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Course: Medical Microbiology, PAMB 650/720

2009/2010

Lecture 34: Nutrition, Growth and Metabolism. Faculty: Dr. Alvin Fox, Phone: 733 3288, E-mail: alvin.fox@uscmed.sc.edu Office: C-19, Building 2 Suggested reading: Murray, 6th Edition, Chapter 3 KEY WORDS Obligate aerobe Obligate anaerobe Aerotolerant anaerobe Facultative anaerobe Microaerophilic Siderophore Mesophile Thermophile Psychrophile Generation time

Growth curve Glycolysis Fermentation Anaerobic respiration Aerobic respiration Tricarboxylic acid (TCA) cycle or Krebs cycle Oxidative phosphorylation Ubiquinone Glyoxylate pathway

Bacterial requirements for growth include oxygen, (or its absence) nutrients, optimal temperature and pH. Oxygen Requirements: Obligate aerobes must grow in the presence of oxygen; they can’t carry out fermentation. Obligate anaerobes do not carry out oxidative phosphorylation. Furthermore, they are killed by oxygen; they lack certain enzymes (e.g. catalase [decomposes + hydrogen peroxide, H2O2, to H2O and O2], peroxidase (H2O2+ NADH + H converted to NAD and 2H2O), superoxide dismutase (superoxide [O2-] to H2O2) which detoxify both H2O2and oxygen free radicals (superoxide) produced as side-products during metabolism in the presence of oxygen. Aerotolerant anaerobes are bacteria that respire anaerobically, but can survive in the presence of oxygen. Facultative anaerobes can perform both fermentation and aerobic respiration. In the presence of oxygen, anaerobic respiration is generally shut down and these organisms respire aerobically. Microaerophilic bacteria grow well in low concentrations of oxygen, but are killed by higher concentrations. Nutrient Requirements: include sources of organic carbon, N, P, S and metal ions (e.g. iron). Bacteria secrete small molecules that bind iron (siderophores, e.g. enterobactin and mycobactin). Siderophores (with bound iron) are then internalized via receptors by the bacterial cell. The human host also has iron transport proteins (e.g. transferrin). Thus bacteria that ineffectively compete with the host for iron are poor pathogens. Temperature: Bacteria may grow at a variety of temperatures from close to freezing to 1


near to the boiling point of water. Those that grow best at the middle of this range are referred to as mesophiles; which includes all human pathogens and opportunists. Those having lower and higher temperature optima for growth are respectively known as psychrophiles and thermophiles. pH: Many bacteria grow best at neutral pH; however certain bacteria can survive and even grow in quite acid or alkaline conditions. Measuring bacterial mass in liquid cultures of bacteria: Common methods include: (a) Turbidity (how cloudy is a liquid culture of bacterial -a measure of total bacteria [live and dead]). This is usually quantitated with a spectrophotometer or (b) Number of viable bacteria in a culture - usually assessed by counting the number of colonies that grow after streaking a known volume on a plate (“plate counting” of colony forming units). In either case plotting the log of turbidity or number of living cells versus time is referred to as the growth curve. In the lag phase, since bacteria are adapting to the media, there is little or no growth. During the log phase – growth is fast (logarithmic). In the stationary phase the bacteria stop growing. Finally, as the nutrient supply is exhausted they start to die (death phase). In many cases (but not always) the bacteria autolyse (during the death phase) and the turbidity decreases. The generation time is defined as the time required for bacterial mass to double.

Growth Curve

Growth Curve Stationary

COLONY FORMING UNITS

Death

Log

Stationary

TURBIDITY (cloudiness)

Autolysis

Log

Lag

Lag

TIME

TIME

METABOLISM OF SUGARS Step 1. Glycolysis (Embden Meyerhof Parnas Pathway) is the most common pathway in bacteria for sugar catabolism (also found in most animal and plant cells). A series of enzymatic reactions convert sugars into pyruvate, generating ATP (adenosine triphosphate) and NADH (nicotinamide adenine dinucleotide). Chemical energy needed for biosynthetic purposes are stored in the newly formed compounds (ATP and NADH).

2


NAD Glucose (C6) *

ADP

NADH ATP

Pyruvate (C3)*

refers to number of carbons in molecule

There are alternatives, for catabolizing sugars, to produce stored energy within ATP including the pentose phosphate pathway (hexose monophosphate shunt) that is found in most animal and plant cells. NADPH is generated using this pathway. A third pathway (the Entner Doudoroff pathway) is found in a few species of bacteria and is not seen in animals. Step 2 –either A or B A. Anaerobic Respiration Anaerobic respiration includes glycolysis and fermentation. During the latter stages of this process NADH (generated during glycolysis) is converted back to NAD by losing a hydrogen molecule. The hydrogen is added to pyruvate and depending on the bacterial species a variety of metabolic end-products can be produced. NADH

NAD

Pyruvate (C3)

Short chain alcohols or fatty acids (e.g. lactic acid or ethanol) (C2-C4)

B. Aerobic Respiration/Catabolism Aerobic respiration involves glycolysis and the tricarboxylic acid cycle (Krebs cycle). Pyruvate is fully broken down to CO2 and in the process NAD converted to NADH. Thus in aerobic fermentation NADH is generated from two sources (glycolysis and the Krebs cycle). Oxidative phosphorylation converts excess NADH back to NAD and in the process produces more ATP (stored energy). Ubiquinones and cytochromes are components of the electron transport chain involved in this latter process. Conversion of oxygen to water is the final step in the process.

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Catabolism/Krebs Cycle (C4-C6 intermediate compounds) NAD

NADH

Pyruvate (C3)

3 CO2 (C1)

Oxidative phosphorylation NADH

NAD

O2

H2O ADP

ATP

The Krebs cycle C2 Acetate -CO2

+

Isocitrate Citrate

C X -CO2 NADH

C6

Alpha-keto glutarate

Oxaloacetate C4

-CO2 NADH

Pyruvate

x

Succinate C

Malate Fumarate

Replenishing biosynthesis Intermediates of the Krebs cycle can be removed for biosynthesis of amino acids (e.g. by conversion of oxaloacetate to aspartic acid). If sugars are the sole carbon source, removing Krebs intermediates would shut down the cycle. In an alternative enzymatic reaction addition of CO2 to pyruvate produces oxaloacetate which replenishes the Krebs Cycle. METABOLISM OF FATTY ACIDS Fatty acids are broken down to acetic acid (C2) which feed into the Krebs Cycle by addition to a C4 intermediate (oxaloacetate) to produce C6 compound (citric acid). During the cycle the added C2 is lost (as 2CO2) and C4 regenerated. Overall no increase in the number of molecules of cycle intermediates occurs.

Thus if fatty acids are the sole carbon source then no Krebs Cycle intermediates can be removed 4


without shutting it down + C2 C4 C6 Instead bacteria utilize the glyoxylate cycle (a modified Krebs Cycle). The 2 enzymatic steps in which CO2 molecules are removed from intermediates of the cycle are by-passed. Instead a C6 intermediate is converted to two C4 compounds (both components of the cycle). Thus for every acetyl group (from fatty acids) an additional cycle intermediate can be produced. The glyoxylate pathway is not generally found in animal cells since pre-formed fatty acids, present in food, are utilized. C6

C4

+ + C2 C2

C4

Take home message the Krebs Cycle functions in a biosynthetic and energy producing fashion. However, if intermediates are to be removed for use in other metabolic pathways they must be replenished. The replenishment process for utilization of sugars and fatty acids is different.

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