires productive interaction with MHC self proteins in the thymus, which stimulates cell division; the lack of interaction with MHC results in cessation of development and cell death (apoptosis). Negative selection occurs next; all positively selected T cells that very strongly interact with MHCs binding thymic self antigens remain in the thymus where they are unable to divide and eventually die.

Positively selected T cells that did not interact too strongly with self antigens migrate to the lymph nodes and spleen. The term clonal deletion is used to describe the overall process of positive and negative selection during T cell maturation. B cells also undergo clonal deletion in the bone marrow but additionally undergo selection via clonal anergy (unresponsiveness).

9. Naive T cells require activation by antigen-presenting cells (APCs) before they can function as effector cells. The first signal in activation is the binding of the TCR of the uncommitted T cell to the MHC–peptide complex on the APC. The second signal for activation involves the interaction of B7 protein on the APC with CD28 protein on the T cell, and this results in the conversion of a naive T cell to an effector Tc cell. B cell activation is more complex and requires more protein interactions. The first signal is antigen binding, which cross-links surface immunoglobulins on the B cell. The second signal requires the interaction of CD40 and MHC class II with CD40L and the TCR of a Th cell, respectively, which culminates in the release of cytokines by the Th cell that activate the B cell and stimulate antibody production.

10. The cytokine IL-2, which is secreted by activated Th cells, causes these same cells to divide and make clonal copies. It also causes production of IL-4 and IL-5 by these cells, which stimulates B lymphocyte activity and antibody synthesis. The cytokines IFN-, GM-CSF, and TNF- are all produced by Th1 cells and affect macrophage activation and the inflammatory response. Other cytokines and their functions are shown in Table 26.3.

Answers to Application Questions 1. The thymus is a primary lymphoid organ that provides an environment for the maturation of antigen-reactive T cells. Immature T cells that do not interact with MHC–peptide (positive selection) or that react strongly with self antigens (negative selection) are eliminated by clonal deletion in the thymus. T cells that survive positive and negative selection leave the thymus and can participate in the immune response. B cell reactivity to self antigens is controlled through clonal deletion and anergy. Clonal selection is the production by a B or T cell of copies of itself after antigen interaction. It selects antigen-reactive cells that respond to foreign antigens while ignoring self antigens. Each antigen-reactive B cell or T cell has a cell surface receptor for a single antigen epitope. When stimulated by interaction with that antigen, each cell can replicate, and antigen-stimulated B and T cells grow and differentiate, producing a pool of cells that expresses the same antigen-specific receptors. Individual B cells, specific for a single antigen, proliferate and expand to form .

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a clone after interaction with the specific antigen. The antigen drives selection and then proliferation of the individual antigen-specific B cell. Clonal copies of the original antigen-reactive cell have the same antigen-specific surface antibody. Continued exposure to antigen results in continued expansion of the clone.

Chemokines are a group of small proteins produced by macrophages, lymphocytes, and other cells in response to bacterial products, viruses, and other cell-damaging agents. Chemokines attract phagocytes and T cells to the site of injury, stimulating an inflammatory response and recruiting the cells necessary to initiate a specific immune response.

2. The following functions would be impaired: IL-2: Proliferation (often autocrine) IFN- γ: Activation in macrophages IL-6: Activation in lymphocytes IL-12: Activation, enhances differentiation to Th1 in NK cells, naive T cells TNF-α: Activation, production of proinflammatory cytokines a. Involved in allergic reactions and parasite immunity b. The IL-4 interaction signals an immunoglobulin class switch leading to IgG1 and IgE synthesis; suggests impaired IL-4 production/interaction. c. Chemokine IL-5 not produced.

3. After a B cell is activated, it no longer needs T cell interactions or cytokines to make antibody; antigen interaction alone can then stimulate antibody production. Some activated B cells will be transformed into plasma cells that will secrete large amounts of antibody in the primary immune response. Others remain as memory B cells and play a major role in the secondary immune response during subsequent exposure to an antigen.

4. Activation of all peripheral T cells that contact antigen would be devastating in that these T cells may not undergo clonal anergy (i.e., may not become unresponsive to self-antigen), and thus they would never be able to differentiate self from nonself antigens. Excessive T cell activation and the associated autoimmunity are prevented by clonal deletion via negative selection, described in Review Question 8 above.

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CHAPTER

27

Diagnostic Microbiology

Summary Chapter 27 covers a variety of diagnostic methods and approaches used in microbiology and immunology laboratories to identify pathogens in clinical and natural samples. This information is essential for forming specific and effective patient treatment plans. A record of specific cases of infectious diseases is also crucial for tracking and preventing their movement through a population, and this is the focus of epidemiology, as discussed in Chapter 28. Therefore, Chapter 27 closes Unit 5 on Pathogenicity and Immunology and helps usher in Unit 6—Infectious Diseases and Their Transmission.

27.1–27.2 | The Clinical Environment It is recommended that you do not omit or understate the importance of safety procedures and protocols in the clinical laboratory (Section 27.1). Many of your students may work in health-related fields in their future careers, and their education regarding the responsibility of employing good laboratory practices may save lives in the future (Table 27.1). Discuss the definitions and properties of biological containment as a means to prevent accidental infections and contamination. Note that the biosafety levels classification scheme (BSL-1 through BSL-4) is based upon the containment potential of a given laboratory (Table 27.2), with BSL-4 laboratories handling the most dangerous pathogens (Figure 27.1). As stated in the text, ignorance and carelessness are the two most common causes of laboratory accidents.

Next, define and discuss healthcare-associated (nosocomial) infections (HAIs) with your students (Section 27.2). Ironically, hospitals are notorious places for the spread of potentially serious infectious pathogens because of several important risk factors associated with healthcare (Table 27.3). To compound the problem, many of these pathogens are resistant to certain antibiotics (see Section 27.17). Most microbiology laboratory courses include an exercise with Staphylococcus spp. to demonstrate hemolysis of red blood cells. A reminder of this laboratory exercise could serve as an excellent introduction for a discussion of healthcare-associated pathogens (Table 27.4). Review some of the most common infection sites of HAIs (Figure 27.2), noting that about one-third of the 1.7 million HAIs that occur annually in the U.S. are urinary tract infections. Furthermore, nearly 100,000 deaths occur annually in the U.S. from HAIs, and about two-thirds of these are caused by pneumonia and bloodstream infections.

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27.3–27.5 | Microbiological Identification of Pathogens Section 27.3 presents a current view of what might be termed the classical methods of clinical microbiology—the safe collection and transport of clinical samples and the isolation and identification of the etiologic agent of disease (Figures 27.3 and 27.4). Many important infectious diseases (e.g., tuberculosis and gonorrhea) can be accurately diagnosed through direct microscopic observation of clinical samples (Figure 27.5). However, this method is insufficient for many other infections. Students should already be familiar with enrichment culture techniques employed to isolate specific metabolic groups of microorganisms from the environment (see Sections 1.9 and 18.1). Here, they will learn that this technique is also useful in clinical diagnostic microbiology (Tables 27.5 and Figure 27.6). Discuss the pathogenic microorganisms often associated with bodily fluids of infected individuals (blood, feces, wound exudate, genital fluids, and urine). Point out to students that while the presence of bacteria in blood (called bacteremia) is not common in healthy individuals, blood is an excellent medium for the growth of bacteria. Septicemia is a blood infection that transfers the pathogen to various tissues to initiate new infections; it is often fatal if not treated quickly. Urinary tract infections are common, with the majority of cases occurring in females. Point out a few examples of the most common urinary tract pathogens, noting that E. coli accounts for the majority of the cases (about 90%). Discuss some of the diagnostic tests that have been developed to identify gram-negative pathogens of the urinary tract, including the use of differential and selective media (Figure 27.7). Note that the culture of strictly anaerobic pathogens, such as species of Clostridium, requires the exclusion of O2 from the growth medium. This may be accomplished in several ways—for example, by adding a reducing agent, such as thioglycolate, to the culture medium (Figure 5.27) or by using an anoxic jar or glove bag (Figure 5.28).

Discuss the importance of the growth-dependent diagnostic methods (Section 27.4) used to identify potential pathogens. Emphasize the difference between the various types of media employed in clinical microbiology: enriched, differential, and selective media. These media can be used for diagnostic purposes based on the metabolic properties of isolated bacteria.

The information describing growth-dependent identification methods may be covered in the laboratory portion of many microbiology courses. Consequently, the amount of detail you include concerning these biochemical tests will depend upon whether the information was already covered elsewhere and the experience level of the students (upper versus lower division, allied health, etc.).

Antimicrobial drug susceptibility testing procedures (Section 27.5) may also be included in the laboratory portion of your course. However, you should discuss the minimum inhibitory concentration (MIC) and Kirby-Bauer disc diffusion assay techniques with students, especially if these techniques will not be included in laboratory activities in the course (Figure 27.8). Point out that the size of the zone of inhibition in the disc diffusion assay is determined by (1) the degree of susceptibility of the microorganism, (2) the concentration of the antimicrobial drug, and (3) the solubility and rate of diffusion of the antimicrobial drug through the agar medium. The standards for comparing the efficacy of different antimicrobial agents against various bacterial pathogens are defined by the Food and Drug Administration or the Clinical and Laboratory Standards Institute. The susceptibility of nosocomial pathogens (those associated with healthcare facilities) to antimicrobial agents is routinely moni-

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tored and documented in reports called antibiograms for the purpose of tracking emerging pathogens and antibiotic resistance.

27.6–27.10 | Growth-Independent Diagnostic Methods These sections address culture-independent methods for detection of specific pathogens or toxins. Differentiate between the concepts of specificity and sensitivity when introducing serology to students. Explain that serological tests are used to determine the presence of antigen-specific antibodies in patient serum. Note that passive agglutination tests (see Section 27.7), while quick and easy to perform, are not nearly as sensitive as the more involved enzyme immunoassays (EIAs), which are able to detect antigen quantities as low as 0.1 ng.

Immunoassays also include the measurement of antibody titer (a type of serological test) and skin tests (e.g., the tuberculin test) to diagnose infections and/or to detect or confirm the presence of (or exposure to) an infectious agent (Section 27.6). Define antibody titer for your students as the highest dilution (lowest concentration) of serum that gives a positive antigenantibody reaction (Figure 27.9). It is important to inform students that the presence of a specific antibody does not necessarily indicate active infection but is indicative only of previous infection with, or exposure to, the pathogen.

Agglutination tests (Section 27.7) are characterized by the visible clumping of particulate antigen in the presence of antibodies specific to the antigen, and they are widely used in clinical and diagnostic laboratories. Note that direct agglutination tests generally target an antigenic component of the cell surface and are used for blood type determinations (Figure 27.10). In contrast, passive agglutination tests detect the agglutination of soluble antigens in the presence of antibodies, usually by coupling the antigen to an insoluble component, such as latex beads or charcoal particles (Figure 27.11). Conversely, antibody-coated latex beads can be used to detect antigens on the surface of bacterial cells. Passive test kits are available for identification of a variety of pathogens or pathogenic products. The extensive use of agglutination tests stems from the fact that they are fast, sensitive (even more so for passive agglutination tests than direct agglutination tests), and inexpensive.

Section 27.8 describes the various applications of immunofluorescence (i.e., the use of fluorescent antibodies). Describe the difference between direct (fluorescent antibody binds to the antigen) and indirect (fluorescent antibody binds to a nonfluorescent antibody that is bound to the antigen) fluorescent antibody staining methods (Figure 27.12). Immunofluorescent techniques have impressive utility, as they can be used for the identification, enumeration, and sorting of a variety of eukaryotic and prokaryotic cell types (Figure 27.13), as well as viral pathogens (Figure 27.14). Spend sufficient time describing the use of fluorescent antibodies in clinical microbiology. For example, a particularly useful application of fluorescent antibodies is to separate mixtures of cells into pure populations or to define the numbers of cell types in complex sample mixtures.

One of the most sensitive immunological assays available is the enzyme immunoassay (EIA; also called enzyme-linked immunosorbent assay, or ELISA) (Section 27.9). Compare and contrast the methodologies and applications of the direct and indirect EIA formats, as well as the highly sensitive antigen sandwich and combination formats (Figure 27.15). Explain the extensive use of EIAs for the detection of clinically important pathogens and toxins, such as Salmonella, Borrelia, Treponema, Yersinia, Candida, cholera toxin, pathogenic my.

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cobacteria and rickettsias, and a variety of viruses. Stimulate student interest by discussing the significance of the various HIV-EIA assays for detecting HIV-infected individuals, blood, and blood products. For example, AIDS is a worldwide public health problem of staggering proportions, and thus sensitive and highly specific tests are essential for restricting the spread of this deadly immunodeficiency disease. Also point out that commercial pregnancy and drug-testing kits are EIA-based assays.

Although not as sensitive or specific as EIAs, rapid tests provide diagnostic aid in a matter of minutes for a variety of conditions, including infection by HIV or influenza virus. Figure 27.16 illustrates a rapid test setup, in which colored molecules called chromophores are used to visualize antibody-antigen complexes on a support matrix composed of paper, nitrocellulose, or a plastic membrane. Rapid test results are often confirmed by more sensitive tests, such as EIA. The immunoblot (Western blot) method is a valuable tool for identifying pathogen-specific proteins, even in complex fluid mixtures. Immunoblots consist of three main components: (1) the separation of a protein mixture in a polyacrylamide gel, (2) protein transfer from the gel to a nitrocellulose membrane (blotting), and (3) identification of target proteins using specific antibodies (Figure 27.17). Note that the detection of bound antibody-protein complexes can be done using either radiolabeled antibodies or the EIA technique. Although not used as an HIV screening tool due to inferior sensitivity and greater cost, the immunoblot technique is often used to confirm positive HIV-EIA results.

Section 27.10 describes the use of the polymerase chain reaction (PCR) to detect specific pathogens in tissues, even in cases where the pathogen is not readily observable or cultivable.

Thus, an important advantage of PCR techniques is that they do not require culturing of the pathogen and do not depend on an immune response. Instead, genotypic characteristics of species-specific genes are used to identify pathogenic species. You may need to review briefly the principles of PCR at this time (Section 11.3), especially if the technique is not demonstrated or carried out in the laboratory component of the course. Discuss the advantages and principles of nucleic acid probe methodologies in clinical diagnostics: • Nucleic acids are easily isolated from infected tissues and clinical samples.

• The uniqueness of genomic sequences allows for certainty in pathogen identification.

• Nucleic acids can be readily amplified to increase quantities for analysis.

• In reasonable quantities, nucleic acids are easily visualized and measured.

Describe for your students the use of quantitative real-time PCR (qPCR), reverse transcriptase PCR (RT-PCR), and qualitative PCR for the detection of pathogens. qPCR uses nucleic acid probes that fluoresce strongly when bound to template DNA, allowing for a quick determination of whether the target DNA sequence is present in the sample (Figure 27.18). In addition, because the intensity of the fluorescence is proportional to the amount of complementary template DNA, this technique can be used to quantify the abundance of a particular pathogen in the original sample. In contrast to qPCR, RT-PCR uses RNA as a template for amplification, and therefore it can be used to monitor the expression of specific pathogenic genes or for the detection of RNA retroviruses, such as HIV. Finally, qualitative PCR expands upon the qPCR technique by using fluorescent hybridization probes to differentiate pathogen subtypes that contain the same target gene. The technique exploits nucleotide polymorphisms, which cause the same gene in different pathogens to have differ.

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ent melting points because of differences in their nucleotide sequences. By comparing the fluorescent melting behavior of a patient sample to that of known DNA standards, one can determine which pathogen subtype has infected the individual (Figure 27.19).

27.11–27.16 | Antimicrobial Drugs These sections discuss the in vivo uses of synthetic and naturally occurring antimicrobial agents. Students that have learned the details of prokaryotic cell structure and protein synthesis will have a greater appreciation and understanding of the mechanisms of action of these agents as well as how resistances develop. In your opening discussion regarding the in vivo use of antimicrobial agents, use Figures 27.20–27.22 to provide an important overview of the modes of action of the most widely used chemotherapeutic agents (Section 27.11). You may also wish to present a brief historical review of the evolution of synthetic chemotherapeutic agents. In the early 1900s, Paul Ehrlich developed the concept of selective toxicity, and his discovery of salvarsan marked the first effective antimicrobial drug for the treatment of syphilis. Students should understand that particular types of antibiotics and antimicrobial agents target specific cellular components or processes. The chemical structures of both synthetic antimicrobial drugs and antibiotics can be quite complex, with the latter requiring numerous and often unique biosynthetic steps by the producing microorganism. In addition, the producing organism must be immune to the effects of its own antibiotic. Using Figures 27.23 and 27.24, distinguish between and provide examples of synthetic antimicrobial drugs in widespread use: growth factor analogs (e.g., sulfa drugs, isoniazid, and nucleoside analogs) and quinolones.

Antibiotics are naturally occurring antimicrobial drugs, the majority of which (>99%) are not clinically useful without structural modifications in the laboratory, a process that creates semisynthetic antibiotics (Section 27.12). Discuss the mode of action and selective toxicity of antibiotics by explaining the concept of narrow- versus broad-spectrum antibiotics (Figure 27.21). Depending upon the background of the students and the focus of your course, you may wish to discuss the structural chemistry of the different classes of antibiotics: β-lactams (e.g., penicillins and cephalosporins), which are produced by fungi; and aminoglycosides, macrolides, and tetracyclines, which are all derived from species of Bacteria (Sections 27.13 and 27.24; Figures 27.25–27.29). Be sure to differentiate the modes of action in your description of these groups. Note that novel antibiotics continue to be discovered and/or synthesized by modifying current drugs. Discuss the two examples of newer classes of antibiotics, both of which are produced by species of Streptomyces: (1) daptomycin depolarizes bacterial cell membranes and is used mainly against gram-positive bacteria (Figure 27.30); (2) platensimycin inhibits a key enzyme in bacterial fatty acid biosynthesis (Figure 27.31).

Continue your survey of in vivo antimicrobial agents with a discussion of antiviral (Table 27.6) and antifungal (Figure 27.32 and Table 27.7) drugs. Inform students that research regarding antiviral drugs has progressed rapidly in the past 25 years due mainly to the prevalence of human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). The nucleoside analogs, such as acyclovir and azidothymidine (AZT), have been the most successful and widely used antiviral drugs to date.

Distinguish between the nucleoside reverse transcriptase inhibitors (NRTIs), which disrupt elongation of viral DNA and include nearly all nucleoside analog drugs, and the nonnucleo.

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side reverse transcriptase inhibitor (NNRTI) nevirapine, which directly binds and inhibits reverse transcriptase. Other important classes of antiviral drugs include protease inhibitors, which obstruct protein processing in HIV, and the fusion inhibitor enfuvirtide, which prevents fusion of the HIV envelope to T lymphocyte membranes. Finally, interferons cause interference of viral replication and help prevent multiple viral infections. They are potentially useful for antiviral and anticancer therapies.

The action of some antifungal drugs is presented in Figure 27.32. Note that highly specific antifungal drugs target cell components not found in other eukaryotic cells or in prokaryotes, such as ergosterol in fungal cell membranes and chitin in the fungal cell wall. Note that ergosterol inhibitors include two classes: (1) the polyenes, which bind directly to ergosterol, and (2) the azoles and allylamines, which inhibit ergosterol biosynthesis. Both classes kill fungal cells by causing their membranes to become excessively permeable. To conclude these sections, refer to the information just presented regarding selective toxicity, and ask students why chronic infections by eukaryotic pathogens are difficult to treat and why relatively few antiviral and antifungal drugs are available. Finally, ask students how this relates to the fact that many antifungal drugs are suitable for use only as topical ointments.

27.17–27.18 | Antimicrobial Drug Resistance Given the serious threat to humankind that antimicrobial drug resistance presents, it is well worth your time to discuss this topic in some detail. Discuss the six reasons why a microorganism may be naturally resistant to certain antibiotics, using Figure 27.33 to show how drugs can be chemically altered by enzymes encoded by resistance genes. The mechanisms of resistance and the factors responsible for its spread have thwarted the large-scale search for new antimicrobial drugs by the biotechnology and pharmaceutical industries.

Table 27.8 presents a good summary of the mechanisms that bacteria employ for resistance to various antibiotics. Note that most drug-resistant bacteria isolated from patients contain R (resistance) plasmids carrying extrachromosomal genes that confer resistance, as opposed to chromosomal resistance factors. The mobility of plasmid DNA explains the rapid horizontal transfer of antibiotic resistance observed in communities and some hospitals (Figures 27.34 and 27.35). Considering the challenge and huge cost of discovering and implementing new antibiotics, adherence to standards for preventing antimicrobial drug resistance is of utmost importance (Table 27.9).

The problem remains that the high biotic potential driving evolutionary change in microorganisms is a formidable opponent to the science and technology we employ to combat the problem, including the production of semisynthetic drug analogs and the use of computers for designing and testing new drugs (Figures 27.36 and 27.37). Promising strategies to combat antimicrobial drug resistance include (1) the development of more sensitive assays that allow identification of drugs that are active against novel targets and (2) drug combination therapy, which depends more on a better understanding of the antimicrobial drugs we currently have than in developing new drugs. Using drug combinations, the efficacy of an antibiotic can be retained when it is administered with a second drug that inhibits the development of resistance. An especially successful example is the combined use of a nucleoside analog with a protease inhibitor (a treatment protocol referred to as HAART, for highly active anti-retroviral therapy) to treat HIV patients. .

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Answers to Review Questions 1. The two most common causes of laboratory-associated infections are ignorance of safety procedures and carelessness. Prevention requires stringent adherence to the safety standards and guidelines listed in Tables 27.1 and 27.2.

2. A nosocomial infection, also called healthcare-associated infection (HAI), is a local or systemic infection acquired by a patient in a healthcare facility, particularly during a stay in the facility. Staphylococcus aureus, Acinetobacter, Burkholderia cepacia, Hepatitis, Vancomycin-resistant Enterococcus and Mycobacterium abscessus are examples of associated pathogens.

Risk factor

Rationale

Patients Newborn infants and the elderly Infectious disease patients Patient proximity Healthcare personnel

Patients are already ill or compromised Not fully immune competent Pathogen reservoirs Increases cross-infection Can transfer pathogens between and among patients Breaching the skin barrier can introduce pathogens Exposes internal organs, may introduce pathogens, and stress lowers resistance to infection Lower resistance to infection May select for resistant and opportunistic pathogens

Medical procedures (blood draws, etc.)

Surgery

Anti-inflammatory drugs Antibiotic treatment

3. Sterile swabs are used to sample the throat. The swab is then used to inoculate the surface of a suitable agar plate or broth culture medium. The proper collection of a blood sample is done by drawing 20 ml of blood from a vein. Before insertion of the needle, the surface of the skin must be prepared using a topical antiseptic, such as alcohol or betadine. The medical practitioner should use gloves during the procedure. Cultures from the blood sample are first prepared in a rich, liquid culture medium, with incubation occurring at 35°C for up to five days under both aerobic and anaerobic culture conditions. After a Gram stain of the primary culture is performed, gram-positive bacteria are subcultured on chocolate agar, whereas gram-negative bacteria are subcultured on MacConkey agar.

4. Rapid processing of the clinical sample reduces the chance that anaerobic bacteria in the sample will die because of exposure to oxygen. The isolation of obligate anaerobes from samples requires careful procedures in which the specimen does not come into contact with air. This possibility is reduced if the sample is obtained by aspiration and immediately put into an oxygen-free tube of dilute salts with a redox indicator. After the specimen is streaked on an appropriate agar medium, plates are incubated in a sealed chamber, purged of air, and incubated under strict anoxic conditions.

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5. A selective medium contains material that inhibits the growth of some microorganisms while allowing other microorganisms to grow. A differential medium allows for microorganisms to be distinguished based on phenotypic properties following growth. EMB agar is both a selective medium (gram-positive bacteria are inhibited) and a differential medium (lactose fermenters may be distinguished from nonlactose fermenters). In the clinical laboratory, a specimen from a kidney infection would be streaked on both EMB agar and blood agar to help distinguish whether an enteric pathogen (e.g., E. coli) or a pseudomonad might be the causative agent.

6. Antimicrobial susceptibility is measured by determining the smallest amount of agent needed to completely inhibit the growth of the tested organism in vitro, a value called the minimum inhibitory concentration (MIC). Antibiograms are used to monitor control of known pathogens, to track the emergence of new pathogens, and to identify the emergence of antibiotic resistance at the local level.

7. The presence of antigen following infection stimulates antibody production. However, high antibody titers do not necessarily indicate that ongoing infection is occurring since antibody titers can remain high well after a previous infection has cleared. It is important to obtain an acute and a convalescent blood sample to monitor infections, as a single measurement does not indicate active infection. To establish that a particular pathogen is causing an acute illness, it is essential to show a rise in antibody titer.

8. Agglutination is a reaction between antibody and particle-bound antigen resulting in visible clumping of the particles. Direct agglutination results when soluble antibody causes clumping due to interaction with an antigen that is an integral part of the surface of a cell or other insoluble particle such as a red blood cell (hemagglutination). Passive agglutination is the agglutination of soluble antigens or antibodies that have been adsorbed or chemically coupled to insoluble particles such as latex beads or charcoal particles. Passive agglutination is up to five times more sensitive than direct agglutination.

9. In direct immunofluorescence, the antibody that interacts with the surface antigen is itself covalently linked to the fluorescent dye. In indirect immunofluorescence, the presence of a nonfluorescent antibody on the surface of a cell is detected by the use of a fluorescent antibody directed against the nonfluorescent antibody. Antibodies can be covalently modified by fluorescent dyes such as rhodamine B, which fluoresces red, or fluorescein isothiocyanate, which fluoresces yellow-green. The attached dyes do not alter the specificity of the antibody but make it possible to detect the complex by use of a fluorescence microscope once it has bound to cell or tissue surface antigens. The cell-bound fluorescent antibodies emit their characteristic fluorescent color when excited with light of particular wavelengths. Fluorescent antibodies can be used to identify a microorganism directly in a patient specimen (in situ), bypassing the need for the isolation and culture of the organism.

10. EIA is a highly sensitive technique because measurements are based on covalently bonded enzymes (EIA), which allow for detection of minute quantities of antigen-antibody complexes.

11. EIA tests for AIDS occasionally yield false positive results. The immunoblot (Western blot) method identifies several HIV proteins (both core and coat) that are virus-specific, essentially eliminating false positives. The immunoblot procedure is more expensive, .

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more time-consuming, and more laborious than EIA tests and therefore is used only for confirmation after a positive EIA test.

12. Quantitative PCR uses nucleic acid probes that fluoresce strongly when bound to template DNA, allowing for a quick determination of whether the target DNA sequence is present in the sample. Because the intensity of the fluorescence is proportional to the amount of complementary template DNA, this technique can be used to quantify the abundance of a particular pathogen in the original sample. Qualitative PCR expands upon the quantitative technique by using fluorescent hybridization probes to differentiate pathogen subtypes that contain the same target gene. The technique exploits nucleotide polymorphisms, which cause the same gene in different pathogens to have different melting points because of differences in their nucleotide sequences. By comparing the fluorescent melting behavior of a patient sample to that of known DNA standards, one can determine which pathogen subtype has infected the individual.

13. Sulfanilamide, the simplest sulfa drug, is an analog of p-aminobenzoic acid, which is a part of the vitamin folic acid, a nucleic acid precursor. Sulfanilamide blocks the synthesis of folic acid, thereby inhibiting nucleic acid synthesis. Sulfanilamide is selectively toxic in bacteria because bacteria synthesize their own folic acid, whereas humans and most animals obtain folic acid from their diet.

Isoniazid is an important growth factor analog with a very narrow spectrum of activity. Effective only against Mycobacterium, isoniazid interferes with the synthesis of mycolic acid, a mycobacterial cell wall component. A nicotinamide (vitamin) analog, isoniazid is the most effective single drug used for control and treatment of tuberculosis.

Quinolones are synthetic antibacterial compounds that disrupt bacterial metabolism by interfering with bacterial DNA gyrase, preventing the supercoiling of DNA, a required step for packaging DNA in the bacterial cell. Ciprofloxacin, a quinolone, is a fluorinated derivative of nalidixic acid with broad-spectrum activity. It is more soluble than the parent compound, allowing it to reach therapeutic levels in blood and tissues.

Because DNA gyrase is found in all Bacteria, fluoroquinolones are effective for treating both gram-positive and gram-negative bacterial infections.

14. Natural antibiotics are produced by a variety of bacteria and fungi, including species of Streptomyces (Bacteria) and Penicillium and Cephalosporium (fungi) in particular.

15. The enzymes (transpeptidases) that cross-link the two glycan-linked peptide chains of peptidoglycan also bind the β-lactam ring. Thus, these proteins are known as penicillinbinding proteins (PBPs). When PBPs are bound to the β-lactam ring, they no longer catalyze the transpeptidase reaction. The cell wall continues to be formed but becomes progressively weaker as autolysins continue to remove the existing cell wall, resulting in lysis. The outer membrane of gram-negative bacteria is impermeable to naturally occurring -lactam antibiotics, and therefore such cells are not susceptible to the effects of these antimicrobial drugs. Gram-positive bacteria have no outer membrane, and therefore -lactam drugs can freely bind transpeptidases.

16. Tetracycline (as well as streptomycin, spectinomycin, and others) is a protein synthesis inhibitor that acts by binding to and inhibiting function of the 30S ribosomal subunit.

Chloramphenicol and erythromycin interfere with the 50S ribosomal subunit. Mupirocin .

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and puromycin interfere with tRNA synthesis. All have the net effect of blocking protein synthesis in bacterial cells.

17. Viruses depend largely on host cell machinery to perform their metabolic functions.

Because of this commonality, it is a considerable challenge to design antiviral drugs that are not also toxic to the host.

18. Unlike other eukaryotes, the cell membranes of fungi contain ergosterol. Drugs designed to disrupt ergosterol or inhibit its synthesis will be selectively toxic. Fungal cell walls contain the polysaccharide chitin. Therefore, drugs that interfere specifically with chitin synthesis will be selectively toxic to fungi.

19. Bacterial antibiotic resistance mechanisms are summarized here (see also Table 27.8): (1) The organism may lack the target structure that the antibiotic inhibits; (2) the organism may be impermeable to the antibiotic; (3) the organism may be able to alter the antibiotic to an inactive form; (4) random mutation may modify the target of the antibiotic; (5) an alteration that circumvents the metabolic pathway that an antibiotic affects may develop or be acquired by the target organism; and (6) the organism may be able to remove the antibiotic by active transport as soon as it enters the cell (i.e., an efflux pump system).

20. Several β-lactam antibiotics can be combined with β-lactamase inhibitors to preserve antibiotic activity in β-lactamase-resistant microorganisms. Ampicillin can be mixed with sulbactam (Unasyn), or clavulanic acid (Augmentin), both β-lactamase inhibitors. The inhibitors bind β-lactamase irreversibly, preventing degradation of the ampicillin. This combination preserves the effectiveness of the β-lactamase-sensitive ampicillin for treatment of β-lactamase producers such as staphylococci. Sulfamethoxazole– trimethoprim (Bactrin), a mixture of two folic acid synthesis inhibitors, is used to prevent the loss of efficacy through mutation and selection for resistance Highly active anti-retroviral therapy (HAART) is designed to target two independent viral functions; nucleoside analogs target virus replication and the protease inhibitors target virus maturation.

Answers to Application Questions 1. Bacillus subtilis: BSL-1 Bacillus anthracis: BSL-4 Ebola virus: BSL-4 The safety standards for Staphylococcus aureus:

Rule

Implementation

Restrict access

Only laboratory workers and trained support personnel have access.

Eating, drinking, and application of cosmetics are forbidden in the lab.

Handwashing prevents spread of pathogens.

Practice good personal hygiene

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Use personal protection equipment

Vaccinate Handle specimens safely Decontaminate

Lab coats, gloves, eye protection, and respirators are recommended or required depending on the pathogens being handled.

Personnel must be vaccinated for agents they may contact.

Consider clinical specimens to be infectious and handle appropriately.

After use or exposure, decontaminate specimens, surfaces, and materials by disinfecting, autoclaving, or incinerating.

2. Isoniazid is effective against tuberculosis. Observation of acid-fast stained Mycobacterium tuberculosis in sputum from the patient can detect the disease. Skin tests such as the tuberculin test, which consists of an intradermal injection of a soluble extract from cells of M. tuberculosis, are routinely used for aiding in diagnosis of tuberculosis. A positive inflammatory reaction at the site of injection within 48 hours indicates current infection or previous exposure to M. tuberculosis. This test identifies responses caused by pathogen-specific inflammatory Th1 cells. Indirect EIAs have been developed for detecting serum antibodies to M. tuberculosis. Nucleic acid amplification (PCR) methods are applied as extremely specific diagnostic tools used for a large number of pathogens. qPCR and qualitative PCR techniques provide quantification and identification of pathogens.

3. Viruses depend heavily on the host machinery for their metabolism, and thus virusspecific targets are relatively few. Fungi, on the other hand, contain their own biosynthetic and metabolic enzymes, but because they are eukaryotes, their cellular machinery is very similar to that of human cells. Fungi do, however, have some unique features, such as chitin-containing cell walls and ergosterol-containing cell membranes, and these signatures provide opportunities for drug development. Nucleoside analogs are antimicrobial agents that can be effective against both viruses and fungi. However, host toxicity is an issue with such nonselective drugs.

4. a. Entry in to host cells: Enfuvirtide, b. Reproduction within the host’s genome: Interferons, Nevirapine c. Release of pathogenic viruses during host-cell lysis: neuraminidase inhibitors oseltamivir (brand name Tamiflu) and zanamivir (Relenza)

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CHAPTER

28

Epidemiology

Summary In this chapter, students will study how pathogens spread throughout populations, focusing particularly on the occurrence, distribution, and determinants of disease. The students will learn that although deaths from noninfectious diseases occur much more frequently than those from infectious diseases in developed countries, this is not the case in many developing countries, where infectious diseases are a more prominent threat to public health. The final four chapters of the text (Chapters 29−32) build upon this introduction to the science of epidemiology by discussing specific diseases, including a description of their causative agents and mechanisms of transmission.

28.1–28.4 | Principles of Epidemiology Many students will view the field of epidemiology as an interesting and exciting career, focused on uncovering clues to identify and contain lethal infectious agents and prevent human catastrophe. Although this scenario is essentially an accurate portrayal of the role of epidemiologists, in fact the important measure of their success is the prevention and control of potential outbreaks. About one-quarter of annual deaths worldwide result from various infectious diseases (Table 28.1). You may want to review Figure 1.8 with students to reinforce the effect of implementing control measures on reducing mortality from infectious disease in the United States from the early 1900s to the present. Impress upon students the reality that newly emerging infectious diseases in both developed and developing countries pose a serious threat to global human health (Figure 28.1). Effective solutions require political, economic, societal, and scientific research efforts.

The terminology of the discipline will probably be new to students (Section 28.1), and therefore it is important for you to define new terms as they are introduced. Differentiate between chronic infections, which are caused by pathogens that are well-adapted to their hosts and often cause minimal damage, and acute infections caused by emerging pathogenic strains that trigger rapid onset of disease. Also define incidence vs. prevalence; epidemic vs. pandemic vs. endemic disease (Figure 28.2); outbreak; reservoirs; and carriers. Differentiate between the terms morbidity (the incidence of disease in populations—whether fatal or nonfatal) and mortality (the incidence of death in a population). Explain the progression of symptoms for a typical acute infectious disease in terms of the following stages: (1) infection, (2) incubation period, (3) acute period, (4) decline period, and (5) convalescent period.

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Emphasize to students the fundamental relationship strategy an obligate parasite must employ to ensure its evolutionary success. This essentially involves the establishment of a balanced equilibrium with the host (Section 28.2). Point out that successful host-parasite interactions require coevolution of each organism. The textbook example of the intentional introduction of myxoma virus into the population of an invasive rabbit species previously introduced into Australia is an excellent example of rapid, successful coevolution between host and pathogen (Figure 28.3). However, for pathogens that do not depend upon host-to-host transmission for survival, there is no selective pressure for the pathogens to curb virulence and develop a coexistence with the host. Such pathogens are typically highly virulent and cause acute disease, as in the following examples: • Infection by the malaria parasite, Plasmodium falciparum, is fatal to the human host. There is no evolutionary pressure to select for human survival because the parasite can successfully reproduce outside of the human body as long as its vector host (mosquitoes) can obtain a blood meal (see Section 32.5).

• Nosocomial infections by enteropathogenic E. coli strains kill infants, and the virulence of the infective strain increases during subsequent transmission from patient to patient.

The death of the host has no effect on the reproductive success of the agent because its transmission occurs from both animate and inanimate routes.

The concept of herd immunity is important to develop with students. Simply defined, herd immunity is a group’s ability to resist infectious disease and potential epidemics due to immunity present in a large proportion of the group members (Figure 28.4). Students should understand from their previous biology courses that evolution selects for populations, not individuals, and thus the terms “host” and “parasite” must be viewed from this perspective.

Emphasize to students that epidemiological methods for following disease transmission (Section 28.3) draw on fields of study outside of the life sciences, such as geography, meteorology (seasonal factors), sociology, and age group incidence (Figure 28.5). Contrast the progression characteristics of common source epidemics and host-to-host epidemics (Figure 28.6), and illustrate these ideas by providing a few examples of each (Table 28.2). Point out that both living (vectors) and nonliving (fomites and vehicles) carriers can perpetuate host-to-host transmission. Also introduce the concept of basic reproduction number (R0), which incorporates mathematical models to define the number of transmissions expected from each case of disease in an entirely susceptible population (Table 28.3). Because R0 is a measure of pathogen transmissibility and the potential for an outbreak, higher R0 values require greater herd immunity to prevent the spread of infection in a population. Note that while R0 is a theoretical value, the observed reproduction number, R, is a truer representation that, if comprehensive disease transmission data are available, can be calculated for actual public health emergencies. See the feature box Explore the Microbial World: SARS⎯A Model of Epidemiological Success for an excellent example of a case in which R was reduced by implementing stringent public health measures to end the threat of an outbreak.

Discuss the reservoirs of common human diseases (Section 28.4) as shown in Table 28.2.

Keep in mind that reservoirs include both living (e.g., humans or other animals) and nonliving (e.g., soil) sources of infection. Stress the important point that many pathogens exist only in humans and are maintained only by person-to-person transmission. By contrast, a disease that primarily occurs in animals but is occasionally transmitted to humans is a zoonosis. Point

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out that although carriers have subclinical infections and show no symptoms or only minor symptoms of disease, they are sources of infection for others.

28.5–28.6 | Epidemiology and Public Health Students interested in medical applications relating to their microbiological/molecular career interests will find the discussions of public health (Section 28.5) and measures of global health disease control (Section 28.6) especially informative. Point out that public sewage treatment, the availability of clean drinking water, and advances in food preparation have drastically decreased the spread of infectious diseases through common vehicles (Figure 28.7).

Discuss control measures directed against disease reservoirs and pathogen transmission.

Discuss the strategies of immunization, quarantine, and surveillance in controlling and/or eradicating infectious diseases (Figure 28.8). Point out that infectious disease eradication is most feasible in treatable or preventable diseases that are restricted to humans, such as smallpox (already eradicated), poliomyelitis, Hansen’s disease (leprosy), syphilis, and diphtheria. Also discuss key points in Tables 28.4 concerning the reporting and tracking of infectious diseases in the United States.

Perhaps the most poignant perspective from which to initiate class discussions on the importance of public health is the global perspective. The most important agency involved in monitoring global health problems is the World Health Organization (WHO). With the goal of collecting and reporting health information, WHO has divided the world into six geographic regions. Point out to students that infectious diseases account for about 25% of all deaths worldwide. Use Figure 28.9 to contrast mortality due to infectious diseases in Africa, a mostly developing region, and in the Americas, a region that is largely developed. It may surprise many students to learn that an individual in Africa is 10 times more likely to die from infection than someone in the Americas. This sad fact is mostly due to a lack of adequate healthcare, insufficient vaccine and antibiotic availability, and limited access to safe food and water sources—deprivations with which most of your students (and their parents) will be unfamiliar. Those who travel to endemic areas will be aware of these issues by the number of immunizations recommended and/or required prior to travel.

28.7–28.8 | Emerging Infectious Diseases Section 28.7 introduces the public health problem of emerging and reemerging infectious diseases. Discuss these ideas in terms of recent disease outbreaks and the factors that contributed to them (Table 28.5). Link these issues with the lack of adequate public health infrastructure in many developing regions and increasing antibiotic resistance in pathogenic strains. The latter problem is attributed in large part to the routine administration of antibiotics as a preventive tool, as opposed to a treatment strategy. Engage students in a discussion of the implications of this scenario. Child mortality rates are extremely high in developing countries compared with industrialized societies. Consequently, the use of antibiotics as a preventive strategy gives children a chance to survive early childhood diseases (pertussis, diphtheria, etc.), but it also selects for antibiotic-resistant pathogens.

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Use Figures 28.10 and 28.11, as well as Table 28.6, to indicate recent outbreaks and summarize the factors responsible for the emergence of new (or reemergence of old) diseases: (1) human demographics and behavior; (2) technology and industry (e.g., foods distributed nationwide become a common source for infection); (3) economic development and land use; (4) international travel; (5) microbial adaptation (evolution); (6) insufficient public health facilities; and (7) abnormal perturbations that affect the usual host-parasite balance. Use the examples presented in the text to illustrate these factors, some of which will be familiar to students (e.g., the 2010 contamination of water supplies in Haiti that led to the most extensive cholera outbreak in that nation in over 100 years). Point out that mobile genetic elements carrying genes encoding virulence factors can also contribute to increased pathogenic virulence and the emergence of new epidemics (Table 28.7).

Section 28.8 addresses the topics of biological warfare and biological weapons. These topics will certainly attract students’ attention, given the acts of terrorism that transpire on an increasingly regular basis. Develop the discussion from the microbiological perspective, focusing on the candidate organisms, route of delivery, and clinical effects (Table 28.8). A few examples presented in the text are illustrated below. You may want to refer to the appropriate sections cited in the text for additional readings and/or incorporate other details for emphasis: • Variola major (smallpox virus): Smallpox is a particularly frightening candidate for a biological weapon due to its contagious transmission and lethal outcome. Its worldwide eradication by the late 1970s due to the production and dissemination of an extremely effective vaccine has now resulted in an inadequately protected (not vaccinated) population.

• Bacillus anthracis: Many students will recall the inhalation anthrax attacks in the United States following the September 11, 2001 terrorist attacks. B. anthracis is an endosporeforming, gram-positive soil bacterium; anthrax obtained by the inhalation route is nearly 100% lethal (Figures 28.12 and 28.13). Reinforce the importance of understanding cell structure, chemistry, and metabolism (discussed in Chapters 2 and 3) as they relate to the activities of this “biological weapon.” A vaccination is available for anthrax, but its administration is usually restricted to those considered at risk, such as military personnel and livestock caretakers.

• Bacterial toxins: Although not contagious, potent toxins such as the botulism toxin (see Sections 23.9 and 23.10) could have tragic results if delivered to select groups.

• Other pathogens: Yersinia pestis (Section 30.7), Salmonella spp. (Section 31.10), and rabies virus (Section 30.1) are all potential biological weapons.

28.9–28.11 | Current Pandemics These sections examine current pandemics and provide students with real-world data acquired by epidemiologists tracking specific diseases. The information gathered allows healthcare workers to develop treatment and prevention strategies while researchers work on eradicating the infectious agent. Perhaps the most significant story to focus on is the HIV/AIDS pandemic (Section 28.9), including the history of this disease from its discovery to its current status, as described in the text (Figure 28.14). A few pertinent facts include the following:

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• The first described cases in 1981 were associated with homosexuality and intravenous drug abuse, suggesting that the causative agent was transferred by the exchange of body fluids.

The seriousness of the disease was not fully realized initially. However, it was soon diagnosed in many who had received blood transfusions, especially in hemophiliacs.

• The initial pattern of AIDS infection indicated that it was a transmissible disease (Figure 28.15). However, the public (including much of the medical establishment in the early stages of the epidemic) erroneously interpreted this accurate description of the epidemiological pattern of the disease as contagious (i.e., transmissible by casual contact, such as through food, water, or particles released into the air; Figure 32.8). Reinforce to students that transmissible and contagious are not synonymous terms.

• Provide students with pertinent geographic and demographic data, shown in Figures 28.14 and 28.15). Table 28.9 lists the number of HIV/AIDS infections by geographic distribution. A few statistical points include the following: • United States: More than one million reported cases, with over 500,000 deaths, through 2011; over 33,000 new AIDS cases have been reported annually in the U.S. since 1989; 35,741 new AIDS cases were diagnosed in 2010 (Figure 28.14).

• Worldwide: Over 80 million cases of HIV infection were reported between 1981 to 2010; about 46 million deaths have occurred from AIDS, with about 34 million currently infected with HIV. One of the most serious and significant waterborne diseases in developing countries is cholera (Section 28.10). Note that cholera is caused by Vibrio cholerae, a gram-negative, curved rod of the phylum Proteobacteria that is usually transmitted via contaminated water (see Section 31.3). Students should find the epidemiology of cholera interesting in that there have been seven pandemics recorded from two biotypes: classic and El Tor (Figures 28.16 and 28.17). Remind students of the Chapter 1 discussions of the history of microbiology and the contribution of Robert Koch (see Section 1.8), who is credited with the isolation of the classic biotype of Vibrio cholerae. The classic strain was responsible for the first six of the seven cholera pandemics, whereas the V. cholerae El Tor biotype is responsible for the seventh (and possibly eighth) pandemic (Figure 28.17). Several epidemiological and pathogenic points of interest to cover with the students are as follows: • The seventh pandemic, caused by the El Tor biotype, started in Indonesia in 1961 and is still in progress. Emphasize the scale and impact of the disease by pointing out the pertinent statistical data presented in the text: 5 million cases and at least one-quarter of a million deaths since 1961; annual reported cases of cholera in the past decade range from 95,560 in 2004 to 589,854 in 2011 (Figure 28.16).

• Endemic cholera in developing countries is primarily the result of insufficient water treatment programs and facilities. Reports of cholera have occurred in the United States, but rather than originating in drinking water, these cases are mostly foodborne and are the result of consuming raw shellfish from coastal areas where V. cholerae is a common inhabitant (Figure 35.14).

• The 2010 cholera outbreak in Haiti following a large earthquake is ongoing, with nearly 600,000 cases reported and over 7000 deaths by summer 2012. The onset of the outbreak may have occurred from V. cholerae-contaminated seawater influx into compromised .

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municipal water treatment facilities. More likely, however, the cholera outbreak in Haiti ensued following improper disposal of sewage from an encampment of United Nations peacekeeping troops that had recently arrived from Nepal (where a cholera outbreak caused by the same V. cholerae serotype had recently occurred).

Influenza pandemics (Section 28.11) occur every 10 to 40 years as a result of genomic reassortment, a concept referred to as antigenic shift, which will be discussed in more detail in Section 29.8. The focus of this section is the recent swine flu (H1N1) pandemic of 2009. In this case, a reassortant virus that originated in Mexico spread from the swine population to the human population, resulting in a large-scale infection of susceptible individuals around the world. Unlike seasonal flu outbreaks, which peak in midwinter, pandemic (H1N1) 2009 spread throughout the summer and culminated in a peak three to four months earlier than usual (Figure 28.18). Perhaps the most closely watched influenza strain currently is H5N1 (avian) influenza A, which is spread to humans primarily from poultry, either from contact or ingestion of contaminated meat. The concern is threefold: First, few have ever been exposed to this strain, and therefore the population consists largely of susceptible individuals.

Secondly, the mortality of the H5N1 virus has been nearly 60% (360 deaths out of 610 reported cases), and therefore H5N1 is a particularly virulent strain. Finally, if the strain is or has been transferred to swine, as some believe, then an entirely new reassortant virus may emerge for which no immunity in humans exists.

Answers to Review Questions 1. Chronic (long-term) infections are the result of a pathogen that is well-adapted to its host in that the two organisms remain in a balanced equilibrium, with the pathogen causing only minimal harm. Acute infections are often the result of a pathogen that does not depend on the host for survival. Such infections lead to rapid and dramatic onset of disease that can result in death of the host. Mortality expresses the incidence of death in a population. Morbidity refers to the incidence of disease in a population. Prevalence is the number of new and existing cases of a disease in a population at a given time. Incidence is the number of new cases of a disease in a population at a given time. Epidemic refers to an unusually large number of cases of a disease within a population. Pandemic refers to a widespread, usually worldwide, epidemic.

2. Malaria parasites (Plasmodium spp.) show antigenic variations in coat proteins that aid in avoiding the immune response of the host. This ability to avoid the host immunity increases pathogen virulence without regard to the susceptibility of the host.

If a high proportion of the individuals in a group are immune to a pathogen, then the whole population will be protected; this resistance to infection is called herd immunity. Assessment of herd immunity is important for understanding the development of epidemics. The more highly infectious a pathogen, or the longer its period of infectivity, the greater the proportion of immune individuals necessary to prevent disease spread.

3. Sexually transmitted diseases, such as gonorrhea, are spread by direct host-to-host contact. Diseases spread by indirect host-to-host contact include malaria and Lyme disease, which are spread by arthropod vectors. Diarrheal diseases caused by pathogenic E. coli and skin infections caused by Staphylococcus can be spread by fomites.

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4. Some pathogens whose reservoirs are not in animals only incidentally infect humans and cause disease, such as the genus Clostridium, common soil bacteria, occasionally infect humans, causing life-threatening diseases such as tetanus, botulism, gangrene, and certain gastrointestinal diseases.

For many pathogens, however, living organisms are the only reservoirs. In these cases, the reservoir host is essential for the life cycle of the infectious agent; maintenance of human pathogens of this kind requires person-to-person transmission. Many viral and bacterial respiratory pathogens and sexually transmitted pathogens require human hosts; certain staphylococci and streptococci are examples of human-restricted pathogens, as are the agents that cause diphtheria, gonorrhea, and mumps.

Carriers are potential sources of infection for others. They may be in the incubation period of the disease, in which case the carrier state precedes the development of actual symptoms.

5. The major methods used to control the spread of infectious diseases include the control of pathogen transmission, the development of vaccines and antibiotics, and quarantine.

Methods of controlling pathogen transmission include decontamination or destruction of the transmission vehicle (including water purification and pasteurization); increased awareness of personal hygiene; and the use of face masks, gloves, and other physical barriers to the spread of infectious agents.

6. In developed countries such as the United States, infectious disease accounts for a relatively low percentage of deaths (usually less than 10%). In contrast, infectious disease may account for up to 60% of mortalities in developing countries.

7. Diseases that suddenly become prevalent are called emerging diseases, infectious diseases whose incidence recently increased or whose incidence threatens to increase in the near future. Reemerging diseases are diseases that were previously under control but suddenly appear as a new epidemic.

Tuberculosis: Antimicrobial drug resistance Dengue: Poor mosquito control; increased urbanization in tropics; increased travel and shipping Candidiasis: Immunosuppression; medical devices (catheters); antibiotic use Virulence-enhancing factors are often carried by bacteriophages (e.g., erythrogenic toxin), plasmids (e.g., pili colonization factor), and transposons (e.g., aerobactin siderophores) as mobile genetic elements that can be transferred between and among members of the same species, and sometimes to other species and genera.

8. Effective candidates for biological warfare weapons must possess the following characteristics: (1) easy to produce and deliver, (2) safe for use by those delivering them, and (3) able to incapacitate or kill the individuals under attack. Smallpox is a strong candidate for bioweapons due to the ease with which it can be spread and its high mortality rate (~30%). Bacillus anthracis has several properties that make it desirable as a biological weapon, including its ability to form endospores that enhance dissemination and allow the organism to remain viable in the potentially harsh conditions it may encounter before reaching a suitable environment for growth. In addition, inhalation anthrax has a mortali-

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ty rate approaching 100%. Other potential bioweapons include Clostridium botulinum toxin, Yersinia pestis, and Francisella tularensis.

9. The major risk factors for acquiring human immunodeficiency virus (HIV) include multiple-partner sexual activity and intravenous drug abuse. Although initially a major risk factor, the acquisition of HIV from contaminated blood transfusion products is now extremely rare. Based on these factors, the source of pathogen exposure is the transfer of body fluids, and multiple-partner sexual activity leading to high incidence of AIDS is the norm in many developing regions.

10. Cholera is endemic in Africa, Southeast Asia, the Indian subcontinent, and Central and South America. The two distinct pandemic strains of V. cholera are the classic and the El Tor biotypes.

11. Influenza A H5N1 is the avian influenza for which few have immunity. It is of significant concern because of its ease of transmission from birds (or swine), its high mortality rate (nearly 60%), and the fact that it could infect swine and cause the emergence of a new reassortant strain for which there is no immunity in humans.

Answers to Application Questions 1. A disease that primarily infects animals but is occasionally transmitted to humans is called a zoonosis. Factors leading to the emergence of zoonotic disease include the existence and propagation of the infectious agent in an animal host, the proper environment for propagation and transfer of the agent, and the presence of the new susceptible host species.

Pasteurization of milk is also important because milk was the main vehicle for transmission of bovine tuberculosis to humans. Carriers can be identified using diagnostic techniques; culture or immunoassay surveys are conducted in populations to identify carriers. Examples where the reservoirs are domestic animals include brucellosis and bovine tuberculosis in humans and bovine spongiform encephalitis (mad cow disease). Immunization or destruction of infected animals may eliminate the disease in animals and in humans. Index diseases are diseases that exhibit unusually high incidence, new syndromes, or are linked to new or evolving pathogens and have high potential for causing new epidemics.

2. Answers will vary. See Section 28.8 and Table 28.8.

3. Prior to 1981, dengue fever was unknown in the Americas as the mosquito vector Aedes aegypti was not present. However, by 2003 the mosquito and dengue virus were present in all Central American and tropical South American countries, possibly imported into tropical population centers from Africa via cargo ships that transported infected mosquitoes along with trade goods. One of the filoviruses was imported into Marburg, Germany, in 1967 with a shipment of African green monkeys used for laboratory work. The virus spread from the primate host to some of the human handlers. Seven of 31 infected people died in this outbreak of what became known as the Marburg virus.

Basic reproduction number (R0) is the number of expected secondary transmissions from each single case of a disease in an entirely susceptible population. Herd immunity is the resistance of a population to a pathogen as a result of the immunity of a .

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large portion of the population. Observed reproduction number, R, calculated from studies of actual disease spread, is a more useful term because it takes into account observed transmissions from infected to susceptible individuals. The mathematical models that predict R0 may not take into account such factors as numbers of recovered individuals, population density (close contact), length of contact time, populations of high-risk individuals, and other variables that may affect disease spread. As a result, R0 can only estimate theoretical infectivity. For the SARS outbreak of 2003, the observed R was 3.6.

Public health officials, recognizing the potential for a serious epidemic, instituted infection controls such as isolation of infected individuals and strict barrier protection for healthcare personnel. These measures reduced the SARS R value to 0.7, ending the threat of further disease spread.

4. The majority of rabies cases in the United States are in wild animals and its eradication would require the immunization or destruction of all wild animal reservoirs, including such diverse species as raccoons, bats, skunks, and foxes, which is impractical. When insects such as the mosquito vectors that transmit malaria are also required hosts, effective control of the disease can be accomplished by eliminating the vector–host with insecticides or other agents. The alternative remedial measures include vaccines, mass immunizations, isolation, quarantine, and surveillance.

The CDC in the United States is the agency of the United States Public Health Service that tracks disease trends, provides disease information to the public and to healthcare professionals, and forms public policy regarding disease prevention and intervention. The CDC operates a number of infectious disease surveillance programs like the National Notifiable Diseases Surveillance System, which provides a database that spots nationwide trends and allows for national healthcare planning; healthcare-associated infections are reported to the National Healthcare Safety Network (NHSN). The Select Agents Program handles reports of potential bioterror incidents; emerging worldwide infectious disease outbreaks are reported to the Global Emerging Infections Sentinel Network (GeoSentinel). The overall goal of surveillance is to formulate and implement plans for diagnosis and treatment of infections.

The CDC’s notable public health achievements are in the areas of immunizations, motor-vehicle safety, workplace safety, control of infectious diseases, declines in deaths from heart disease and stroke, safer and healthier foods, healthier maternity care, family planning, fluoridation of drinking water, and tobacco as a health hazard. Its challenges for the future include concerns about obesity, heart diseases, diabetes, eradication of polio, multi antibiotic resistant strains, cancer drugs, and HIV treatment.

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CHAPTER

29

Person-to-Person Bacterial and Viral Diseases

Summary Chapters 29–32 discuss common human pathogens within the contexts of pathology, treatment, and prevention. Introduce Chapter 29 by first reminding students of the beneficial and essential roles played by microorganisms in nature, industry, and as commensal microflora in the human body (Chapters 11, 20, and 23). Emphasize that only a small percentage (<0.1%) of microorganisms actually cause disease. However, of the microorganisms that are pathogenic, many are serious threats to human health, and therefore the importance of research and education in this area cannot be overstated. The discussions in this chapter will focus on (1) airborne transmission, (2) direct contact transmission, and (3) sexual transmission of prevalent diseases caused by bacteria and viruses.

29.1–29.2 | Airborne Pathogens and Streptococcal Diseases Figure 29.1 shows a dramatic example of the instant dissemination into the air of up to 100,000 infectious bacteria by a single, ordinary human action⎯a sneeze. Point out the scale of airborne transmission, as the size of the particles containing a given microorganism dictates the degree to which it can penetrate the respiratory tract and also determines the kind of infections that can occur in a given region of the respiratory system (Figure 29.2).

The significance of extremely small particle size to the airborne dispersal of an infectious agent became shockingly apparent to the general public with the discovery of anthrax spores disseminated through the mail in 2001 (Section 28.8). Based on the velocity of air moving into the lungs, only particles having a diameter of less than 3 m are able to travel deep into the bronchioles of the lower respiratory tract (Figure 29.2). Most respiratory pathogens are transmitted person-to-person via the respiratory route or through direct contact and are dependent upon human hosts for propagation.

Discuss the important streptococcal pathogens (Section 29.2), including Streptococcus pneumoniae, and Streptococcus pyogenes (Figure 29.3). Some streptococci, such as Streptococcus equi, are -hemolytic and can cause serious pyogenic (pus-forming) wounds (Figure 29.4).

You may want to review the general characteristics of streptococci with the students (nonsporulating, homofermentative, aerotolerant anaerobic, gram-positive cocci; Section 15.6). Focus your discussion on the epidemiology and pathogenesis of S. pyogenes, also called group A streptococci (GAS). Students will be surprised by the number of clinical conditions this bacterium is capable of causing: streptococcal pharyngitis (strep throat; Figure 29.5), otitis media .

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(middle ear infection), mastitis (mammary gland infection), impetigo (Figure 29.6; can also be caused by S. aureus), erysipelas (Figure 29.7), scarlet fever (see Figure 29.9), rheumatic fever, toxic shock syndrome, acute glomerulonephritis (kidney infection), and some of the infamous necrotizing fasciitis (“flesh-eating bacteria”) cases (see Figure 29.10). List the important genetic and physiological properties possessed by streptococci that account for their virulence and diverse pathogenic effects: • Production of -hemolytic toxins that lyse red blood cells (Figure 29.8) • The presence of a lysogenic bacteriophage that encodes a pyrogenic exotoxin that is responsible for producing symptoms of streptococcal toxic shock syndrome (STSS) and scarlet fever • Necrotic tissue destruction resulting from the combined action of superantigens, including exotoxins A and B and the M protein (see Chapters 24 and 25) • Follow-up diseases (i.e., delayed sequelae) due to untreated infections of S. pyogenes strains possessing humanlike cell-surface antigens, resulting in immune response crossreactivity with self antigens. Examples of these autoimmune diseases include rheumatic fever and acute poststreptococcal glomerulonephritis.

• No effective vaccine is available for S. pyogenes, although an effective multivalent vaccine is available for the most common of the 90 known S. pneumoniae serotypes (strains having variation in capsular antigens). Antibiotic treatments (particularly vancomycin, penicillin and its derivatives, and the macrolide erythromycin) are effective against most streptococci.

• Many strains of S. pneumoniae are encapsulated, which increases invasiveness and allows for evasion of phagocytosis (Figure 29.11)

29.3 | Diphtheria and Pertussis Corynebacterium diphtheriae, the causative agent of diphtheria (Figure 29.12), induces severe, and often fatal, respiratory disease, primarily in children (Section 29.3). Active infection results in destruction of tissues of the pharynx by diphtheria exotoxin (an inhibitor of protein synthesis; Figure 23.20) and eventual death by suffocation and tissue destruction from pseudomembrane formation in the throat (Figure 29.13). Students may be familiar with the childhood toxoid DTaP (diphtheria, tetanus, and acellular pertussis) vaccine but will have little knowledge of its significance in reducing child mortality rates in the United States. Although the worldwide incidence is currently low due to vaccination efforts, there are still over 5000 fatal cases per year. Effective treatment includes the administration of antibiotics (e.g., penicillin, erythromycin, or gentamicin) and/or antitoxin derived from antibodies produced in horses (given for acute cases only).

Bordetella pertussis, the causative agent of pertussis, or “whooping cough,” produces both a potent cyclic AMP synthesis-inducing exotoxin and an endotoxin primarily responsible for the painful cough. Although a vaccine is available (a component of the DTaP vaccine), incidence of this highly communicable disease has been steadily increasing in the United States, perhaps due to slackened vaccination protocols (Figure 29.14). Antibiotic treatment using ampicillin, tetracycline, or erythromycin is beneficial. However, a proper immune response appears to be necessary to rid the body completely of the infectious agent.

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29.4 | Tuberculosis and Leprosy Tuberculosis (TB) as an airborne disease is a particularly interesting story, primarily due to its ease of transmission. Point out to students that TB is one of the most prevalent and dangerous diseases in the world—over one-third of the world’s population has been infected with Mycobacterium tuberculosis! Note that person-to-person transmission can occur even from normal conversation. Discuss the pathology of infection with this gram-positive, acidfast bacterium (Figure 29.15), including its ability to survive and grow within the lysosomes of macrophages. An acute pulmonary infection occurs in persons with low resistance and is invariably fatal following the spread of the pathogen from the lungs to other parts of the body. Fortunately, most infections remain localized in the form of tubercles, which are aggregates of activated macrophages (Figure 29.16), and from this response the tuberculin test was developed. Point out to students that immunity from M. tuberculosis is lifelong.

Because of the highly contagious nature of TB, prevention and treatment strategies must be strictly followed and monitored. Describe the treatment strategy for eradicating the infection, stressing the point that failure to complete the 9-month antibiotic course (a combination of rifampin and isoniazid; Figure 29.17) often results in reactivation of the disease and antibiotic resistance of the resident pathogen.

Discuss the pathology of leprosy (also called Hansen’s disease), caused by Mycobacterium leprae. Differentiate between lepromatous leprosy and tuberculoid leprosy, the former being the much more severe form that leads to disfigurement and loss of motor control (Figure 29.18). Note that although the incidence of the disease is low in many parts of the world, including the United States, about 750,000 new cases are reported annually worldwide (only 100 cases are reported in the United States annually). Considering that more than 2 million people are permanently disabled from leprosy and an estimated 12 million cases may go unrecognized due to the long latent period of the disease, M. leprae should still be considered a serious threat to human health.

29.5 | Meningitis and Meningococcemia Students may remember news stories of outbreaks of meningococcal meningitis on college campuses. However, they probably know little about the epidemiology and pathology of the infectious agent, Neisseria meningitidis, a gram-negative, obligately aerobic, oxidasepositive, encapsulated diplococcus (Figure 29.19a). At least thirteen different strains exist and are distinguished by antigenic variations in capsular polysaccharides.

The nasopharynx of about 30% of individuals harbors this organism asymptomatically, and this level rises to 80% during epidemics. Meningitis is a general term describing inflammation of the meninges, a condition that may be caused by bacterial, viral, fungal, or protozoan infections. Upon entry into the bloodstream, N. meningitidis bacteremia resulting from meningococcemia can lead to coagulation in vascular tissue, gangrenous tissue destruction (Figure 29.19b), shock, and death in over 10% of cases. Besides N. meningitidis, causes of acute meningitis include pyogenic bacteria, such as Staphylococcus spp., Streptococcus spp., and Haemophilus influenzae, as well as several viruses. In general, viral meningitis is less severe than bacterial meningitis.

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29.6–29.8 | Airborne Viral Diseases Students usually find the information presented in these sections relevant and interesting.

Although viral respiratory diseases are highly infectious and potentially life-threatening, many common childhood viral diseases are controllable with proper immunizations (e.g., measles, mumps, rubella, and chickenpox; Section 29.6). Discuss the dramatic results obtained from the use of the MMR (measles, mumps, and rubella) vaccine on the prevalence of these major childhood diseases in the United States (Figures 29.20–29.23). Chickenpox is a common childhood disease and thus will be familiar to many students. The causative agent, the varicella-zoster virus (VZV), is a dsDNA herpesvirus and is highly contagious. Decades ago, it was common practice for parents to “self-inoculate” their children with the virus by purposely exposing them to an infected child as a protective measure against shingles, the manifestation of zoster virus in adults. However, an attenuated vaccine is now available in the United States, and as a result, there are about one-fourth the number of cases reported each year since the vaccine was licensed for use in 1995.

Colds (Section 29.7) occur more commonly than all other infectious diseases combined.

The “common cold” is appropriately named because each of us averages more than three colds per year. Discuss the array of causative agents responsible for colds: (1) rhinoviruses (~75%), (2) coronaviruses (~15%), and (3) other viruses (~10% adenoviruses, coxsackieviruses, respiratory syncytial virus, and orthomyxoviruses) (Figure 29.24). Although antirhinovirus drugs that inhibit adherence of virions to host cell binding sites have been developed, most medications simply treat the symptoms of colds. Point out to students that cold symptoms are distinct from those of influenza infection, as this is often a point of confusion (Table 29.1).

If Chapter 9 has been covered in the course, students will already be somewhat familiar with the influenza virus structure (an RNA virus of the orthomyxovirus group; Section 29.8 and Figures 29.25 and 29.26). Review the segmented genome of influenza A virus, the most important variant from a pathogenic standpoint, and discuss the mechanisms by which the virus undergoes antigenic shift and antigenic drift. Provide an overview of the epidemiology, pathogenesis, and treatment for infection by influenza virus with the students. Antigenic determinants on the virus, hemagglutinin (H) and neuraminidase (N) proteins (Figure 29.26) stimulate strain-specific secretory IgA antibody.

Explain to your students that influenza epidemics occur in two- to three-year cycles due to antigenic drift, and pandemics occur every 10 to 40 years as a result of antigenic shift (Table 29.2). The major pandemics of 1918 and 1957 likely originated from virulent mutant strains of the original virus that underwent gene reassortment in an animal reservoir (Figures 29.27 and 29.28). The prevailing thought is that the strains originated from the reassortment of avian and human viruses in swine, which have receptors for both avian and human orthomyxoviruses (Figure 29.27). A new avian influenza strain (Influenza A H5N1) appeared in Hong Kong in 1997 and has been the focus of news stories reporting the threat of a potential “bird flu” pandemic. As discussed in Chapter 28, through 2012, 610 cases of human H5N1 infections have been reported, with a mortality rate of almost 60% documented.

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Treatment options for influenza are somewhat limited due to the plasticity of the influenza genome, and therefore most efforts focus on surveillance and immunizations. Polyvalent vaccines prepared against the major emerging strains (identified through worldwide surveillance) allow for yearly influenza immunization. Annual immunization is especially recommended for those over 50 years of age, sufferers of chronic debilitating diseases (e.g., AIDS), and healthcare workers. Antiviral drugs that inhibit viral replication (e.g., amantadine and rimantadine) or block virion release from the host cell (e.g., oseltamivir and zanamivir) are also effective at preventing the spread and reducing the severity of influenza.

29.9–29.11 | Direct-Contact Diseases These sections discuss pathogens transmitted by direct contact. Note that this route can also spread some of the respiratory diseases described above. Staphylococcus epidermidis and Staphylococcus aureus are normal flora inhabitants of the skin and upper respiratory tract that can also function as opportunistic pathogens (Section 29.9). The most common staphylococcal diseases include acne, boils (Figure 29.29), impetigo, pneumonia, osteomyelitis, carditis, meningitis, and arthritis. Discuss the factors responsible for invasion and pathogenicity in staphylococci (hemolysins, enterotoxin, coagulase, leukocidin, and toxic shock syndrome toxin-1 [TSST-1 superantigen]). Note that S. aureus infections are common in hospitals, an environment that selects for strains with resistance to multiple antibiotics. Most carriers (including hospital personnel) are asymptomatic and easily transmit the strain to patients. One of the most dangerous of these strains is methicillin-resistant S. aureus (MRSA), of which more than 100,000 cases of infection are reported each year (Figure 29.30). Culture and identification of S. aureus and other staphylococci are aided by the use of selective and differential media. For example, mannitol-salt agar contains 7.5% NaCl, a property that inhibits most microorganisms associated with the human body but allows for growth of Staphylococcus spp., which are halotolerant (Figure 29.31).

Helicobacter pylori is a gram-negative, motile spirillum (Figure 29.32) that is associated with gastritis, ulcers, and gastric cancers (Section 29.10). H. pylori is the most commonly found species in the microflora of the stomach. The colonization of the stomach wall at pH 2 by H. pylori results in infection leading to peptic ulcers. The medical community largely dismissed the first suggestion of a microbial cause for this disease and assumed ulcers to be caused by stress and/or other psychologically induced factors. We now know that the host’s immune response to colonization by H. pylori, in addition to the resulting inflammation and tissue damage, is the actual cause of ulcers. Point out to students that H. pylori is not invasive in the strict clinical sense, but it is found in ~80% of patients with ulcers. The mode of transmission has not been confirmed but may involve host-to-host contact and contaminated food and water. Some evidence suggests that it may be able to be spread from pets, such as cats living in close contact with the host. Note also that incidence of H. pylori infection is high in some families and increases with age. Tissue destruction and ulceration of gastric mucosa result from the effects of a cytotoxin (vacA), urease, and lipopolysaccharide. Chronic gastritis occurs in untreated infections, and this may eventually develop into gastric cancer.

However, a combination treatment of metronidazole, tetracycline (or amoxicillin), and an antacid preparation administered over a two-week period provides a long-term cure.

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Infection by hepatitis viruses can lead to acute or chronic (e.g., cirrhosis) disease that may cause liver cancer in some cases (Section 29.11). Discuss the biology and epidemiology of the hepatitis viruses shown in Table 29.3 and Figure 29.33. Note that the different classes of hepatitis viruses are not genetically related, but all share the ability to infect liver cells. HBV (Figure 29.34) and HCV cause chronic and oncogenic disease, and an effective vaccine exists for the former but not the latter. A vaccine is also available for HAV. Discuss the prevention and treatment strategies for these diseases with your students. A summary of the general characteristics of hepatitis viruses is provided below: • HAV (infectious hepatitis): ssRNA genome; transmitted person-to-person or by ingestion of fecally contaminated food (often shellfish) and water. Historically, HAV has had the highest incidence of any hepatitis virus, although this is declining because of the development of an effective vaccine.

• HBV (serum hepatitis): dsDNA genome (hepadnavirus); transmitted person-to-person by the transfer of body fluids (e.g., via blood transfusions, sexual intercourse, or shared hypodermic needles). Causes acute disease that can lead to liver failure and/or cancer and death. An effective vaccine is available.

• HCV: ssRNA genome; transmitted by a parenteral route (outside the gut). Causes the development of chronic liver disease that can eventually cause liver cancer after an extended latent period. No vaccine is available.

• HDV: ssRNA genome; a defective virus lacking genes for its own protein coat; replication occurs only with coinfection by HBV. No vaccine is available.

• HEV: ssRNA genome; transmitted via an enteric route and causes an acute but selflimiting hepatitis. Endemic in tropical and subtropical regions. No vaccine is available.

29.12–29.14 | Sexually Transmitted Infections The topic of sexually transmitted infections (STIs) is particularly relevant to the student population, given the reality that about one-third of all STIs occur in teenagers having multiple sexual partners. Discuss the social reasons for the failure of individuals to obtain treatment, resulting in the continuing spread of these diseases. Stress the fact that most STIs are curable or treatable if treatment is started early and that failure to treat can lead to very serious medical conditions (birth defects, stillbirth, degenerative nerve disease, cancer, heart disease, and infertility). Introduce the array of causative organisms and their diseases, shown in Table 29.4. Direct students to material in Chapter 15 for more information on each organism, and provide a detailed discussion of the prevalent STIs presented in these sections.

Compare the different incidence patterns of gonorrhea and syphilis (Section 29.12). Note the downward trend in the incidence of these two diseases since the introduction of antibiotics, followed by an upward trend in gonorrhea incidence after the introduction of birth control pills during the 1960s (Figure 29.35). The story of Neisseria gonorrhoeae (Figure 29.36) is a real-world example of the problems incurred by the overuse of antibiotics. In this case, lateral transfer of a plasmid-encoded penicillinase selected for increasing numbers of resistant strains of N. gonorrhoeae during the 1980s, prohibiting the use of penicillin to effectively treat the disease. The general downward trend of gonorrhea incidence has leveled off in re.

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cent years, and more than 350,000 new cases of gonorrhea are still reported annually in the United States. The prevalence of N. gonorrhoeae infection is partly due to periods in which the disease may be asymptomatic, especially in women. Because coinfection with Chlamydia trachomatis is common and difficult to diagnose (see Section 29.13), about one-half of gonorrhea patients are also given antichlamydial antibiotics, such as azithromycin, during treatment.

Syphilis, caused by the spirochete Treponema pallidum (Figure 29.37), is much more serious than gonorrhea and more difficult to treat. For example, syphilis kills about 100,000 people per year, whereas gonorrhea kills about 1000 per year. As is the case with Neisseria gonorrhoeae, T. pallidum is also extremely sensitive to environmental stress (e.g., drying, ultraviolet radiation, and sunlight), and therefore syphilis is transmitted via intimate sexual contact through tiny breaks in the epidermal layer of the skin. Note also that this organism can be transmitted from an infected woman to the fetus, causing congenital syphilis in the latter. Summarize the pathobiology of this complex disease by first pointing out the characteristic lesion called a chancre that develops at the initial site of infection during the primary syphilis stage (Figure 29.38a and b). Spread of the pathogen to other parts of the body produces a skin rash during secondary syphilis (Figure 29.38c). At this point, the patient may be cured spontaneously, endure a low-level chronic infection, or, as is usually the case, may develop tertiary syphilis, which can lead to fatal infections of the cardiovascular system or central nervous system. Refer the students to Section 14.20 for a review of the microbiology of T. pallidum.

Three STIs that are often more difficult to diagnose and treat than gonorrhea and syphilis are chlamydia, herpes, and human papillomaviruses (HPVs). Summarize the major characteristics of each disease and causative agent (Section 29.13): • Chlamydia trachomatis: obligate intracellular bacterium; the most prevalent cause of sexually transmitted disease; diagnosed with EIA tests and monoclonal antibodies; treatment with azithromycin or doxycycline (Figure 29.39).

• Herpes simplex viruses (HSV-1 and HSV-2): HSV-1 usually causes fever blisters and cold sores but occasionally infects the anogenital region (Figure 29.40); HSV-2 primarily infects the anogenital region (Figure 29.41); long-term infection correlates highly with cervical cancer; presently incurable but symptoms (blistering lesions) are successfully treated with nucleoside analogs, such as acyclovir (Figure 29.42).

• Human papillomaviruses (HPVs): a family of dsDNA viruses; about 30 strains are transmitted sexually, with several that cause genital warts and/or cervical cancer. Note that about 20 million people in the United States are infected and over 6 million new infections occur annually. Almost 10,000 women develop cervical cancer, resulting in about 3700 deaths per year. Because most individuals are asymptomatic, the virus is easily transmitted via sexual contact. A vaccine has been developed that could provide immunity from most oncogenic strains and is recommended for females 11–26 years of age.

The epidemiology of AIDS was discussed in Section 28.9, and the disease is addressed in more detail in Section 29.14. Provide students with a detailed discussion of HIV infection, including its pathogenesis, immune system interactions, and treatment (Figures 29.43–29.48).

Point out that two forms of the virus exist⎯HIV-1 and HIV-2. More than 99% of AIDS cases result from HIV-1 infection. HIV-2 was discovered in West Africa in 1985, is less virulent than HIV-1, and causes a milder form of AIDS. .

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Pathogenesis involves infection by HIV of cells containing the CD4 cell-surface protein, typically macrophages and T-helper (Th) cells (Figure 29.43). Note that in addition to CD4, HIV infection requires interaction with target cell coreceptors. The final result of HIV infection is the progressive decline in CD4 Th lymphocyte number (Figure 29.44). Point out to students that CD4-displaying cells in healthy individuals make up about 70% of the T cell pool, but in AIDS patients, these cells may be nearly absent.

The original correlation of AIDS to compromised immune system function came from the large number of opportunistic infections observed only in individuals with a defective immune system (Figures 29.45 and 29.46). Discuss with students the current accepted definition of acquired immunodeficiency syndrome, as documented by the Centers for Disease Control and Prevention. Note the quantitative and specific nature of the AIDS definition, indicative of the extensive amount of data collected over the past ~30 years that has allowed a precise description of AIDS and HIV infection.

Also discuss the present methods of diagnosis and treatment of HIV, in addition to the methods used to monitor HIV load (the number of viral particles present in the blood; Figure 29.47). Discuss the chemotherapeutic agents currently used for HIV/AIDS treatment: nucleoside reverse transcriptase inhibitors (NRTIs; Figure 29.48a), nonnucleoside reverse transcriptase inhibitors (NNRTIs; Figure 29.48b), protease inhibitors (Figure 29.48c), fusion inhibitors, and integrase inhibitors, a newer category of anti-HIV drugs that prevents the integration of retroviral DNA into the host genome. The primary tools used to prevent HIV/AIDS continue to be public education and avoidance of high-risk behavior.

Answers to Review Questions 1. Upper respiratory tract: Haemophilus influenza, common cold virus, Streptococcus pyogenes, Corynebacterium diphtheria Lower respiratory tract: Mycobacterium tuberculosis, Streptococcus pneumonia, Bordetella pertussis, Influenza virus 2. Streptococcal respiratory infections caused by S. pneumoniae or S. pyogenes are usually initiated due to a weakening of host defenses, as these organisms are normally found in the respiratory tract of up to 40% of normal individuals. Symptoms of streptococcal pharyngitis are sore throat, enlarged tonsils, mild fever, and general malaise. Prompt treatment is essential because encapsulated strains of S. pneumoniae are extremely invasive and resist phagocytosis, and systemic infections by either organism lead to serious complications (scarlet fever, rheumatic fever, bone infections, toxic shock syndrome, etc.).

3. Diphtheria is caused by the gram-positive, aerobic, nonmotile bacterium Corynebacterium diphtheriae. The organism enters the body via the respiratory route, lodging in the throat and tonsils. The inflammatory response of throat tissues to C. diphtheriae results in a characteristic lesion called a pseudomembrane, which may block the air passage and result in death. Some C. diphtheriae strains are lysogenized and produce a powerful exotoxin (diphtheria toxin). Pertussis is caused by the gram-negative, aerobic coccobacillus Bordetella pertussis. This highly infectious respiratory disease is initiated by attachment of the pathogen via a specific adherence factor, followed by production of pertussis exotoxin. This exotoxin induces synthesis of cyclic AMP, which is partly responsible for tissue damage. An effective combined vaccine exists for diphtheria and .

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pertussis that is part of the toxoid DTaP vaccine. Because pertussis is more infectious than diphtheria, a higher percentage of vaccinated individuals is required to achieve herd immunity. Therefore, a lack of appropriate vaccination of all ages may cause the observed increases in incidence of pertussis in recent years (Figure 29.14b).

4. Mycobacterium tuberculosis is initially introduced into the lungs by inhalation of droplets containing the organism or by inhalation of dust particles that have formed from dried droplets. The bacteria settle in the lungs and grow. In some cases, aggregates of activated macrophages called tubercles form, and this may cause the development of an acute pulmonary infection. This leads to extensive destruction of lung tissue, spread of the pathogen to other parts of the body, and death. In most cases, however, an acute infection does not develop from the initial exposure. These individuals develop hypersensitivity to the pathogen, and upon further exposure to the organism, may develop chronic infections that involve destruction of lung tissue followed by partial healing and spread of the lesions within the lungs. A diagnostic skin test called the tuberculin test is used to detect the hypersensitivity that develops from previous exposure to M. tuberculosis (Figure 24.6).

5. The symptoms of meningococcemia include septicemia, intravascular coagulation, shock, and death in over 10% of cases. Meningitis is another possible serious outcome of infection and is characterized by sudden onset of headache, vomiting, and stiff neck; it can progress to coma and death in hours. Penicillin G is the drug of choice for Neisseria meningitidis, and chloramphenicol is given to penicillin-sensitive individuals.

6. During the first three months of pregnancy, rubella virus can infect the fetus by placental transmission and cause serious fetal abnormalities including stillbirth, deafness, heart and eye defects, and brain damage, events called congenital rubella syndrome. Mumps are measles are caused by paramyxoviruses. Attenuated vaccines are used for rubella, mumps, measles, shingles, and chicken pox.

VZV remains dormant indefinitely in nerve cells, and sometimes migrates from this reservoir to the skin surface, often years or decades later, causing shingles (zoster). It strikes immunosuppressed individuals or the elderly. A fairly effective shingles vaccine containing concentrated attenuated virus (Zostavax) is available for individuals over 50 years of age. The vaccine stimulates antibody and cell-mediated immunity to VZV, keeping VZV from migrating out of nerve ganglia to skin cells and triggering shingles.

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7. Rhinoviruses are single-stranded plus-sense RNA viruses of the picornavirus group and are the most common causes of colds. About one-quarter of all colds are due to infections with other viruses. These include in particular the coronaviruses. Adenoviruses, coxsackie viruses, respiratory syncytial viruses (RSV), and orthomyxoviruses are collectively responsible for only a small percentage of common colds. There are three classes of influenza viruses: influenza A, B, and C, of which influenza A is the most important human pathogen. Each virus has one type of HA and one type of NA on its viral capsid.

The HA antigen is important in attaching the influenza virus to host cells while the NA antigen is instrumental in releasing the virus from host cells; each antigen is composed of several individual proteins.

Influenza viruses are particularly dangerous pathogens because they are easily spread in infectious droplets and their genetics allow them to rapidly become transmissible in new hosts. Health officials today are concerned that new highly virulent influenza viruses may be formed from the exchange of genes between different strains. It is well known that influenza viruses that infect humans can also infect birds and swine. When an animal is infected by more than one strain, their genes can mix—a process called reassortment—to form viruses with new properties. Unique reassortant viruses trigger the phenomenon known as antigenic shift, a major change in a surface antigen resulting from the total replacement of the RNA that encoded it.

Slight variation in the structure of influenza viral surface antigens is at the heart of a phenomenon in influenza biology called antigenic drift. As a result of these subtle yet important changes, host immunity to a given virus strain diminishes as the strain mutates, and reinfection with the mutated strain can occur. Cold symptoms include rhinitis (inflammation of the nasal region, especially the mucous membranes), nasal obstruction, watery nasal discharges, muscle aches, and a general feeling of malaise, usually without fever. Symptoms of influenza include a low-grade fever lasting up to a week, chills, fatigue, head and muscle aches, a cough and/or a sore throat, and general malaise.

8. Influenza is common primarily because of the plasticity of the influenza genome. This allows new strains to develop rapidly via antigenic drift. Polyvalent vaccines are developed yearly based upon three inactivated, candidate viral strains (two influenza A and one influenza B) identified from global surveillance data.

9. Staphylococci are common pathogens of humans and animals, and most infections result from transfer of these species from the normal flora of an infected but asymptomatic individual to a susceptible individual. Although both Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens, S. aureus is the more virulent of the two and is most commonly associated with human disease. The types of harmful products produced by the particular Staphylococcus strain determine virulence (e.g., hemolysins, enterotoxin, coagulase, leukocidin, and toxic shock syndrome toxin-1).

10. Pathogen products such as the cytotoxin VacA (an exotoxin), urease, and an autoimmune response triggered by H. pylori lipopolysaccharide all contribute to localized tissue destruction and ulceration. A simple diagnostic test for the H. pylori enzyme urease is used for a noninvasive diagnosis. In this test, a small amount of 13C− or 14C−labeled urea (H2N–CO–NH2) is ingested; if H. pylori is present, the bacterium will hydrolyze the urea,

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forming labeled CO2 and ammonia. Hence, the presence of labeled CO2 in the patient’s breath is highly suggestive of H. pylori infection.

11. The hepatitis viruses are grouped into six major classes, five of which are pathogenic.

Hepatitis viruses are a diverse group, and none of them are phylogenetically related to one another. However, all infect cells in the liver, causing various forms of hepatitis that often lead to similar symptoms. Depending on the class of virus, they are spread by direct contact or from a common source, such as contaminated food or water.

12. In general, sexually transmitted diseases have risen markedly during the past 30 years due to changing perceptions of acceptable sexual behavior. This, coupled with the widespread use of oral contraceptives and the availability of effective antibiotic therapy, has led to increased sexual activity and carelessness. In the case of syphilis, visibly apparent symptoms (e.g., the formation of chancre lesions) develop in infected individuals, and there is a greater fear of the consequences of untreated infections, which are lifethreatening. Hence, treatment is usually promptly sought, and the infection is controlled with penicillin. By contrast, although the gonococcus causes a painful urethral infection in the male, symptoms in the female are often not particularly noticeable. Thus, one female with multiple partners who does not seek treatment may infect a large number of males. Another important factor is that the gonococcus does not confer immunity, and this lack of acquired immunity permits reinfection.

13. Chlamydia:

Bacterial; very high incidence; effective treatment possible using azithromycin or doxycycline.

Genital herpes:

Viral; moderate incidence; incurable, but symptoms are treated us ing nucleoside analog drugs, such as acyclovir.

HPV:

Viral; very high incidence; oncogenic; no effective treatment is available for active infections, but a vaccine is now available to provide immunity from some strains.

14. An AIDS patient is one who tests positive for HIV in immunological and/or nucleic acidbased tests and meets at least one of the following two criteria: a. A CD4 T cell number of less than 200/μl of whole blood (the normal count is 600– 1000/μl) or a CD4 T cell/total lymphocytes percentage of less than 14%. b. A CD4 T cell number of more than 200/μl and any of the following diseases: candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, isosporiasis, Pneumocystis jiroveci pneumonia, cryptosporidiosis, or toxoplasmosis of the brain (all fungal diseases); pulmonary tuberculosis or other mycobacterial infections, or recurrent Salmonella septicaemia (bacterial diseases); cytomegalovirus infection, HIV-related encephalopathy, HIV wasting syndrome, chronic ulcers, or bronchitis due to herpes simplex (viral infections); or certain malignant diseases such as invasive cervical cancer, Kaposi’s sarcoma, Burkitt’s lymphoma, primary lymphoma of the brain, or immunoblastic lymphoma, or recurrent pneumonia due to any agent.

Effective anti-HIV drugs fall into four categories, including two classes of reverse transcriptase inhibitors, various protease inhibitors, fusion inhibitors, and integrase inhibitors. Highly active anti-retroviral therapy (HAART) is carried out by administering at

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least three anti-retroviral drugs at once to inhibit the replication of HIV and prevent the development of drug-resistant strains.

Answers to Application Questions 1. Many strains of viruses from multiple virus families are capable of causing the symptoms associated with the common cold, and therefore immunity to all of them never develops.

In contrast, a case of measles usually confers lifelong immunity upon recovery of the patient since the disease is caused specifically by rubeola, a paramyxovirus.

2. Mycobacterium leprae, which causes leprosy (Hansen’s disease), is grampositive bacteria and has the waxy mycolic acid as its cell walls constituent. Pathogenicity in the disease is due to a combination of delayed-type hypersensitivity and the highly invasive activities of M. leprae, which can grow within macrophages and lead to the characteristic lesions.

Tuberculoid leprosy is characterized by a vigorous immune response and a good prognosis for spontaneous recovery. It is treated using a multiple drug therapy protocol, which includes some combination of extended therapy of up to 1 year with dapsone (4,4 ′sulfonylbisbenzeneamine, an inhibitor of folic acid synthesis), rifampin, a bacterial RNA polymerase inhibitor, and clofazimine, a drug that targets bacterial respiration and ion transport.

3. If a roommate is diagnosed with meningococcal meningitis, and you have no symptoms, you have probably already acquired strain-specific antibodies, which prevent infection in most adults. However, it is still advisable to inform university health officials of the situation so that they can monitor your condition and be aware of the very real possibility that other students on campus may become infected and develop serious symptoms.

4. Hepatitis D virus (HDV) is a defective virus that lacks genes encoding its own capsid and thus relies on HBV to produce capsid proteins (which are the same as those used by HBV) to form infectious virions. All four pathogens infect the liver. Hepatitis B (HBV) and Hepatitis C (HCV) are oncogenic. Diagnosis of hepatitis is based on a combination of clinical symptoms and laboratory tests that assess liver function, especially key liver enzymes. Cirrhosis is diagnosed by visual examination of biopsied liver tissue. The immunological and molecular diagnostic tools include enzyme immunoassays that target viral-specific proteins or antiviral antibodies in a blood sample, immunoblots (Western blots), and immunofluorescence (microscopic) methods. Polymerase chain reaction (PCR) tests are also used to detect hepatitis viral genomes in blood or in liver tissue obtained by biopsy.

5. MRSA causes pyogenic diseases. At least four different hemolysins (proteins that lyse red blood cells) have been recognized, and a single strain often produces several. One of its many virulence factors is coagulase, an enzyme that converts fibrin to fibrinogen, forming a localized clot. Clotting induced by coagulase results in the accumulation of fibrin around the bacterial cells, making it difficult for host immune cells to contact the bacteria and initiate phagocytosis. Most S. aureus strains also produce leukocidin, a protein that destroys white blood cells. Production of leukocidin in skin lesions such as boils and pimples results in host cell destruction and is one of the factors responsible for pus.

Some strains of S. aureus also produce other virulence proteins including hyaluronidase, fibrinolysin, lipase, ribonuclease, and deoxyribonuclease. The symptoms of TSS result .

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from an exotoxin called toxic shock syndrome toxin-1. This very potent toxin is a superantigen that is released during cell growth and recruits large numbers of T cells to the site of infection.

For specific identification of methicillin-resistant strains of S. aureus (MRSA), a special selective and differential medium containing 7.5% NaCl, mannitol, and phenol red, a pH indicator (mannitol–salt agar) is used along with PCR protocol that identifies mecA, the gene that encodes methicillin resistance in MRSA strains.

6. Nongonococcal urethritis (NGU) due to C. trachomatis is one of the most frequently observed sexually transmitted diseases. Both Neisseria gonorrhoeae and C. trachomatis are often transmitted to a new host simultaneously. However, treatment of gonorrhea does not eliminate the chlamydia. In this case the patient suffers from Lymphogranuloma venereum (LGV), an STD caused by distinct strains of C. trachomatis (LGV 1, 2, and 3).

The disease occurs most frequently in males and is characterized by infection and swelling of the lymph nodes in and about the groin. From the infected lymph nodes, chlamydial cells may travel to the rectum and cause a painful inflammation of rectal tissues called proctitis. Thus, patients treated for gonorrhea with drugs such as cefixime or ceftriaxone are also given azithromycin or doxycycline to treat a potential coinfection with C. trachomatis.

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CHAPTER

30

Vectorborne and Soilborne Bacterial and Viral Diseases

Summary This chapter focuses on diseases transmitted via animal, insect, and soil routes. The host for pathogens transmitted by animals is a nonhuman vertebrate, and the infected animals then transmit the pathogen to humans. Humans acquire vectorborne infections by serving as either accidental hosts or important reservoirs in the life cycle of the vectorborne pathogen. The final topic in the chapter discusses important soilborne pathogens, including species of the genera Clostridium and Bacillus.

30.1–30.2 | Animal-Transmitted Viral Diseases Vectorborne diseases, such as malaria and bubonic plague, have significantly influenced human populations over the course of history. This chapter discusses diseases transmitted by nonhuman reservoirs, many of which continue to emerge as serious human health problems. An animal disease that is transmissible to humans is referred to as a zoonosis. Animal diseases can be enzootic (endemic in certain populations) or epizootic if the incidence of the disease in the animal population reaches epidemic proportions.

Discuss the biology and epidemiology of rabies (Section 30.1), which cycles between enzootic and epizootic incidence as wild animal reservoir populations fluctuate (Figure 30.1).

The major enzootic reservoirs in the United States are raccoons, skunks, coyotes, foxes, and bats, although sporadic cases have been observed in some domestic animals. Remind students that rabies is caused by a rhabdovirus, a negative-stranded RNA virus (Section 9.9) that infects cells in the central nervous system and is fatal if left untreated (Figure 30.2). Summarize the guidelines for dealing with possible rabies virus exposure. Note that the key to rabies prevention in the United States lies in controlling the disease in wild animal reservoirs (Figure 30.1b). Eradication of the disease may be possible by delivering a vaccine, via food baits, to wild animal populations. This vaccine consists of genetically engineered strains of vaccinia or canarypox viruses that express rabies coat proteins as immunogens.

Hantavirus syndromes are caused by hantavirus transmission from infected rodents (Section 30.2) and include hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Note that the hantavirus group is a family of single-stranded RNA viruses (Section 9.9; Figure 30.3) related to the more notorious Ebola virus and Lassa Fever virus (see the feature box Explore the Microbial World: Handling Viral Hemorrhagic Fever Viruses). Biosafety level 4 (BSL-4) precautions are necessary for the study and manipu.

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lation of hantaviruses due to their especially lethal nature. A hantavirus outbreak in 1993 in the southwestern United States killed 32 people out of 53 infected individuals (>60% mortality) from acute respiratory failure caused by inhalation of the virus, which was carried by the deer mouse and present in mouse droppings. Pathogenesis of hantaviruses includes cardiac complications and shock due to pulmonary edema, and victims usually die within a few days. No treatment is currently available, and therefore preventive measures should be taken by limiting exposure to rodents and controlling rodent populations by destroying their habitats.

30.3–30.7 | Arthropod-Transmitted Bacterial and Viral Diseases Summarize the major characteristics of arthropod-transmitted pathogens. Recall that rickettsias are obligate intracellular parasitic bacteria that have life cycle stages in blood-sucking arthropods such as fleas and ticks (Section 30.3). Rickettsias have been divided into three groups according to the clinical disease they produce: (1) the typhus group includes Rickettsia prowazekii and Rickettsia typhi, which are transmitted through human lice (Figure 30.4a); (2) the spotted fever group includes Rickettsia rickettsii, which is transmitted through dog and wood ticks (Figures 30.4b, 30.5 and 30.6); and (3) the ehrlichiosis group includes a variety of rickettsias, such as Rickettsia sennetsu and Ehrlichia chaffeensis, that are transmitted through ticks (Figure 30.7). Discuss the pathology of each disease caused by these bacteria, noting that the only effective control methods require control of the arthropod vectors themselves.

You should also discuss Q fever, a pneuomia-like disease caused by Coxiella burnetii.

Domestic livestock animals serve as a reservoir for the pathogen, and thus infections are more common in agricultural areas, such as in significant parts of the Midwestern and Western United States.

Lyme disease (Section 30.4) is caused by the tickborne spirochete Borrelia burgdorferi (Figure 30.8). Discuss the epidemiology of the disease, and emphasize the variety of animals that the deer tick vector (Figures 30.9 and 30.10) parasitizes. Cases of Lyme disease in the United States have increased dramatically since the early 1980s, with the upper Midwest and East Coast states having the highest incidence (Figure 30.11). Transmission occurs while the tick is obtaining a blood meal (Figure 30.9a), and the resulting systemic infection develops into the acute symptoms of Lyme disease, including headache, backache, chills, and fatigue; often a characteristic circular bull’s eye rash referred to as erythema migrans develops at the site of the tick bite (Figure 30.9b and c). Pathogenesis includes neurological and cardiac damage and long periods of dormancy, followed by chronic seizures, vision problems, and facial paralysis. Prevention and treatment strategies require an accurate and timely diagnosis, and the treatment itself usually involves an intravenous antibiotic regimen.

Yellow fever and dengue fever (Section 30.5) are diseases caused by flaviviruses that are transmitted through the bite of infected mosquitoes (Figure 30.12). Because of their shared mode of transmission, these and several other encephalitic and hemorrhagic viruses are often called arboviruses (arthropod-borne viruses), although the term does not represent a taxonomic grouping. Yellow and dengue (pronounced deng-gay) fever are most prevalent in developing countries that have a tropical or subtropical climate. Cases of yellow fever range from asymptomatic to deadly, with the major diagnostic symptoms being jaundice and hemorrhaging from the mouth, eyes, and gastrointestinal tract. More serious symptoms may follow, including bloody vomit, toxic shock, and organ failure, and one in every five of such .

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cases is fatal. Although an effective vaccine for yellow fever is available, nearly 200,000 cases are estimated to occur every year. Although generally milder (but still potentially fatal), dengue fever is much more prevalent than yellow fever, with up to 100 million cases occurring worldwide per year. In more serious cases, dengue hemorrhagic fever can develop, and this can cause a fatal drop in blood pressure. Unlike yellow fever, there is no vaccine for dengue fever. Probably the best means of controlling the transmission of arboviruses is by limiting either the population of or contact with the mosquito vector. Eliminating sources of stagnant water, which are breeding grounds for mosquitoes, and using insect repellant and mosquito-proof bed netting greatly limits the possibility of arbovirus outbreaks.

Numbers of reported cases of West Nile fever (Section 30.6) in the United States have fluctuated in recent years, from less than 500 in 2009 to over 5600 in 2012. Like the viruses that cause yellow fever and dengue fever, West Nile virus (WNV), the causative agent of West Nile fever, is transmitted through the bite of a mosquito (Figure 30.13a). WNV is an enveloped flavivirus with a positive-sense RNA genome (Figure 30.13b), and it invades the nervous system of its warm-blooded host (Figure 30.14). The epidemiology of WNV infection in humans is an interesting story. It was first identified in Uganda in 1937, spread to Israel and Egypt by the 1950s, and by the 1990s outbreaks in humans across Africa and Europe were reported. The first cases in the United States were reported in 1999 in upstate New York, and it has now spread to almost every state (Figure 30.15). Although most human infections by WNV are asymptomatic and undiagnosed, the elderly and the very young are most susceptible to its most serious consequences; reported cases of WNV have a mortality rate of 4%.

Primary control measures revolve around reducing exposure to the vector using the same means as described above for yellow fever and dengue fever.

The final arthropod-transmitted pathogen discussed in these sections is Yersinia pestis, the causative agent of plague (Section 30.7). Material in this section will be at least somewhat familiar to most students from their history classes since the disease (also known as the black death) killed over one-third of Europe’s population in the Middle Ages. Y. pestis is a gramnegative, facultatively aerobic bacterium (Figure 30.16) classified within the enteric bacteria group (Section 15.3). Y. pestis reproduces in the gut of the rat flea (Figure 30.17), which obtains the bacterium after taking a blood meal from an infected rodent. Symptoms of plague are often characterized by the development of buboes (swollen lymph nodes full of Y. pestis (Figure 30.18a), followed by necrosis of tissue caused by localized hemorrhaging (Figure 30.18b). The natural reservoir is rodents, whereas fleas are intermediate hosts (Figure 30.19).

Humans who come into contact with rat fleas are accidental hosts. Discuss the characteristic pathologies resulting from Y. pestis infection, noting that pneumonic and septicemic infection generally lead to rapid death. However, full recovery from bubonic plague is >95% successful if antibiotic treatment is started quickly.

30.8–30.9 | Soilborne Bacterial Diseases Discuss and summarize the major characteristics of diseases caused by pathogenic bacteria found in soil. These diseases result from accidental interactions between pathogen and host.

Anthrax was discussed in Section 28.8 as a potential weapon for bioterrorism, but Section 30.8 focuses more on the biology and pathology of the causative agent of anthrax, the endospore-forming Bacillus anthracis (Figure 30.20). The bacterium is a common soil inhab.

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itant that is usually transmitted to humans from livestock, either through a break in the skin or via ingestion or inhalation of endospores (Figure 30.21). Although all three forms of anthrax are rare in humans in the United States, they show increasingly high mortality rates, and death occurs as a result of several potent toxins produced by the bacteria (see Section 28.8). A human vaccine is available for those considered to be at high risk for the disease, and livestock are commonly vaccinated. Discuss the characteristics of each form of anthrax with your students: • Cutaneous anthrax: most common form in humans; endospores enter the body through a skin abrasion; a painless, black pustule forms at the site of the wound (Figure 30.21b); 20% mortality rate if untreated • Gastrointestinal anthrax: very rare in humans; endospores enter the body via ingestion of undercooked meat from diseased animals; symptoms include abdominal pain, bloody diarrhea, and intestinal lesions; 50% mortality rate • Inhalation anthrax: most severe form of anthrax; cases usually observed in those who process wool or animal hides; airborne endospores enter the body via inhalation and move from the lungs to the bloodstream; systemic infection causes septic shock and rapid death in nearly 100% of cases Tetanus (Section 30.9) is another serious, and often life-threatening, disease caused by a soilinhabiting, endospore-forming bacterium–the anaerobe Clostridium tetani (Figure 30.22a).

The pathology from infection by C. tetani results from the production of a potent exotoxin that inhibits control of the relaxation phase of muscle contraction (see Section 23.9 for molecular details). This results in rigid paralysis of voluntary muscles (initial paralytic symptoms of the face and mouth are commonly referred to as lockjaw), and death due to respiratory failure (Figure 30.22b). The mortality of this disease is close to 10%, even in developed countries.

However, a toxoid vaccine is available that effectively prevents disease. Gas gangrene is also caused by species of Clostridium, usually C. perfringens or C. novyi (Figure 30.23a and b, respectively). It is characterized by gaseous necrosis of body tissues that are O2-depleted, often from decreased blood flow caused by a deep wound, such as a puncture (Figure 30.23c). Most cases require surgical removal of the affected part of the body to prevent invasion into healthy tissues, and severe cases of gas gangrene can be fatal if C. perfringens alpha toxin disseminates and causes systemic toxemia. However, in some cases, a hyperbaric chamber can be used to enrich hypoxic/anoxic tissues with pure, pressurized O2, which inhibits growth of the anaerobic bacteria. This, in combination with antibiotic treatment and skin grafts, may allow for restoration of damaged tissues.

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Answers to Review Questions 1. Diseases in animals may be enzootic when present endemically in certain populations.

Rabies occurs in wild animals, and the major enzootic reservoirs of rabies virus in the United States are raccoons, skunks, coyotes, foxes, and bats. Rabies is diagnosed by examining tissue samples for the virus. Fluorescent antibodies that bind to rabies virus in brain tissues are used to confirm a case of rabies in a post-mortem examination. Viral inclusions called Negri bodies are seen in the cytoplasm of nerve cells stained for light microscopy and these characteristic structures confirm rabies virus infection as well.

The rabies virus proliferates in the brain, especially in the thalamus and hypothalamus. Infection leads to fever, excitation, dilation of the pupils, excessive salivation, and anxiety. A fear of swallowing (hydrophobia, an early name for rabies) develops from uncontrollable spasms of the throat muscles, and death eventually results from respiratory paralysis. In humans, an untreated rabies infection in which symptoms have begun is almost always fatal. If a wild or stray animal is suspected of being rabid, it should be immediately examined for evidence of the rabies virus. If a domestic animal, generally a dog, cat, or ferret, bites a human, especially if the bite is unprovoked, the animal should be held in quarantine for 10 days to check for signs of rabies. If the animal exhibits rabies symptoms, or a definitive diagnosis of its illness cannot be made after 10 days, the human should be passively immunized with rabies immune globulin (purified human antibodies to rabies virus) injected at both the site of the bite and intramuscularly. The patient should also be actively immunized with a rabies virus vaccine. Because of the very slow progression of rabies in humans, this combination of passive and active immune therapy is nearly 100% effective, stopping the onset of the disease.

2. Hantavirus pulmonary syndrome emerged in 1993 and was traced to the deer mouse.

The virus is found in rodents throughout the world. Excessive rodent populations and rodent habitat can lead to hantavirus emergence. Humans seem to be an accidental reservoir. Avoiding contact with rodents and minimizing rodent habitats effectively prevents the spread of HPS. 3. The three major rickettsial categories are the typhus group, the spotted fever group, and the ehrlichiosis group. Typhus fever, caused by Rickettsia prowazekii, is transmitted from human to human via the common body or head louse. Rocky Mountain spotted fever, caused by Rickettsia rickettsii, is transmitted to humans by various species of ticks—most commonly dog and wood ticks. Ehrlichiosis is a tickborne disease caused

by Ehrlichia chaffeensis and Rickettsia sennetsu, whose animal reservoirs include deer, some rodents, and humans.

4. Lyme disease is caused by infection with a spirochete, Borrelia burgdorferi, transmitted by a tick bite. Antibodies to B. burgdorferi appear 4–6 weeks after infection and can be detected by various immunological assays. A PCR assay is also used to detect B. burgdorferi DNA in body fluids and tissues.

5. Yellow fever and dengue fever are both tropical to subtropical hemorrhagic fevers caused by flaviviruses that are transmitted by mosquitoes of the genus Aedes. Yellow fever is not nearly as prevalent as dengue fever, but it is more serious, with an estimated 15% mortality rate. A vaccine is available for yellow fever but not dengue fever.

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6. The West Nile virus (WNV) is a flavivirus and has an enveloped capsid containing a plus-sense, single-stranded RNA genome. The WNV causes West Nile fever, a human viral disease that is transmitted through the bite of a mosquito (Culex species). Infected birds develop a systemic viral infection (viremia) that is often fatal. Mosquitoes feeding on viremic birds are infected and can then infect other susceptible birds, renewing the cycle. In contrast to birds, humans and other animals are dead-end hosts for the virus because they do not develop the viremia necessary to infect mosquitoes.

After an incubation period of 3–14 days, about 20% of infected individuals develop West Nile fever, a mild illness lasting 3–6 days. The fever may be accompanied by headache, nausea, myalgia, rash, lymphadenopathy (swelling of lymph nodes), and malaise. Less than 1% of infected individuals develop serious neurological diseases such as West Nile encephalitis or West Nile meningitis from viral replication in neural tissues.

7. Because bubonic plague can be successfully treated with antibiotics, and the number of cases in the United States and worldwide is low, there is no need to administer vaccinations that have some inherent risk themselves. Controlling rodent populations and destroying rodent habitat is probably the most effective means of controlling the disease.

This is especially important in densely populated, urban areas.

8. The fact that Bacillus anthracis can form endospores allows it to survive long periods of time in inhospitable conditions. If environmental conditions once again become suitable for growth, the endospores can quickly germinate to become dividing, vegetative cells even after years of dormancy. With a mortality rate near 100%, inhalation anthrax is the most serious form.

9. Tetanus is caused by an exotoxin produced by Clostridium tetani, an obligately anaerobic, endospore-forming rod. Cells of C. tetani normally gain access to the body through a soil contaminated wound, typically a deep puncture. In the wound, anoxic conditions develop around the dead tissue and allow germination of endospores, growth of the organism, and production of a potent exotoxin, the tetanus toxin. The toxin directly affects the release of inhibitory signaling molecules in the nervous system causing lockjaw, mild spasms, paralysis, and death due to respiratory failure.

Gas gangrene is caused by Clostridium perfringens, which is also a common cause of foodborne illness. C. perfringens alpha toxin, which is distinct from the toxins it produces in perfringens food poisoning, is a major virulence factor in gangrene. Alpha toxin is a phospholipase that hydrolyzes the membrane phospholipids of host cells, leading to cell lysis and the typical accumulation of gas and fluids that accompanies gas gangrene.

The tetanus toxoid vaccine is highly effective, and thus virtually all tetanus cases occur in individuals who were inadequately immunized. If a deep wound is severe or heavily contaminated by soil, treatment might also include administration of a tetanus antitoxin preparation, especially if the patient’s immunization status is unknown or is out of date. Antitoxin is injected intramuscularly (or into the sheath surrounding the spinal cord if necessary) to prevent binding of newly released toxin to cells. A tetanus antitoxin preparation might be administered if a deep wound is severe or heavily contaminated by soil, especially if the patient’s immunization status is unknown or is out of date.

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Answers to Application Questions 1. You have probably contracted Lyme disease. Untreated cases of Lyme disease may progress to a chronic stage weeks to months after the initial tick bite, causing arthritis and serious neurological problems such as palsy, weakness in the limbs, and heart damage.

Prevention of Lyme disease begins by avoiding contact with the vector. Insect repellents containing DEET or the wearing of snug-fitting clothing is helpful, as is a thorough body exam following walks in tick-infested environments. Lyme disease vaccines are available for domestic animals.

2. See Review Question 3 above. In addition, note that outdoor activities in tick-infested areas are the biggest predisposing factors in acquiring ehrlichiosis. The emergence of this disease is similar to that of Lyme disease because of its arachnid vector and because of an increasing awareness, and thus a larger number of reported cases, of the disease. In addition, tick populations have been high in recent years, thus providing a large reservoir for the pathogen.

3. The yellow fever virus replicates in lymph nodes and certain immune system cells, and travels to the liver. It can cause major organ failure and death. It is controlled through isolation and quarantine, and is preventable by an effective vaccine.

Rabies infection leads to fever, excitation, dilation of pupils, excessive salivation, anxiety, hydrophobia, and death from respiratory paralysis. Vaccination of domestic animals is the key to the control of rabies.

Hemorrhagic fever with renal syndrome is characterized by a sudden onset of fever, muscle pain, a reduction in the number of blood platelets along with an increase in the number of circulating leukocytes, and hemorrhaging. Death, if it occurs, takes several days, and is usually a result of systemic shock and cardiac complications precipitated by leakage of fluid into the lungs, causing suffocation and heart failure. It can be prevented by avoiding rodent contact and rodent habitats.

Dengue begins with a high fever and headache or joint pains and in some patients, severe eye pain and a systemic rash. A dengue infection can take a more severe course of events and proceed to dengue hemorrhagic fever, which is fatal. It is controlled by eliminating either the vector or contact with the vector.

Rickettsias can cause typhus fever, spotted fever rickettsiosis (Rocky Mountain spotted fever), and ehrlichiosis. It is controlled by vaccines and prompt antibiotic treatments.

Q fever infection can progress to include prolonged fever, headache, chills, chest pains, pneumonia, and endocarditis. Infected animals or contaminated animal products such as wool, meat, and milk are potential sources for human infection. Q fever can be controlled through vaccination.

West Nile fever–infected individuals develop serious neurological diseases such as West Nile encephalitis or West Nile meningitis from viral replication in neural tissues. It can be controlled by limiting exposure to mosquitoes, using insect repellents, and wearing tight-fitting clothing.

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CHAPTER

31

Water and Food as Vehicles of Bacterial Diseases

Summary Chapter 31 reviews the important public health methods used to monitor water quality for the purpose of minimizing the dissemination of waterborne biological and chemical contaminants. Common waterborne diseases and the pathogens that cause them are then discussed.

Next, the authors present an overview of the activities of microorganisms that directly affect the food sources of humans and other animals. Epidemiological characteristics of select foodborne diseases are considered, and the final sections of the chapter describe the major causative agents of microbial food poisoning and food infections. Methods of diagnosis, treatment, and prevention of foodborne infections are described.

31.1–31.2 | Water as a Disease Vehicle Students interested in public health will find this chapter particularly informative because it discusses microorganisms as agents of disease that affect human health through the quality of our water and food. Emphasize to students that water is the most significant pathogen delivery vehicle worldwide (Section 31.1). Therefore, the use of methods for identifying potential pathogens and treating water supplies is essential to public health. Waterborne diseases are classified as common source infectious diseases, and waterborne pathogens (Table 31.1) may come from drinking water or recreational water sources (Table 31.2); consequently, these sources are strictly regulated and monitored. Point out that, in the United States, outbreaks of waterborne diseases occur at nearly equal frequency in drinking water (~25 outbreaks per year) and recreational waters (~20 outbreaks per year). Impress upon students that waterborne infections are a much larger problem in developing countries than in the United States and other developed countries. Most individuals in developed countries take the availability of potable water for granted, but contaminated water sources contribute significantly to morbidity and mortality by infectious diseases.

Because natural water sources are not sterile, determination of the quality of water used for drinking, bathing, and other uses is an important public health issue (Section 31.2).

Explain to students that water supplies are tested for the presence of indicator organisms, rather than for specific pathogens, as a measure of water quality. The most common indicators are intestinal inhabitants, which indicate fecal contamination, belonging to the coliform group of organisms. Methods of detecting these microorganisms were implemented around 1905.

Discuss the use of membrane filters and defined substrate tests to identify coliforms in water .

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samples (Figure 31.1). Point out that the use of culture media that is both selective and differential, such as eosin-methylene blue (EMB) agar, allows for the identification and enumeration of coliforms and, specifically, Escherichia coli. In addition to coliform-counting culture methods, the next most important advance in public health monitoring of drinking water, which occurred around 1913, was the use of chlorine as a disinfectant in water supplies. This water purification strategy resulted in a dramatic decrease in the number of cases of waterborne disease in the early part of the twentieth century.

31.3–31.5 | Waterborne Diseases One of the most serious and significant waterborne diseases in developing countries is cholera (Section 31.3). Cholera is caused by Vibrio cholerae, a gram-negative, curved rod of the phylum Proteobacteria that is usually transmitted via contaminated water (Figure 31.2). The epidemiology of cholera was discussed in Section 28.10, and therefore this section focuses on the biology, pathology, treatment, and prevention of the disease: • Cholera disease requires a large inoculum of V. cholerae (108–109) due to the bacterium’s sensitivity to the low pH of the stomach and the fact that cells must reach the small intestine to become established by attaching to intestinal epithelia. Lower numbers are required if the bacterium is ingested along with food because the food will provide a protective buffer against the low stomach pH. • Enterotoxin production by V. cholerae was discussed in Section 23.9, but stress to students that the toxin’s effects lead to a rapid and volumetric loss of water (and electrolytes) of up to 20 liters (44 pounds) a day—an obvious reason for the high mortality rates (~60%) during an epidemic (Figure 31.3).

• The treatment required to reduce the mortality rate to less than 1% is a simple one—electrolyte replacement therapy. This method requires the frequent administration of a high sugar/salts drink. Students may be curious as to why the mortality rates from cholera remain high in certain regions of the world if the solution is so simple. You might use this as an opportunity to discuss the geographic and demographic problems with availability and dissemination of supplies in areas where no real healthcare exists.

• The key to preventing cholera infection in endemic areas is to avoid untreated water (and ice) and raw or undercooked foods, especially fish and shellfish, which can ingest the pathogen by feeding on contaminated phytoplankton (Figure 31.4).

Most students (and many instructors) will be too young to remember the “Legionnaires’ disease” outbreak at an American Legion convention in Philadelphia, Pennsylvania (USA), in the summer of 1976, but the story is an interesting example of the epidemiological detective work required to identify and control an epidemic (Section 31.4). The initial news reports suggested that an unknown toxin was delivered through air conditioning ducts because the nutrient-poor environment in those areas would not appear to be able to support enough pathogen population to cause infection. Because of the diligent work done by scientists from the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia (USA), the causative agent, Legionella pneumophila (Figure 31.5), was identified. We now know much more about the biology of this pathogen, including its prevalence in residential waters, its

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high iron requirement, and its ability to invade and reproduce in macrophages and monocytes present in lung tissues. Discuss the epidemiology and incidence of legionellosis in the United States, noting that although incidence peaks in the summer months, infections occur at all times of the year. Symptoms of mild cases of infection called Pontiac fever are not treated and include a mild cough, sore throat, headache, and fever. Some serotypes of L. pneumophila cause serious cases of legionellosis, which are characterized by pneumonia, high fever, chills, and muscle aches. The treatment of choice for such cases is intravenous administration of erythromycin.

Section 31.5 discusses two other serious waterborne infections—typhoid fever and norovirus gastrointestinal illness. Like Vibrio cholerae discussed above, Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is one of the most significant waterborne pathogens globally (Figure 31.6a). The pathogen enters the lymphatic system and bloodstream through the wall of the small intestine and may then colonize various organs, including the liver and spleen. Symptoms include high fever, malaise, headache, diarrhea, and abdominal pain. In some cases, perforation of the small intestine can lead to life-threatening sepsis. Because typhoid is primarily transmitted through feces -contaminated water, effective water treatment methods, including filtration and chlorination, have essentially eliminated typhoid fever in developed countries, and fewer than 400 cases occur per year in the United States. Much more common is waterborne illness due to norovirus infection (Figure 31.6b).

Symptoms are similar to, but milder than, typhoid fever and include vomiting, diarrhea, and malaise. Exposure to as few as 10 virions can cause disease, and detection of either viral RNA (via RT-PCR) or viral antigens (via enzyme immunoassay) allows for clinical diagnosis.

31.6–31.7 | Food as a Disease Vehicle From their study of metabolism early in the course, students should understand that the chemical nature of foods selects for growth of a wide variety of chemoorganotrophic bacteria.

Thus, microbial growth can result in food spoilage (Section 31.6), which may or may not render food unsafe to eat but which becomes an economical issue for both producers and consumers if not controlled. Relate the three categories of food spoilage (i.e., perishable, semiperishable, and nonperishable) to the relative moisture content of foods, a function of water activity (aw; see Section 5.15). Review this concept with students, as it illustrates the rationale of using salt and sugar as preservatives before refrigeration. Table 31.3 shows the preference of different types of bacteria and fungi for different fresh food products, an ecological situation analogous to the normal flora distribution in the human body. Discuss the various methods of food preservation with regard to the physiological effects on the microorganism. The method chosen depends on several factors, including food quality, shelf life, and food classification.

• Temperature: Lower storage temperatures retard the rate of spoilage; freezing at –20°C is good for weeks or months, but microbial growth may still occur in tiny pockets of liquid water in the food.

• Acidity: These methods usually employ vinegar (low pH) and salt or sugar to decrease aw.

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• Drying and dehydration (aw): Low water availability adversely affects metabolic rates.

Dehydration by heat or freeze-drying, called lyophilization, is used most often. Spray drying is a technique used to make powdered liquids (e.g., powdered milk) by atomizing (spraying in a fine mist) the liquid in a heated atmosphere.

• Heating: Pasteurization is a technique in which liquids are heated to a specific temperature for a set period of time to reduce total numbers of viable microorganisms and increase the shelf life of the liquid. Note that while standard pasteurization does not sterilize the liquid, ultra-high temperature (UHT) processing (liquids heated to 138C for 2–4 seconds) kills all microorganisms. In canning, foods are heated to at least boiling temperature and sealed, which should, theoretically, preserve foods indefinitely. However, this method does not always sterilize foods. Figure 31.7 shows the results of anaerobic fermentation and gas production in a sealed can, probably by toxin-producing species of Clostridium (see Section 31.9).

• Aseptic processing: Foods are prepared by flash heating under aseptic conditions; a rapid, high-temperature cooking procedure sterilizes foods.

• High-pressure processing: Foods are subjected to very high hydrostatic pressures (up to 100,000 psi) to kill microbial cells. However, because the process does not kill endospores, it is not a sterilization procedure.

• Chemical food preservation: Several antimicrobial chemical additives have been approved for use in controlling microbial growth on different foods. Some of these additives are still controversial—animal studies have shown possible deleterious effects for some additives that may affect humans (e.g., nitrites and cancer; ethylene and mutagenesis). Additives considered safe by the Food and Drug Administration (FDA) are labeled GRAS (generally recognized as safe).

• Irradiation: Ionizing radiation using 60Co or 136Cs is a standard method in the United States for reducing contamination of foods by bacteria, fungi, and some insects.

Students are probably aware that many common foods and beverages are produced, preserved, or enhanced through direct microbial activities. Fermented foods are any foods in which significant alterations in raw products are produced by microbial processes, and a variety of microorganisms take part in their production (Table 31.4). Discuss the varieties of fermented foods and their associated microorganisms, as summarized below.

• Yeast products: Yeast breads and alcoholic beverages are produced by the fermentation of simple sugars and other carbohydrates by Saccharomyces cerevesiae. Carbon dioxide produced by the fermentation accounts for bread rising (and the pockets in the finished loaf; Figure 31.8) and carbonation in fermented beverages.

• Dairy products: Lactic acid bacteria carry out the primary fermentation of dairy products.

Lactose in milk is hydrolyzed to produce glucose and galactose, and lactic acid is produced by the fermentation of these products. By controlling the incubation times, temperatures, and microbial constituents, a variety of dairy products can be produced (e.g., yogurt, cheeses, sour cream, and buttermilk; Figure 31.8).

• Meat products: Fermented meat products include dry sausages, such as pepperoni and salami, and semidry sausages, such as bologna (Figure 31.8). As in the production of dairy

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foods, the pH of the meat mixture is reduced by lactic acid fermentation before the sausages are dried and/or smoked.

• Vegetables and vegetable products: A huge number of vegetables and fruits can be preserved by fermentation, but the most economically important examples are sauerkraut (fermented cabbage) and pickles (fermented cucumbers). Soy sauce is produced using a mixture of soybeans and wheat that is inoculated with the fungus Aspergillus, incubated for 2−3 days, and then mixed with brine (17%−19% NaCl solution) for extended fermentation. The final soy sauce product is filtered and pasteurized. Vinegar is a product of acetic acid bacteria (primarily Acetobacter and Gluconobacter), which convert ethanol to acetic acid. The source of ethanol is usually wine (or other fermented fruit juices) or fermented rice. Point out that while acetic acid can easily be produced from ethanol chemically (in the absence of microorganisms), the distinctive flavor of vinegar is a result of components in the starting material and products of the fermentation process. Therefore, vinegar production is still dependent upon microbiological processes.

Each of the pathogens discussed in the following sections causes foodborne disease, some of which can be fatal if not treated promptly. It is important to differentiate between food poisoning (or food intoxication) and food infection. Food poisoning does not require viable cells and microbial growth to cause disease because presynthesized microbial toxins cause the symptoms and pathology (e.g., Clostridium exotoxins in Figures 23.21–23.22 and S. aureus superantigen toxins in Section 24.9). In contrast, food infection is caused by ingestion of live, active cells (Sections 31.10–31.14 below). Point out the fact that food infections are quite common, occurring about ten times more often than food poisonings. Detection of specific pathogens in foods usually begins with processing of the food sample in a stomacher to suspend embedded microorganisms (Figure 31.9). Following this, nucleic acid probes, PCR, and other techniques (as discussed in Chapter 27) may be used for detection and quantification. Because this can be expensive on a large scale, incubation and growth on selective and differential media can be employed as a first step in monitoring the safety of food products.

31.8–31.9 | Food Poisoning Most students know that leaving potato salad and other foods out too long on the picnic table can result in “food poisoning” in the generic sense, but most of them will not be able to explain why or how this occurs. Point out that the number of annual cases of staphylococcal food poisoning (Section 31.8) in the United States (nearly 250,000) suggests that many people assume that once refrigerated, a “few hours” on the picnic table would not be enough time for bacteria to grow. At this point in their microbiology education, students should understand microbial growth dynamics (e.g., generation time) and the fact that even at relatively low cell numbers, the enterotoxins produced by Staphylococcus aureus (Figure 31.10) are synthesized rapidly and secreted into foods. In addition, these toxins are quite heat-stable and remain active even after 16 hours at 60C. Remind students that the source of S. aureus can be any individual because it is part of the normal microflora and is easily transmitted while handling foods. Low temperatures dramatically inhibit growth of S. aureus. A few important points and concepts regarding staphylococcal food poisoning are summarized below:

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• Staphylococcus enterotoxins include SEA, SEB, SEC, and SED. The genes encoding these toxins are located on the chromosome (SEB and SEC), on a lysogenic bacteriophage (SEA), or on a plasmid (SED).

• Qualitative assays for S. aureus enterotoxin or exonuclease can detect the presence of S. aureus in foods, but quantitative data require plate counts on selective media.

• The disease can be severe, but it is self-limiting and usually resolved within 48 hours. Ask students why antibiotics are not prescribed for staphylococcal food poisoning.

From students’ previous understanding of bacterial endospore structure and properties, it should not surprise them that food poisonings caused by Clostridium species represent some of the most common and serious foodborne diseases known (Section 31.9).

• Food poisonings by C. perfringens (Figure 31.11) are among the most commonly reported in the United States, with nearly one million cases annually. Symptoms require ingestion of a large number of cells or endospores (>108) from contaminated foods, especially meats.

• Sporulation and subsequent enterotoxin production by C. perfringens occur in the intestinal tract about 7–15 hours after consumption. Symptoms, which include diarrhea, nausea, and intestinal cramps, usually resolve within 24 hours.

• Diagnosis of perfringens food poisoning is usually done by selective isolation and growth from fecal samples or by enzyme immunoassay (EIA) against C. perfringens enterotoxin.

• Botulism food poisoning by C. botulinum is often fatal, and the mechanism of action of its neurotoxin was described in Section 23.9. Seven types of botulism toxins are known and most cases occur due to ingestion of foods that are not cooked following processing (e.g., home-canned foods and raw honey). Only about 150 cases of foodborne botulism occur every year in the United States. Although botulism mortality is high without treatment, fewer than 5% of all cases are fatal due to efficient clinical diagnosis.

31.10–31.14 | Food Infection Continuing with pathogens causing foodborne diseases, discuss those responsible for food infections. Stress to students that unlike food poisoning, food infection requires colonization and growth of the pathogen for the onset of disease symptoms. If necessary, refer students to (or review with them) the mechanisms of entry, colonization and growth, and virulence, as presented in Sections 23.6–23.8.

Mention to students that although salmonellosis is often informally referred to as “Salmonella food poisoning,” it is actually a classic example of food infection (Section 31.10).

Discuss the biology and epidemiology of Salmonella enterica, being sure to differentiate S. enterica serovar Typhi (the causative agent of typhoid fever) from S. enterica serovar Typhimurium, which is the most common agent of foodborne gastroenteritis. Several interesting and conceptually important points to present to students are summarized below: • Transmission of Salmonella to humans can occur from contact with animals or animal products known to harbor the bacterium, including poultry, reptiles, and amphibians (Figure 31.12).

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• Over 1400 Salmonella serotypes are human pathogens, and they can be readily isolated using selective and differential media, such as Hektoen agar (Figure 31.13). The incidence and prevalence of salmonellosis has been steady over the past decade, with approximately one million cases per year. Considering that food production animals are a common source of the pathogen, the key to preventing outbreaks includes thorough handwashing by food handlers and proper cooking and food storage temperatures.

• Colonization requires relatively large numbers of cells (105–108), and the onset of disease is within 8–48 hours. Salmonellosis is characterized by the presence of a variety of toxins, including endotoxins, enterotoxins, and cytotoxins. Recovery occurs in 2–5 days, but it is important to stress to students that even after symptoms disappear, cells of pathogenic Salmonella are shed in feces for weeks.

• Although not the case for septicemia or typhoid fever, antibiotic treatment of gastrointestinal salmonellosis is unnecessary and does not reduce the duration of disease symptoms.

Several pathogenic strains of E. coli (~200) are capable of causing serious diarrheal disease and urinary tract infections (Section 31.11). Remind students of the essential role played by the human normal flora strains of E. coli that occupy the large intestine (see Section 23.4).

Contrast this to Shiga toxin-producing E. coli (STEC), which possess the genetic potential to produce various toxins and diseases. Based on news reports of outbreaks occurring from ingestion of contaminated hamburger meat, E. coli O157:H7 is probably the most notorious STEC strain (Figure 31.14). E. coli O157:H7 infection is characterized by the production of verotoxin following attachment and growth on the small intestine epithelium, resulting in bloody diarrhea and kidney failure. E. coli O157:H7 causes about 50 deaths per year and more than 60,000 infections; it is the leading cause of kidney failure in children. Diagnosis of strain O157:H7 infection is performed using traditional culturing methods and by serological identification of the O (lipopolysaccharide) and H (flagellar) antigens and toxins. Discuss the characteristics of other pathogenic classes of E. coli, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). Stress that the most effective means of preventing outbreaks of pathogenic E. coli include practicing proper methods of food handling and preparation, water purification, and personal hygiene.

Most cases of bacterial foodborne infection in the United States are caused by species of Campylobacter (Section 31.12). These bacteria are responsible for about 2 million cases of diarrhea annually, mostly by infection with C. jejuni and C. coli (Figure 31.15). The source for most infections is poultry, in which C. jejuni is part of the resident microbiota. Pork and shellfish are also common vehicles for this pathogen, but its presence is rare in beef. Diagnosis, treatment, and prevention of Campylobacter infection are similar to that of pathogenic E. coli.

Spend some time discussing the foodborne pathogen Listeria monocytogenes (Figure 31.16), the causative agent of the gastrointestinal infection known as listeriosis (Section 31.13).

L. monocytogenes is becoming an increasingly important pathogen due to its metabolic and physiological versatility: it is acid-tolerant, psychrotolerant, and halotolerant.

Considering the food preservation techniques described in Section 31.6 and the presence of L. monocytogenes in water and soil, this bacterium can be expected to be present in every fresh food source, and even more alarming, invasive infection can lead to bacteremia and meningitis. Discuss the significant points regarding the epidemiology, pathology, diagnosis, and treatment of listeriosis. Remind students that this bacterium is an intracellular pathogen

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that induces phagocytic macrophages and monocytes to engulf it, wherein it survives, multiplies, and is transmitted to neighboring cells through cytoplasmic extensions called filopods (Figure 31.17). This mode of transmission allows the pathogen to invade other tissues without being exposed to other immune defenses, such as antibodies and complement proteins. Most individuals experience only gastrointestinal symptoms upon ingestion of a suitable inoculum.

However, immunocompromised individuals, the elderly, pregnant women, and infants are more susceptible to chronic and acute infections that can lead to bacteremia and meningitis, the latter of which has a mortality rate of about 20%. Listeriosis is diagnosed by the positive culture of L. monocytogenes from blood or spinal fluid, and treatment using certain -lactam antibiotics or trimethoprim plus sulfamethoxazole is effective.

Summarize for students examples of other infectious agents that contribute to foodborne diseases (Section 31.14): • Bacteria: Yersinia enterocolitica (Figure 31.18a and b), Bacillus cereus (Figure 31.18c and d), Shigella spp., and some Vibrio spp.

• Viruses: Note for students the important differences between viral foodborne illnesses and foodborne illnesses of bacterial origin; viral infections typically cause nausea and vomiting in addition to diarrhea, and recovery is usually spontaneous and rapid (24–48 hours).

Emphasize that noroviruses (Figure 31.19) account for ~70% of foodborne infectious disease in the United States (Table 31.5). Other viral agents include rotavirus, astrovirus, and hepatitis A. • Protists: Giardia lamblia, Cryptosporidium parvum, and Toxoplasma gondii (these pathogens are discussed more thoroughly in Chapter 32).

• Prions: transmission of “new variant Creutzfeldt-Jakob disease” has been traced to eating the meat of cattle afflicted with bovine spongiform encephalitis (BSE, or “mad cow disease;” Section 9.13).

Answers to Review Questions 1.

Pathogen Bacteria Vibrio cholerae Legionella pneumophila Salmonella enterica serovar Typhi Escherichia coli Pseudomonas aeruginosa Viruses Norovirus Hepatitis A virus Parasites Cryptosporidium parvum Giardia intestinalis .

Disease

Cholera Legionellosis Typhoid fever Gastrointestinal illness Nosocomial pneumonia, septicemia, and skin infections Gastrointestinal illness Viral hepatitis Cryptosporidiosis Giardiasis

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Schistosoma

Schistosomiasis

2. Fecal coliforms are defined as gram-negative, aerobic and facultatively aerobic, nonspore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 hours at 35°C. The coliform test is used to assess drinking water purity because the presence of coliform bacteria indicates fecal contamination.

3. Antibiotics are ineffective for the treatment of cholera because by the time symptoms are apparent, the cholera toxin has already been produced in the body, and antibiotics have no effect on the toxin itself. Oral rehydration therapy, initiated as soon as possible, can replenish lost fluids and electrolytes in the patient and reduce the mortality rate of an epidemic to ~1%. Antibiotics can only shorten the duration of cholera and prevent the shedding of live Vibrio cholerae cells in the feces of the victim.

4. Legionellosis, caused by Legionella pneumophila, is transmitted through humidified aerosols rather than through drinking or recreational water. L. pneumophila is a free-living bacterium capable of invading and multiplying in macrophages, and it is fairly resistant to chlorine and heat. It is common in cooling towers and evaporative condensers of large air-conditioning systems, and it can also form biofilms inside water distribution pipes and reside inside cells of some microbial parasites. Infection by this pathogen often causes mild symptoms, such as sore throat, headache, and fever, and the infection is self-limiting. Serious pneumonia-type infections occur from infection by certain serotypes, often occurring in elderly or immunocompromised individuals.

5. Typhoid fever, caused by Salmonella enterica serovar Typhi, is typically transmitted through feces-contaminated water, although foodborne transmission does occasionally occur. By contrast, salmonellosis, caused by Salmonella enterica serovars Typhimurium and Enteritidis, is a common foodborne infection that is only rarely transmitted through water. While typhoid has been virtually eliminated in developed countries by effective water treatment procedures, primarily chlorine disinfection, salmonellosis remains the most common bacterial food infection in the United States. Whereas salmonellosis is usually self-limiting and resolves without treatment after several days, typhoid fever can lead to potentially fatal sepsis and septic shock.

6. Psychrotolerant organisms spoil refrigerated food as they can grow at 3–5°C. The environment inside a can or sealed jar is anoxic, and an important genus of anaerobic bacteria that can grow in canned foods is the endospore-forming Clostridium, one species of which causes botulism. Nitrites, sulfites, propionate, and benzoate are widely used in the food industry as preservatives.

7. The major methods used to preserve foods are (1) low temperature (freezing of meats, many fruits, and vegetables); (2) low pH (pickling of cucumbers, peppers, fish, or fruits with vinegar, salt, or sugar); (3) water activity reduction (drying or adding salt or sugar, as in preservation of jams, meat, fish, vegetables, etc.); (4) canning (following proper heating and sealing, many foods can last for years); (5) chemicals (FDA-approved preservatives); and (6) irradiation (use of gamma rays to preserve spices, hamburger, and poultry). The major classes of fermented foods include yeast breads and alcoholic beverages; dairy products (e.g., yogurt, buttermilk, and cheeses); meat products (e.g.,

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sausages, bologna, and pepperoni); vegetable products (e.g., sauerkraut and pickles); and some liquid products (e.g., soy sauce and vinegar).

8. Presynthesized microbial toxins (e.g., Clostridium exotoxins and S. aureus superantigen toxins) cause food poisoning, and therefore the presence of viable cells and microbial growth is not necessary to cause disease. In contrast, food infection is caused by ingestion of sufficient quantities of live, active cells, as occurs in salmonellosis.

9. Staphylococcal enterotoxins are fairly heat-stable and all are stable to stomach acidity.

However, staph enterotoxins are also superantigens and can lead to potentially lethal toxic shock syndrome. SEA is the most common cause of staph food poisoning worldwide.

S. aureus enterotoxins are given acronyms beginning with “SE” (staphylococcus enterotoxin): SEA, SEB, SEC, and SED. These are encoded by the genes sea, seb, sec, and sed.

When consumed, the toxins cause gastrointestinal symptoms characterized by one or more of the following: nausea, vomiting, diarrhea, and dehydration. The onset of symptoms is rapid; within 1–6 h of ingestion, depending on the amount of enterotoxin consumed, but the symptoms usually pass within 48 hours.

10. Clostridial food poisoning can be categorized into two types: (1) C. perfringens food poisoning and (2) botulism, the causative agent of which is C. botulinum. C. perfringens is the most prevalent type of food poisoning in the United States, causing nearly one million annual cases. However, food poisoning by C. botulinum is far more serious and has a mortality of up to 25% due to the extreme potency of botulinum neurotoxin.

11. The sources of foodborne Salmonella spp. are the intestinal tracts of humans and warmblooded animals. Infection may occur from fecal contamination by food handlers and food production animals, such as chicken or cattle.

12. Cells of E. coli O157:H7 are normally present in the intestines of cattle and enter ground beef through unsanitary processing practices. E. coli O157:H7 is an enterohemorrhagic Shiga toxin-producing Escherichia coli (STEC) strain.

13. Azithromycin.

14. Listeria monocytogenes is widely found in soil and water. It is halotolerant and psychrotolerant (it grows slowly even under refrigeration), and thus virtually any food can become contaminated at any stage during food growth and production. L. monocytogenes is able to escape phagosomes after being taken up by phagocytes. It then proliferates in the host cell and is transmitted to neighboring host cells through cytoplasmic extensions called filopods. In this way, the pathogen avoids opsonization by antibody coating and complement binding.

15. Enteric bacteria: Salmonella Virus: norovirus Protist: Giardia intestinalis 16. Variant CJD (vCJD) is caused by a foodborne prion infection. Unlike norovirus, a plussense RNA virus related to poliovirus, prions are infectious agents that consist only of a single misfolded protein that is capable of converting normal forms of the protein to the infectious form that propagates neural degeneration.

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Answers to Application Questions 1. Swiss cheese: Propionibacterium Dry sausages (pepperoni): Pediococcus, Lactobacillus, Micrococcus, Staphylococcus Cucumbers (pickles): Lactic acid bacteria Soy sauce: Aspergillus, Tetragenococcus halophilus, yeasts Yogurt: Lactobacillus, Streptococcus thermophilus Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, and Salmonella indicate severe food poisoning.

2. The high level of organic acids or alcohol generated from these fermentations prevents the growth of both spoilage organisms and pathogens in the fermented food product.

Non-acidic food such as corn and beans and raw foods directly used for making salad will give easy access to pathogens.

3. PCR, FISH, CARD-FISH, q-PCR, blotting experiments, RFLP, and ARDRA.

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CHAPTER

32

Eukaryotic Pathogens: Fungal and Parasitic Diseases

Summary Chapter 32 is a new chapter in this reorganized edition of the text. It presents and describes a variety of important eukaryotic pathogens and the diseases they cause. A discussion of the major mycoses (fungal diseases) opens the chapter. The authors then present a series of key parasitic infections that target either visceral tissues or certain components of the circulatory and organ systems. Diseases caused by both protists and helminths are considered.

32.1–32.2 | Fungal Infections Sections 32.1 and 32.2 discuss the major characteristics of diseases caused by pathogenic fungi (Figure 32.1). These diseases result from accidental interactions between pathogen and host, and they can range from superficial and self-limiting to systemic and life-threatening (Table 32.1). Pathogenic fungi exist as either single-celled yeasts or multicellular molds that form filaments called hyphae, which can develop into macroscopic entangled masses called mycelia. Some fungi are dimorphic and can take on either a yeast-like form or a filamentous form based on environmental conditions. Show this to your students by comparing the Histoplasma hyphae that develop in laboratory culture (Figure 32.1e) to the yeast-like form that cells of Histoplasma assume in human tissues (Figure 32.5a).

Fungi cause disease by three major mechanisms: (1) inappropriate immune responses, such as allergic reactions; (2) toxin production, such as the carcinogenic aflatoxins produced by Aspergillus niger (Figure 32.2); and (3) causing mycoses (fungal growth in or on the body).

Mycoses can be categorized as superficial (Figure 32.3), subcutaneous (Figure 32.4), or systemic (Figure 32.5). Systemic mycoses are the most serious and are usually caused by opportunistic fungi that are often associated with individuals having compromised immune systems, such as those with AIDS. The most prevalent systemic mycoses in the U.S. are histoplasmosis and coccidioidomycosis, both of which can occasionally cause serious respiratory diseases (Table 32.1). However, symptoms of these infections are typically mild with a proper immune response. Students should be able to explain why superficial and subcutaneous fungal infections are usually treated fairly easily using topical ointments, whereas it is difficult to develop effective chemotherapeutic drugs against systemic fungal infections due to issues with host toxicity.

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32.3–32.4 | Visceral Parasitic Infections Parasitic infections are symbiotic relationships in which one organism (the parasite) benefits at the other organism’s (the host) expense (Table 32.2). Sections 32.3 and 32.4 describe visceral parasitic infections, which are characterized by gastrointestinal symptoms that include vomiting and diarrhea.

Discuss the key protozoan parasitic infections, which are usually waterborne and include diseases caused by amoebae and ciliates. From an evolutionary standpoint, the protozoan Entamoeba histolytica, which causes the serious and sometimes fatal amebic dysentery, is interesting because its actively growing form, the trophozoite, lacks mitochondria and thus employs an anaerobic metabolism for ATP production (Figure 32.6a). Globally, approximately 100,000 deaths per year are attributed to infection by this organism. Another amoebozoan, Naegleria fowleri, infects humans who swim or bathe in warm, soil-contaminated waters and causes a different form of amebiasis (Figure 32.6b). N. fowleri is a free-living amoeba that enters the nasopharynx, invades the nasal mucosa, and burrows into the brain. The resulting damage and hemorrhaging causes an inflammatory condition called meningoencephalitis.

Although rare, the mortality rate is nearly 100%, and infection results in death within one week. Trophozoites of the ciliate Balantidium coli can assume a cyst form that infects humans and swine through ingestion of feces-contaminated water (Figure 32.6c). B. coli infections are rare, and because symptoms are similar to E. histolytica amebiasis, it is often misdiagnosed. B. coli is the only ciliate known to infect humans.

Section 32.4 discusses a variety of other important, and more common, visceral parasitic infections. Some of these diseases may sound familiar to students due to occasional news reports of outbreaks in U.S. cities. The flagellated protozoan Giardia intestinalis (also called Giardia lamblia) causes the intestinal disorder giardiasis (Figure 32.7; see Section 17.3).

Like several other parasitic protozoa discussed in this section, Giardia is a common inhabitant of surface waters, and it can form cysts that aid the organism in its transmission to a potential host. Although not nearly as durable as bacterial endospores, the cysts are, nevertheless, resistant to chlorine disinfection, UV radiation, and desiccation. Ingestion of Giardia cysts in insufficiently treated water can result in acute or chronic symptoms that include foulsmelling diarrhea, intestinal cramps, nausea, and weight loss. Treatment for acute giardiasis may require the drugs quinacrine, furazolidone, and metronidazole.

Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted infection that may also be spread through fomites such as toilet seats, clothing, and towels (Figure 32.8). Unlike other protozoa in this section, T. vaginalis does not differentiate into a cyst form. Although infected men are usually asymptomatic carriers of T. vaginalis, women often experience a yellowish vaginal discharge associated with itching, burning, and dysuria (painful urination). Up to 25% of sexually active females are infected, with only about 5% of males infected. Administration of the drug metronidazole is an effective treatment for trichomoniasis.

Cryptosporidiosis, toxoplasmosis, and cyclosporiasis are foodborne or waterborne diseases caused by the alveolates Cryptosporidium (Figure 32.9), Toxoplasma (Figure 32.10), and Cyclospora, respectively (see Section 17.5). Cryptosporidiosis spreads through fecescontaminated water, whereas cyclosporiasis usually propagates through contaminated fruits and vegetables. These diseases cause self-limiting diarrhea or gastroenteritis that typically .

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lasts for about two weeks. Other than replenishing lost fluids, treatment is unnecessary in healthy adults. However, infections can become serious in immunocompromised individuals, such as infants and the elderly. Toxoplasma gondii is transmitted to humans through undercooked meats, contact with cats or their feces, or occasionally through contaminated water.

Cats are obligate hosts that allow the pathogen to complete its life cycle, and therefore humans that have frequent interaction with cats are at higher risk of infection. Probably most infections are asymptomatic, but flu-like symptoms sometimes occur. Some infections are more severe and may become systemic; those with compromised immune systems can experience damage to the brain and other organs. T. gondii infections in pregnant women can cause severe birth defects in newborns, and therefore expectant mothers should avoid contact with cats and, especially, cat litter boxes.

32.5–32.7 | Blood and Tissue Parasitic Infections Malaria is a widespread infectious disease that threatens the entire tropical and subtropical human population (Section 32.5). The infection, caused by four separate species of the protist Plasmodium, occurs in 350–500 million people annually worldwide, and one million of those individuals will die from the disease each year. The characteristic symptoms of malaria are cyclic, with rounds of chills, high fever (40°C), and, sometimes, severe headache and vomiting occurring every two or three days. Considering the extensive prevalence of malaria in warmer regions around the world, you should spend some time discussing the complex life cycle of this parasite by explaining the mechanism that leads to eventual infection and destruction of red blood cells (Figures 32.11 and 32.12). Also discuss current methods of diagnosis and treatment (usually administration of the drugs chloroquine and primaquine), as well as prevention and eradication strategies targeted at the Anopheles mosquito vector. Regarding the latter, insecticides, mosquito-proof bed netting, and the removal of sources of stagnant water are key to controlling the mosquito population. Finally, students may be surprised to learn that malaria is present in the United States, with the majority of cases being imported from other areas and occurring in Southern and Southwestern states.

Leishmaniasis, trypanosomiasis, and Chagas’ disease are three other parasitic infections that are transmitted through insect vectors that access the bloodstream of humans (Section 32.6).

Leishmaniasis is caused by species of the flagellated protozoan Leishmania and is transmitted through the bite of the sandfly (Figure 32.13). In the more common cutaneous leishmaniasis, large, ulcerated lesions often form on the skin weeks or months after initial infection. These lesions eventually heal spontaneously, but they usually cause permanent disfigurement. Visceral leishmaniasis is a much more serious form of the disease in which the causative agent, Leishmania donovani, invades internal tissues, including the liver, spleen, and bone marrow, resulting in abdominal distention and loss of blood cells. This form of the disease is usually fatal if not treated. African trypanosomiasis (sleeping sickness) and Chagas’ disease (also called American trypanosomiasis) are related diseases caused by species of the flagellated protozoan Trypanosoma (Figure 32.14). Both diseases are transmitted through an arthropod vector: the former by the tsetse fly and the latter by the reduviid bug (called the “kissing bug” because it often bites near the lips; Figure 32.14b). Discuss the pathology of these tropical diseases for which treatment is difficult and no vaccines are available. Point out that death

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can result from either disease due to organ system complications, including the central nervous system and the heart.

The final section of the chapter (Section 32.7) describes important infectious diseases caused by helminths (parasitic worms), including schistosomiasis and several filariases.

Schistosomiasis is a tropical disease caused by flatworm species of the genus Schistosoma (Figure 32.15). It is colloquially called snail fever because the worms depend on snails to complete their life cycle. Human infection occurs when cercaria, the motile form of the worm, burrow through the skin (Figure 32.15d). They then move into the circulatory and organ systems, where they reproduce by laying eggs and establish a long-term infection that often begins at childhood and continues to adulthood. The infection can cause an inflammatory response characterized by edema (swelling) and distention of the abdomen, especially in children (Figure 32.16a). Although extremely prevalent (second only to malaria with an estimated 250 million cases worldwide), schistosomiasis is rarely fatal, and it can be successfully treated with the antihelminthic drug praziquantel. Filariases are diseases caused by parasitic roundworms. Provide a description of the filariases described in the text, highlighting the following points: • Bancroft’s filariasis (also called “elephantiasis”): A chronic disease of the lymphatic system that causes severe edema, especially in the lower legs (Figure 32.16b). Infection occurs when larval microfilariae of the roundworm Wuchereria bancrofti are transmitted to humans through the bite of a mosquito; the accumulation of adult worms in lymph vessels causes the characteristic swelling.

• Onchocerciasis (also called “river blindness”): A chronic disease in which the parasitic roundworm Onchocerca volvulus is introduced into the body through the bite of a fly. The parasite then migrates to and causes inflammation in the eyes. Onchocerciasis is second only to trachoma as a leading cause of blindness due to infectious disease.

• Trichinellosis (also called “trichinosis”): A disease in which larvae of the roundworm Trichinella become embedded in the muscle tissues of animals. It is endemic in wild mammals but also commonly infects swine. Humans become infected through the consumption of undercooked meat. Although probably asymptomatic in most cases, some humans experience fever, aches, and swelling around the eyes. Severe cases can lead to permanent organ damage and death, but chemotherapeutic treatments are available.

Answers to Review Questions 1. Superficial mycoses are those in which the fungus infects only the surface layers of skin, hair, or nails (e.g., athlete’s foot). Subcutaneous mycoses are infections of deeper layers of skin (e.g., sporotrichosis). Systemic mycoses are the most serious category of fungal infections. These are characterized by fungal growth in internal organs of the body (e.g., candidiasis).

2. Cryptococcosis causes fever, chest pain, cough, swelling of the abdomen, headache and blurred vision. It can occur in virtually any organ of the body and is the major mycosis seen in HIV/AIDS patients. Histoplasma, Coccidioides, Candida, and Cryptococcus are opportunistic pathogens.

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3. All of these are pathogenic protists cause dysentery. Balantidium coli is the only known ciliated parasite of humans, and only its cysts are infective. The genera Entamoeba and Naegleria are amoebic human parasites that cause gastrointestinal and brain infections, respectively. Entamoeba is transmitted in focally contaminated waters, whereas Naegleria inhabits warm, soil-contaminated waters. Balantidium is an intestinal parasite transmitted by fecally contaminated water.

4. In contrast to trichomoniasis, giardiasis and cryptosporidiosis are both typically waterborne diseases that cause gastrointestinal distress. Trichomoniasis is sexually transmitted.

5. P. vivax, P. falciparum, P. ovale, and P. malariae. The chill–fever pattern coincides with the release of merozoites from the erythrocytes during the synchronized reproduction cycle. The human host is infected by plasmodial sporozoites, which travel to the liver where they infect liver cells. Quinine-resistant strains of Plasmodium are now widespread and so combination therapy, where the malaria patient is treated with several antimalarial drugs at once, is now a common form of treatment.

6. Sb5+ in some way stimulates or activates the immune response to better attack Leishmania parasites.

Trypanosoma multiplies in the blood and later infects the central nervous system and grows in spinal fluid. Neurological symptoms soon begin, including sleep patterns that are no longer diel. The parasite produces the aromatic alcohol tryptophol, a derivative of the amino acid tryptophan, which triggers a sleep response.

7. Schistosomiasis, also called snail fever, is a chronic parasitic disease caused by species of trematodes (flatworms) of the genus Schistosoma; the major species is S. mansoni. The eggs of the pathogen are shed in the urine and also pass through the intestinal wall and are shed in the feces. Large egg masses also become trapped along with fluids in the bladder, liver, and other organs, triggering an inflammatory response and a major distention of the abdomen, a condition commonly seen in infected children. Other symptoms include bloody urine, diarrhea, and abdominal pain. Schistosomiasis is a disease of tropical countries, primarily those in Africa, but some cases also occur in subtropical countries such as Latin America and the Caribbean region.

Bancroft’s filariasis (also called “elephantiasis”) is a chronic infection of the lymphatic system by Wuchereria bancrofti. Once in the host, microfilariae develop into adult worms and these interrupt lymph flow, leading to major accumulation of fluids (edema).

Fluid accumulation in lower regions of the body can cause massive enlargement of the legs. It is primarily found in the tropics.

W. bancrofti is transmitted to humans in tiny microfilariae in a mosquito bite. Onchocerciasis (also called river blindness) is due to a chronic infection by the large parasitic roundworm Onchocerca volvulus. Humans are the only known host for this parasite, but flies are vectors when they become infected with microfilariae in a blood meal and transmit them to uninfected humans in a bite. Over 120 million people in the tropics suffer from W. bancrofti infection, but the microfilarial stage of the disease is readily treatable. The microfilariae of parasitic roundworm Onchocerca volvulus invade the cornea and from there the iris and retina, triggering an inflammatory response that causes scarring and partial to total loss of vision .

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Answers to Application Questions 1. Several lots of methylprednisolone formulated by a small pharmaceutical company in Massachusetts were found to be contaminated with fungi, including the mold Exserohilum rostratum. E. rostratum is common in soil and can naturally infect several human tissues, in particular the cornea, lungs, and lining of the heart (pericardium). By early December of 2012 there were 590 fungal infections—mostly cases of meningitis—linked to injections of the contaminated drug, with 37 of these being fatal.

Only about 50 species of fungi cause human diseases, and in healthy individuals, the incidence of serious fungal infections is low although certain superficial fungal infections (for example, athlete’s foot) are fairly common.

2. Systemic mycoses are characterized by fungal growth in internal organs of the body and can be either primary or secondary infections. A primary infection occurs when an otherwise normal, healthy individual is infected with the fungal pathogen; these are rather uncommon. By contrast, a secondary infection occurs in a host that harbors a predisposing condition, such as antibiotic therapy or immunosuppression, which makes the individual more susceptible to infection.

In the United States, the three major systemic mycoses are histoplasmosis, coccidioidomycosis, and blastomycosis. Mortality from these is high, about 10%. Chemotherapy against systemic fungal infections is more difficult because of issues with host toxicity. For example, one of the most effective antifungal agents, amphotericin B, is widely used to treat systemic fungal infections but can also affect kidney function and have other unwanted side effects.

3. Malaria, leishmaniasis, and trypanosomiasis are primarily restricted to tropical or subtropical regions because their transmission is dependent upon insect vectors that are endemic to areas having a warm climate. Human-induced climate change may be causing an expansion of tropical regions, and this will allow disease vectors to infiltrate new areas, thereby broadening the geographical ranges in which these diseases occur.

4. He is likely infected with Naegleria fowleri and should be prescribed amphotericin B.

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