International Microbiology

Volume 16 路 Number 3 路 September 2013 路 ISSN 1139-6709 路 e-ISSN 1618-1905

INTERNATIONAL MICROBIOLOGY www.im.microbios.org

16(3) 2013

Official journal of the Spanish Society for Microbiology

Publication Board

Editorial Board

Coeditors-in-Chief José Berenguer (Madrid), Autonomous University of Madrid Ricardo Guerrero (Barcelona), University of Barcelona

Ricardo Amils, Autonomous University of Madrid, Madrid, Spain Shimshon Belkin, The Hebrew University of Jerusalem, Jerusalem, Israel Albert Bordons, University Rovira i Virgili, Tarragona, Spain Albert Bosch, University of Barcelona, Barcelona, Spain Victoriano Campos, Pontificial Catholic University of Valparaíso, Chile Josep Casadesús, University of Sevilla, Sevilla, Spain Rita R. Colwell, Univ. of Maryland & Johns Hopkins Univ.., Baltimore, MD, USA Katerina Demnerova, Inst. of Chem. Technology, Prague, Czech Republic Esteban Domingo, CBM, CSIC-UAM, Cantoblanco, Spain Mariano Esteban, Natl. Center for Biotechnol., CSIC, Cantoblanco, Spain Mariano Gacto, University of Murcia, Murcia, Spain Juncal Garmendia, Institute of Agrobiotechnology, Pamplona, Spain Olga Genilloud, Medina Foundation, Granada, Spain Steven D. Goodwin, University of Massachusetts, Amherst, MA, USA Juan C. Gutiérrez, Complutense University of Madrid, Madrid, Spain Johannes F. Imhoff, University of Kiel, Kiel, Germany Juan Imperial, Technical University of Madrid, Madrid, Spain John L. Ingraham, University of California, Davis, CA, USA Juan Iriberri, University of the Basque Country, Bilbao, Spain Roberto Kolter, Harvard Medical School, Boston, MA, USA Germán Larriba, University of Extremadura, Badajoz, Spain Rubén López, Center for Biological Research, CSIC, Madrid, Spain Michael T. Madigan, Southern Illinois University, Carbondale, IL, USA Beatriz S. Méndez, University of Buenos Aires, Buenos Aires, Argentina Diego A. Moreno, Technical University of Madrid, Madrid, Spain Ignacio Moriyón, University of Navarra, Pamplona, Spain Juan A. Ordóñez, Complutense University of Madrid, Madrid, Spain José M. Peinado, Complutense University of Madrid, Madrid, Spain Antonio G. Pisabarro, Public University of Navarra, Pamplona, Spain Carmina Rodríguez, Complutense University of Madrid, Madrid, Spain Fernando Rojo, Natl. Center for Biotechnology, CSIC, Cantoblanco, Spain Manuel de la Rosa, Virgen de las Nieves Hospital, Granada, Spain Carmen Ruiz Roldán, University of Murcia, Murcia, Spain Claudio Scazzocchio, Imperial College, London, UK James A. Shapiro, University of Chicago, Chicago, IL, USA John Stolz, Duquesne University, Pittsburgh, PA, USA James Strick, Franklin & Marshall College, Lancaster, PA, USA Gary A. Toranzos, University of Puerto Rico, San Juan, Puerto Rico Antonio Torres, University of Sevilla, Sevilla, Spain José A. Vázquez-Boland, University of Edinburgh, Edinburgh, UK Antonio Ventosa, University of Sevilla, Sevilla, Spain Tomás G. Villa, Univ. of Santiago de Compostela, Santiago de C., Spain Miquel Viñas, University of Barcelona, Barcelona, Spain Dolors Xairó, Biomat, S.A., Grifols Group, Parets del Vallès, Spain

Associate Editors Mercedes Berlanga, University of Barcelona Mercè Piqueras, Catalan Association for Science Communication Wendy Ran, International Microbiology Secretary General Jordi Mas-Castellà, International Microbiology Webmasters Nicole Skinner, Institute for Catalan Studies Jordi Urmeneta, University of Barcelona Managing Coordinator Carmen Chica, International Microbiology Specialized editors Josefa Antón, University of Alicante Susana Campoy, Autonomous University of Barcelona Ramón Díaz, CIB-CSIC, Madrid Josep Guarro, University Rovira i Virgili Enrique Herrero, University of Lleida Emili Montesinos, University of Girona José R. Penadés, Institute of Mountain Livestock-CSIC, Castellon Jordi Vila, University of Barcelona

Addresses Editorial Office International Microbiology Poblet, 15 08028 Barcelona, Spain Tel. & Fax +34-933341079 E-mail: int.microbiol@microbios.org www.im.microbios.org Spanish Society for Microbiology Vitruvio, 8 28006 Madrid, Spain Tel. +34-915613381. Fax +34-915613299 E-mail: sem@microbiologia.org www.semicrobiologia.org Publisher (electronic version) Institute for Catalan Studies Carme, 47 08001 Barcelona, Spain Tel. +34-932701620. Fax +34-932701180 E-mail: int.microbiol@microbios.org © 2013 Spanish Society for Microbiology & Institute for Catalan Studies.

Printed in Spain ISSN (print): 1139-6709 e-ISSN (electronic): 1618-1095 D.L.: B.23341-2004

p2

CONTENTS International Microbiology (2013) 16:133-210 ISSN 1139-6709 www.im.microbios.org

Volume 16, Number 3, Setember 2013 RESEARCH REVIEW

Guerrero R, Margulis L, Berlanga M Symbiogenesis: the holobiont as a unit of evolution

133

RESEARCH ARTICLES

Molina MC, Divakar PK, Zhang N, González N, Struwe L Non-developing ascospores in apothecia of asexually reproducing lichen-forming fungi

145

Zahran HH, Chahboune R, Moreno S, Bedmar EJ, Abdel-Fattah M, Yasser MM, Mahmoud AM Identification of rhizobial strains nodulating Egyptian grain legumes

157

Guzmán K, Campos E, Aguilera L, Toloza L, Giménez R, Aguilar J, Baldoma L, Badia J Characterization of the gene cluster involved in allantoate catabolism and its transcriptional regulation by the RpiR-type repressor HpxU in Klebsiella pneumoniae

165

Plasencia A, Gich F, Fillol M, Borrego CM Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

177

Barros J, Grenho L, Manuel CM, Ferreira C, Melo LF, Nunes OC, Monteiro FJ, Ferraz MP A modular reactor to simulate biofilm development in orthopedic material

191

PERSPECTIVES

Abadal E Gold or green: The debate on Open Access policies

199

MEETINGS

Fusté E, Viñas M The 24th Congress of the Spanish Society for Microbiology (L’Hospitalet de Llobregat, Barcelona, 10–13 July 2013)

205

A1

Spanish Society for Microbiology The Spanish Society for Microbiology (SEM) is a scientific society founded in 1946 at the Jaime Ferrán Institute of the Spanish National Research Council (CSIC), in Madrid. Its main objectives are to foster basic and applied microbiology, promote international relations, bring together the many professionals working in this science, and contribute to the dissemination of science in general and microbiology in particular, among society. It is an interdisciplinary society, with about 1800 members working in different fields of microbiology.

International Microbiology Aims and scope International Microbiology, the official journal of the SEM, is a peer-reviewed, open access journal whose aim is to advance and disseminate information in the fields of basic and applied microbiology among scientists around the world. The journal publishes research articles and complements (short papers dealing with microbiological subjects of broad interest such as editorials, perspectives, book reviews, etc.). A feature that distinguishes it from many other microbiology journals is a broadening of the term “microbiology” to include eukaryotic microorganisms (protists, yeasts, molds), as well as the publication of articles related to the history and sociology of microbiology.

International Microbiology, offers high-quality, internationally-based information, short publication times (<3 months), complete copy-editing service, and online open access publication available prior to distribution of the printed journal.

The journal encourages submissions in the following areas:

• Microorganisms (prions, viruses, bacteria, archaea, protists, yeasts, molds) • Microbial biology (taxonomy, genetics, morphology, physiology, ecology, pathogenesis) • Microbial applications (environmental, soil, industrial, food and medical microbiology, biodeterioration, bioremediation, biotechnology) • Critical reviews of new books on microbiology and related sciences are also welcome.

Journal Citations Reports The 2012 Impact Factor of International Microbiology is 2,556.

The journal is covered in several leading abstracting and indexing databases, including the following ones: AFSA Marine Biotechnology Abstracts; Biological Abstracts; Biotechnology Research Abstracts; BIOSIS Previews; CAB Abstracts; Chemical Abstracts; Current Contents – Agriculture, Biology & Environmental Sciences; EBSCO; Embase; Food Science and Technology Abstracts; Google Scholar; IEDCYT; IBECS; Latíndex; MedBioWorld; PubMed; SciELO-Spain; Science Citation Index Expanded; Scopus.

Cover legends Front cover Center. Gaudí’s dragon at the entrance of Park Güell, Barcelona.

With its bright scales of small tiles, it represents Python, Delphian guardian of the underground waters, the source of wisdom. Python, Gaia’s son, spelled oracles, early symbol of the communication of knowledge and science. This symbol of Barcelona also was a adequate, artistic representative of the 24th Congress of the Spanish Society for Microbiology (SEM), that was held in L’Hospitalet de Llobregat and in Barcelona, during 10–13 July 2013 (See article by E. Fusté and M. Viñas, pp 205-209, this issue.)

Upper left. Particles of the turnip mosaic virus, a Potyvirus that infects mainly cruciferous plants and is one of the most damaging viruses in plant crops. It causes chlorotic ringspots in young leaves; as the leaf ages, yellow or brownish spots surrounbded by circular or irregular necrotic rings appear. Micrograph by Fernando Ponz, CBGP, UPM-INIA, Madrid. (Magnification, ca. 100,000×) Upper right. Transmission electron micrograph of a group of cells of the polyhydroxyalcanoate-producing Halomonas venusta MAT-28.

The cells are growing as a microcolony in an artificial biofilm of alginate beads. In this situation, cells maintain a metabolic state equivalent to that of the planktonic culture. Micrograph by M. Berlanga, Faculty of Pharmacy, University of Barcelona, and Carmen López, CCiT, University of Barcelona. [See article by Berlanga M., et al., Int Microbiol (2012) 15:191-199.] (Magnification, ca. 6,000×) Lower left. Scanning electron micrograph of Minorisa minuta, a bacterivorous protist described in 2012. With an average size of 1.4 mm, it is one of the smallest bacterial grazers known to date. It has a worldwide distribution and can account for 5 % of heterotrophic protists in coastal waters. Micrograph by Javier del Campo, Institute for Marine Sciences, CSIC, Barcelona, Spain. [For more information, see article by del Campo J, et al., ISME J (2013) 7:351-358] (Magnification, ca. 40,000×) Lower right. SEM micrograph of Saksenaea vasiformis (Saksenaeaceae, Mucorales, Mucoromycotina) sporangiophores isolated from human tissue. It is a filamentous fungus with characteristic flask-

shaped sporangia that can cause severe human infections in both immunocompromised and immunocompetent hosts. Micrograph

by José F. Cano, University Rovira i Virgili, Reus-Tarragona, Spain. (Magnification, ca. 1000×) Back cover Portrait of Miguel Ángel Ugarte Vega (1862-1898), a pioneer of medicine in Honduras. Born in Tegucigalpa in May 8, 1862, from Miguel Ugarte and Manuela Vega, he attended medical school in Guatemala, but had to leave that country due to his revolutionary ideas, and was denied any documentary evidence of his academic record. He then moved to San Salvador city, El Salvador, where he successfully passed the official medical practitioner examination. He was a brilliant student of Professor Emilio Álvarez, a Colombian who promoted and modernized medical training in El Salvador. At only 19, Ugarte graduated in Medicine and Surgery.

He then worked abroad and returned to Honduras in 1893, already married; within a year he was appointed Chief Surgeon and Director of the General Hospital of Tegucigalpa. Although he died young in 1898, the last years of his life were very fruitful, and he modernized medical practice and training in his country. As a follower of Koch’s principles, he introduced the concepts of asepsis and antisepsis, and the use of corrosive sublimate and iodoform; he also convinced Dr. Policarpo Bonilla, president of Honduras, to buy an X-ray machine—the first to be used in Central America.

As director of the General Hospital of Tegucigalpa, Ugarte renovated the operating theaters to fully comply with advice on asepsis, and was the first surgeon from Honduras to perform major surgery successfully. He was also the first physician in Honduras to use microscopy techniques to study intestinal parasitosis with a microscope he brought from El Salvador. Although by his professional career Ugarte was not a microbiologist (in fact he is considered the developer of surgery in Honduras), because of his introducing microbiology concepts that advanced medicine in his country, he can be considered one of the pioneers of Microbiology in Latin America.

Front cover and back cover design by MBerlanda & RGuerrero

A2

RESEARCH ARTICLE International Microbiology (2013) 16:133-143 doi: 10.2436/20.1501.01.188 ISSN 1139-6709 www.im.microbios.org

Symbiogenesis: the holobiont as a unit of evolution Ricardo Guerrero,1 Lynn Margulis,§ Mercedes Berlanga2* 1 Department of Microbiology, University of Barcelona, Barcelona, Spain.

Department of Microbiology and Parasitology, University of Barcelona, Barcelona, Spain

2

Received 15 August 2013 · Accepted 15 September 2013 Summary. Symbiogenesis is the result of the permanent coexistence of various bionts to form the holobiont (namely, the host and its microbiota). The holobiome is the sum total of the component genomes in a eukaryotic organism; it comprises the genome of an individual member of a given taxon (the host genome) and the microbiome (the genomes of the symbiotic microbiota). The latter is made up of the genes of a variety of microbial communities that persist over time and are not eliminated by natural selection. Therefore, the holobiome can also be considered as the genomic reflection of the complex network of symbiotic interactions that link an individual member of a given taxon with its associated microbiome. Eukaryotic individuals can be analyzed as coevolved, tightly integrated, prokaryotic communities; in this view, natural selection acts on the holobiont as if it were an integrated unit. The best studied holobionts are those that emerged from symbioses involving insects. The presence of symbiotic associations throughout most of the evolutionary history of insects suggests that they were a driving force in the diversification of this group. Support for the evolutionary importance of symbiogenesis comes from the observation that the gradual passage from an ancestral to a descendant species by the accumulation of random mutations has not been demonstrated in the field, nor in the laboratory, nor in the fossil record. Instead, symbiogenesis expands the view of the point-mutationonly as the unique mechanisms of evolution and offers an explanation for the discontinuities in the fossil record (“punctuated equilibrium”). As such, it challenges conventional paradigms in biology. This review describes the relationships between xylophagous insects and their microbiota in an attempt to understand the characteristics that have determined bacterial fidelity over generations and throughout evolutionary history. [Int Microbiol 2013; 16(3):133-143] Keywords: symbiogenesis · symbiosis · holobiont · holobiome · microbiota · microbiome · coevolution

Introduction Symbiosis was an essential evolutionary mechanism in the origin of the eukaryotic cell [33,34]. The first living beings were prokaryotes. In fact, bacteria are the only organisms that

Corresponding author: M. Berlanga Department of Microbiology and Parasitology Faculty of Pharmacy, University of Barcelona Av. Joan XXIII, s/n 08028 Barcelona, Spain Tel. +34-934024497. Fax +34-934024498. E-mail: mberlanga@ub.edu

*

§

Lynn Margulis (née Lynn Petra Alexander), Distinguished Professor at the Uni­ versity of Massachusetts-Amherst, MA, USA, died on 22 November, 2011.

are not dependent on others for their survival; on the contrary, they are necessary for the survival of other living beings. The mechanism that made it possible for a group of randomly gathered compounds to become autopoietic—that is, to form a distinct entity separated from the environment by a boundary and able to both maintain itself actively and autoreplicate [19,20,36]—has yet to be identified. The autopoietic unit, whether a bacterial biont (minimal autopoietic unit) or a holobiont (integrated biont organisms, i.e., animals or plants, with all of their associated microbiota), is capable of selfmaintenance by sensing the environment and is able to adapt to new circumstances. Complex autopoietic units acquire novel properties when the assembly of their components results in higher functional-structural complexity (Fig. 1). However,

134

Int. Microbiol. Vol. 16, 2013

guerrero et al.

Fig. 1. Endosymbiosis: Homage to Lynn Margulis, a painting by Shoshanah Dubiner. A six-foot wide reproduction of the painting occupies a hallway in the Morrill Science Center at the University of Massachusetts-Amherst, MA, USA where Lynn Margulis was a Distinguished Professor from 1988 until her death in 2011. (Image courtesy of the artist [http://www.cybermuse.com].)

autopoiesis alone, while necessary, is not a sufficient condition for life. Rather, living organisms constantly interact with their habitats, selectively taking up compounds from their surroundings to meet their particular needs but also excreting metabolic products and thus modifying their environment.

The term “symbiosis” was coined by Heinrich Anton de Bary (1831–1888) to describe the living together of “differently named organisms” [12]. Symbiosis is a long-term physical association of two or more partners, and symbiotic relationships can only occur under certain environmental conditions. In endosymbiosis, a topological condition, one partner lives inside the other. Symbiogenesis refers to the appearance of new morphologies, tissues, metabolic pathways, behaviors, or other recognizable evolutionary novelties in holobionts. The term was first introduced into the literature by the Russian Konstantin Sergeivich Mereschkovsky (18551921) and is equivalent to “symbionticism” or “microsymbiotic complexes”, both of which were independently coined by the Swedish-American Ivan Emanuel Wallin (1883−1969) [56].

Evolutionary biologists have viewed mutations within individual genes as the major source of phenotypic variation.

Mutations lead to adaptation through natural selection and

ultimately generate diversity among species. Symbiogenesis, however, is another mechanism able to drive evolutionary innovation, as the holobiont is better adapted to the environment than its individual components. Joshua Lederberg (1925– 2008) defined the ho­lobiont as “the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space:” [28]. As such, the holobiome, i.e., the assembly of genetic information contributed by the animal or plant and its associated microbiota, is an essential aspect of the evolving holobiont. It is an essential life-changing force that has resulted in a complex coordinated coevolution of life forms.

Symbiogenesis could provide support for the theory of punctuated equilibrium proposed by Niles Eldredge and Stephen J. Gould [16], based on their observation that according to the fossil record evolution is largely static but then acts suddenly and often dramatically during brief (in geological terms) intervals of time, as evidenced by the periodic large changes in fossil registration. In evolving holobionts, symbiogenesis confers cellular-tissue, and organlevel developmental and morphological complexity [37,47].

Accordingly, a unified theory of evolution can be considered

Symbiogenesis and evolution

135

The role of hindgut microbes: the case of Dictyoptera Insects account for most of the species-richness of the animal clade.

Within this group, termites (Isoptera), cockroaches, and mantids form a well-established lineage of insects, the Dictyoptera. In termites, the omnivorous family Termitidae com­prises 80 % of all termite species, and the six remaining wood-eating families (Mastotermitidae, Kalotermitidae, Hodo­­­termitidae, Termopsidae, Rhinotermitidae and Serriter­mitidae), often misleadingly called “lower termites,” the remaining 20 %. Termites can be considered as “social cockroaches,” with the family Cryptocercidae as their closest relative. Thus, “lower” termites share with wood-feeding cockroaches (family Cryptocercidae; Blattaria, Dictyoptera) the unusual ability to efective degradation of lignocellulosic plant materials [2]. Depending on the species, the food preferences of termites range from wood to leaves, humus, detritus, and herbivore dung. By degrading lignin, cellulose, and hemicelluloses to fermentable carbohydrates, the diverse populations of prokar­ yotes and flagellated protists that inhabit the intestines of all wood-feeding lower termites are indispensable to their respective hosts. In the Termitidae, whose digestive system typically lacks protists, intestinal cellulolytic activity is due either to prokaryotic microbiota or to fungi, grown by the termites themselves or endogenous to the termite intestine [42].

In most insects, mating is the only “social behavior” as females abandon their eggs after depositing them.

Consequently, opportunities for the direct transfer of gut microbes between members of the same species are for the

Int Microbiol

in which the nuclear genome (of the eukaryotic component) and the microbiome (the genetic donation of the symbiotic microorganisms) are the interacting components that give rise to new species and varieties thereof.

Support for this view comes from the following: First, microbial symbionts are universally present in eukaryotes, and prokaryotic microorganisms are widespread in all environments on Earth. Consistent with their ecological ubiquity, many prokaryotic species establish close and in many cases persistent relationships with members of a wide range of eukaryotic taxa [20,60]. Second, hosts typically have strong specificity for microbial symbionts and their functions.

Third, symbiotic relationships have enhanced the limited metabolic networks of most eukaryotes by contributing several prokaryotic metabolic capabilities, such as methanogenesis, chemolithoautotrophy, nitrogen assimilation, and essentialnutrient anabolism [17]. Also, many prokaryotes defend their symbiotic partners against natural enemies and promote their adaptation to specific ecological conditions [45,46]. Others may have parasitic or pathogenic effects on their hosts, causing attenuated host fitness and aberrations in reproduction [58]. Finally, host immune genes evolve rapidly in response to microbial symbionts and as a gene family are frequently involved in hybrid incompatibilities [4,5].

This review examines the characteristics that determine bacterial fidelity to certain groups of animals over generations and throughout evolution, by examining the relationships between two xylophagous insects (namely, the termite Reti­ culi­termes grassei, and the social cockroach Cryptocercus punc­tu­latus) and their microbiota (Fig. 2).

Int. Microbiol. Vol. 16, 2013

Fig. 2. Termites (left) and xylophagous cockroaches (right) mantained in laboratory conditions (Photographs by M. Berlanga and R. Duro.)

136

Int. Microbiol. Vol. 16, 2013

most part limited, unlike, for example, mammals and birds, which have prolonged parent–offspring connections [17].

However, several insect species, including cockroaches, termites, ants, and some wasps and bees, show gregarious or social behaviors. Among social insects, termites, which undergo incomplete metamorphosis, display a diversified caste polyphenism. Termite workers, soldiers, reproductive adults, and undifferentiated immature forms cooperate within their communities in an integrated manner. Each caste plays a significant role within the colony. Reproductive adults maintain the population; soldiers protect the colony from invaders; workers (the most numerous life stage in a colony) build and maintain the galleries, take care of the larvae, and feed the other colony members. Workers transfer food stomodeally (by regurgitation) and/or proctodeally (by excretion with the hindgut contents) (Fig. 3). Both oral trophallaxis and coprophagy allow the direct or indirect transmission of microorganisms and thus promote the coevolution of specialized host-dependent symbionts [1,22,35]. Proctodeal trophallaxis (feeding) is also the means by which microorganisms are vertically transmitted from workers to other individuals of the colony. In many insects, the proctodeal part of the intestine, i.e., the hindgut, is shed during ecdysis.

Consequently, the re-establishment of the gut microbiota of newly molted termite workers and soldiers depends on the contributions of fellow workers [3]. While young soldiers, before developing large mandibles, can chew wood, adult soldiers, owing to their large mandibles, cannot. Nevertheless, adults are able to digest proctodeal wood particles that have already passed through the gut of workers and are thus partially digested.

Therefore, in wood-eating termites, the gut of soldiers, like that of workers, harbors protists and bacteria throughout the insect life cycle [3]. Note that an unexplained finding is that the genus Wolbachia is totally absent from workers but rather abundant in soldiers [3]. Based on the 16S rRNA gene phylogenies of Wolbachia, there are eight major clades (A–H). Clades A and B include most of the parasitic Wolbachia found in arthropods.

Clades C and D include the majority of the mutualistic Wolbachia present in filarial nematodes, and clades E–H include Wolbachia from various arthropods. For clades E–H, the effects on the host are currently unknown; clade F is notable in that its members infect arthropods—especially termites—, but also nematodes.

Phylotypes obtained from soldiers of R. grassei were affiliated with clade F [3]. Wolbachia species are associated with four distinct reproductive phenotypes in a wide range of Arthropoda: feminization, parthenogenesis, male killing, and cytoplasmic incompatibility. However, nothing is known about possible phenotypes linked to Wolbachia in Isoptera [58].

guerrero et al.

Examination of the protist population dynamics in the species Reticulitermes speratus during colony foundation has shown that protist numbers increase dramatically in termite queens and kings during the first 50 days of the colony foundation, but then begin to decrease by day 100, finally having disappeared by day 400. Ultimately, both kings and queens lose their protists entirely and become completely dependent upon their offspring for feeding. Protists are abundant in soldiers from mature colonies but absent in neotenics. This probably reflects the feeding of soldiers by workers via proctodeal trophallaxis and of reproductive members of the colony via stomodeal trophallaxis [50].

Termites and the Cryptocercus gut micro­­­biota The basic structure of the digestive tract is similar across insects, although a diversity of modifications associated with adaptation to different feeding modes can be found.

The insect gut has three primary regions, foregut, midgut, and hindgut [17]; the foregut and hindgut originate from the ectoderm and the midgut from the endoderm. The Malpighian tubules comprising the excretory system in some insects and other animals extend into the body cavity and absorb wastes, such as uric acid, which are sent to the anterior hindgut, from which this system developmentally derives [17,43]. Many herbivorous insects have a tubular hindgut with several dilated compartments that harbor a dense gut microbiota.

In these dilated compartments, or “fermentation chambers,” the prolonged residence time of food allows its degradation by microbial symbionts, a situation analogous to that in the rumen or colon of mammals. With their ability to digest the major structural polymers of plant cell walls (cellulose and hemicelluloses), microorganisms are an important supplement of the digestive capacities of their hosts. The fermentation products, mainly acetate and other short-chain fatty acids, are reabsorbed by the insect and contribute substantially to its nutrition [42,43]. Termite guts are axially and radially structured habitats containing many chemical microniches, reflecting the combination of host and microbial activities [7]. All insect guts are surrounded by tissues aerated by the insect’s tracheal system. Oxygen penetrates the peripheral hindgut contents to a depth of up to 150–200 μm below the epithelium. The removal of oxygen by the respiratory activity of the gut microbiota creates a microoxic periphery around an anoxic center [6]. In the case of termites, whose abdomen can be less than 1 mm wide, the redox potential forms an extreme

Int. Microbiol. Vol. 16, 2013

Symbiogenesis and evolution

137

A

C

Int Microbiol

B

Fig. 3. (A) External appearance of the termite Reticulitermes grassei, soldier caste. (B,C) Microbiota from the wholehindgut of a worker caste individual: (B) Protists and (C) spirochetes. (Photographs by R. Duro.)

gradient, ranging from ca. Eh = +840 mV to Eh = –420 mV over a few dozen micrometers (and returning to atmospheric conditions over the same tiny length).

The diversity of the hindgut microbiota of termites depends on several factors, including the variety of specialized structures present in the gut, the effect of pH, the sharp redox gradient, and the type of food ingested [9,14].

An indigenous biota is one that is present in all individuals, colonizes the gut habitat, and is maintained in “stable” climax communities. The microbiota detected in the guts of lower termites and Cryptocercus greatly differ from the microbiota of the environment (e.g., soil), and the anoxic and extremely low Eh conditions are in dramatic contrast to the oxic and positive Eh that surrounds these insects [2,3,14].

The hindgut bacteria of wood-feeding lower termites and cockroaches belong to several phyla, including Proteobacteria, Bacteroidetes, Firmicutes, Actinomycetes, Spirochetes, Verru­ comicrobia, and Actinobacteria, as detected by 16S rRNA [17]. Of these, spirochetes may account for up to 50 % of all prokaryotes in the hindguts of some lower termites (Table 1, Fig. 4). Spirochete populations provide nitrogen, carbon

sources, and electron donors to other resident microbial populations and to the host [27,32,57]. As specific symbionts that have coevolved with their respective termite hosts, spirochetes are stably harbored by several species and closely associated with members of the same termite family [1].

Termites preserved in amber provide direct paleontological evidence for the stable relationship between termites and their intestinal symbionts (protists and spirochetes) throughout at least 20 million years [59]. This ancient coevolution of unique and diverse spirochetes with xylophagous social insects explains the essential function contributed by these microbial symbionts for wood digestion.

Protists: unique inhabitants in termites and Cryptocercus hindguts At least 15 % of all existing protist species depend on a sym­biotic way of life; indeed, the symbiotic membership of nine separate protistan phyla (in four different kingdoms) is virtually 100 % [10]. Protists found in the digestive tract of

138

Int. Microbiol. Vol. 16, 2013

guerrero et al.

Table 1. Phyla composition (%) of the gut microbiota in several xylophagous insects* Termites

Cockroaches

Wood-feeding beetle

Coptotermes formosanus

Reticulitermes speratus

Reticulitermes grassei

Nasutitermes spp.

Cryptocercus punctulatus

C. punctulatus (fasting)

C. punctulatus (dead)

Actinobacteria

2.0

5.3

4.0

1.5

6.6

10.5

45.8

31.0

Firmicutes (ClostridiaBacilli)

10.8

39.5

12.0

7.0

32.1

10.6

29.2

27.3

Firmicutes (Mollicutes)

4.8

2.6

Phylum

2.2

Acidobacteria Bacteroidetes

0.1 72.4

12.0

13.0

Fibrobacteres

4.0

25.5

15.7

8.6

5.0

Planctomycetes

0.8

1.5

Proteobacteria

1.6

13.4

18.0

4.0

20.7

42.2

Spirochetes

6.0

23.0

25.5

58.0

5.0

5.3

Synergistetes

0.8

3.2

10.5

TG1

2.0

TG2

18.0

25.0

32.0

2.2

3.0

TG3

17.5

TM7 Verrucomicrobia

Anoplophora glabripennis

4.5 0.8

0.7

5.2

2.0

Other phyla

2.5

0.4 0.6

3.0

*Data from Coptotermes formosanus (Rhinotermitidae) are based on Shinzato et al. [51]. Data from Reticulitermes speratus (Rhinotermitidae) are based on Hongoh et al. [21] and Nakajima et al. [38]. Data from Reticulitermes grassei (Rhinotermitidae) are based on Berlanga et al. [3]. Data from Nasutitermes spp. (Termitidae) are based on Köler et al. [26]. Data from Cryptocercus punctulatus (two different physiological states) are based on Berlanga et al. [2].

Data from Anoplophora glabripennis are based on Scully et al. [49].

termites and Cryptocercus cockroaches belong to the orders Trichomonadida, Cristamonadida, Hypermastigida, and Oxymonadida [15]. Hypermastigids are unique in nature, as they are found only in lower termites and Cryptocercus [8]. In fact, Cryptocercus cockroaches retain more diverse flagellate species than any extant termite species [15], with a single gut containing approximately 103–105 protistan cells/ ml, accounting for at least 90 % of the volume of the hindgut.

Each wood-feeding termite species harbors specific protists and hosts between 1 and 20 morphologically distinguishable species [3,31]. Yet, while termites support a characteristic community of gut protists, many protist species are not necessarily restricted to one termite species. Furthermore, many protist species are simultaneously associated with

different bacterial ectosymbionts, such as Spirochaetes, Bacteroidetes, and/or Synergistetes [43].

Ectosymbiotic spirochetes attach to the cell surfaces of protists in the termite gut, forming complex coevolutionary relationships (Fig. 5). A single protist cell usually sustains multiple spirochete species, and different protist genera share the same spirochete species [2,39]. Bacteroidetes are also involved in associations with a wide variety of gut protist species, as either intracellular endosymbionts or surfaceattached ectosymbionts [40]. The close relationships of the ectosymbionts between related protist species suggest that the symbionts were acquired before the diversification of their protist hosts [13]. The Bacteroidetes ectosymbiont ‘Candidatus Symbiothrix’ is distributed among various

Int. Microbiol. Vol. 16, 2013

139

Int Microbiol

Symbiogenesis and evolution

Fig. 4. Phylogenetic tree of the bacteria associated with the termite Reticulitermes grassei, worker and soldier castes, and their GenBank relatives of the most prevalent OTUs (with a distance threshold of 0.03), as generated by 454-pyrosequencing. Phylogenetic tree obtained by Interactive Tree of Life, a web-based tool [http://itol.embl.de], based on the guidelines by Letunic and Bork [30].

termites that harbor the protist Dinenympha. Another example of cospeciation is the endosymbiont Bacteroidetes from the protist Pseudotrichonympha [2,41].

‘Endomicrobia’ represent a special case in the coevolution of symbiont microorganisms, forming a separate line of descent in the bacterial tree and belonging to the termite group 1 (TG1) phylum. They are host-specific intracellular symbionts of termites and Cryptocercus gut flagellates [2, 24], with ‘Endomicrobia’ sequences from each flagellate host representing distinct phylotypes. Accordingly, the diversity of ‘Endomicrobia’ is thought to reflect the diversity of

their flagellate hosts [53]. Thus, ‘Endomicrobia’ phylotypes associated with Trichonympha species constitute a monophyletic group that is phylogenetically distinct from the phylotypes recovered from all other flagellates. The specific lineage of ‘Endomicrobia’ harbored by Trichonympha flagellates has been inherited by vertical transmission from their common ancestor [25]. The complete genome sequence of ‘Endomicrobia’ endosymbionts suggests that their association with their host flagellates stems from their ability to provide amino acids and cofactors [25]. The study of protists from the hindguts of both lower termites and the wood-feeding cockroach Cryptocercus

Int. Microbiol. Vol. 16, 2013

guerrero et al.

Int Microbiol

140

Fig. 5. Protists from the termite Reticulitermes grassei. (Photographs by R. Duro.)

provides evidence that bacteria–protists associations are very ancient, as are microbial–insect associations [24,44].

The colonization of prokaryotes on protist cells often results in an unusual ultrastructure of the junctional complexes [11,43,52]. Motility symbioses between prokaryotes and protist bionts have been reported between the flagellate Mixotricha paradoxa (Cristamonadida), found in the gut of the termite Mastotermes darwiniensis, and its ectosymbiotic spirochetes, and between the flagellate Caduceia versatilis (Cristamonadida), which inhabits the gut of the termite Cryptotermes cavifrons, and its ectosymbiotic bacteroidales [23]. However, the primary functions of symbiotic bacteria in protists are still unkown. Nonetheless, holobiont survival depends on both the maintenance of tripartite interactions (protist–bacteria–wood-feeding insect) and the preservation of the respective holobiome for the offspring is ensured by natural selection in a given environment.

Obligate endosymbiosis Bacterial endosymbionts are found in many insect orders and their presence provides a molecular clock for estimating divergence times among taxa [18]. The endosymbiotic

bacteria of cockroaches (including Cryptocercus) and lower termites (e.g., Mastotermes darwiniensis) are harbored within specialized cells—the bacteriocytes of the fat body—that are transferred vertically through the eggs. In fact, there is a close relationship between the endosymbionts of Mastotermes and those of Cryptocercus. One such endosymbiont is Blattabacterium, which based, on the analysis of its 16S rDNA genes, belongs to the class Flavobacteria in the phylum Bacteroidetes [29].

From the metabolic point of view, uric acid is a major nitrogenous waste product excreted by animals, including terrestrial insects, birds, and certain reptiles. Because of its poor solubility in water (only 60 mg/l at 20°C), uric acid, a non-toxic solid, is excreted to minimize water loss. However, in addition to being a nitrogenous waste, uric acid is apparently utilized as a nitrogen source or metabolic reserve in some insects, particularly those existing on a nitrogenpoor diet [54]. In the termite gut, uric acid degradation is an anaerobic process carried out primarily by gut bacteria [48,55]. The members of Blattabacterium that inhabit the fat bodies of most cockroaches are thought to participate in uric acid degradation, nitrogen assimilation, and nutrient provisioning. Genomic analysis and metabolic reconstruction indicate that Blattabacterium, despite lacking recognizable

Int. Microbiol. Vol. 16, 2013

Symbiogenesis and evolution

uricolytic enzymes, is able to recycle nitrogen from urea and ammonia (both of which are uric acid degradation products) into glutamate by using the enzymes urease and glutamate dehydrogenase [48]. The genome of Blattabacterium cuenoti, whether from the termite Mastotermes darwiniensis or the social wood-feeding cockroach Cryptocercus punctulatus, lacks most of the pathways for the synthesis of essential amino acids found in the genomes of relatives of this bacterium isolated from non-wood-feeding hosts. This deficit is compensated by the presence of other members of the complex gut microbiota, which provide their host with all essential amino acids. In both C. punctulatus and M. darwiniensis, the hindgut microbiota are passed on to their offspring [55]. Previous claims suggesting that Blattabacterium is capable of uricolysis are not supported by genomic evidence, raising the question why these bacteria are located in such close proximity to uric acid deposits in fat bodies. A possible, albeit still speculative explanation is that a uricolytic Blattabacterium ancestor, in the course of genome reduction, may have lost its uricase, transferring this function to uric acid degraders among the gut microbiota.

Final considerations The examples cited in this brief review illustrate the ubiquity of combinatorial modes of evolutionary innovation. A major change in evolution took place with the appearance of eukaryotic cells, which contained a nucleus and several organelles. Independent prokaryotes might have been spared elimination by interacting with other bionts to provide them with useful functions. Over time, these prokaryotic cells probably adjusted their reproductive needs such that they coevolved with other bionts, becoming deeply interdependent communities of microorganisms that eventually formed a holobiont, the functional equivalent of a single organism [33,34]. Extant symbioses likewise combine the development potential of two or more genomes. Selection pressures on the associates lead them to interact strongly and eventually to exploit niches where the presence of other life forms is ruled out, e.g., in extreme environmental conditions or where important nutrients are lacking. Symbiogenesis is a theory of speciation more comprehensive than that suggested by neo-Darwinian tenants: accretions of single-gene mutations in a given nucleus are strongly enhanced by the remarkable competitive advantages of holobionts. Symbiogenesis induces cyclical morphogenesis, which is an underestimated mode of evolutionary innovation. From our anthropocentric vision, it

141

may seem to us that the evolutionary pathway leading from the Cambrian explosion to humans was a major step in the history of life. Nevertheless, since the origin of nucleated cells—the latest major evolutionary step—evolution has only produced different variations of the same essential type of organisms, i.e., eukaryotes.

Acknowledgements. This work was supported by grant CGL200908922 (Spanish Ministry of Science and Technology) to RG. RG and MB are members of the Consolidated Research Group “Ecogenetics and microbial diversity” of the AGAUR of the Autonomous Government of Catalonia, Ref. 2009SGR00721.

Competing interests. None declared.

References 1. Berlanga M, Paster BJ, Guerrero R (2007) Coevolution of symbiotic spirochete diversity in lower termites. Int Microbiol 10:133-139 2. Berlanga M, Paster BJ, Guerrero R (2009) The taxophysiological paradox: changes in the intestinal microbiota of the xylophagous cockroach Cryptocercus punctulatus depending on the physiological state of the host. Int Microbiol 12:227-236 3. Berlanga M, Paster BJ, Grandcolas P, Guerrero R (2011) Comparison of the gut microbiota from soldier and worker castes of the termite Reticulitermes grassei. Int Microbiol 14:83-93 4. Brucker RM, Bordenstein SR (2012) Speciation by symbiosis. Trends Ecol Evol 27:443-451 5. Brucker RM, Bordenstein SR (2013) The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus Nasonia.

Science 341:667-669 6. Brune A, Emerson D, Breznak JA (1995) The termite gut microflora as an oxygen sink: microelectrode determination of oxygen and pH gradients in guts of lower and higher termites. Appl Environ Microbiol 61:2681-2687 7. Brune A, Friedrich M (2000) Microecology of the termite gut: structure and function on a microscale. Curr Opinion Microbiol 3:263-269 8. Carpenter KJ, Horak A, Chow L, Keeling PJ (2011) Symbiosis, mor­ phology, and phylogeny of Hoplonymphidae (Parabasalia) of the wood-feeding roach Cryptocercus punctulatus. J Eukaryot Microbiol 58:426-436 9. Colman DR, Toolson EC, Takacs-Vesbach CD (2012) Do diet and taxonomy influence insect gut bacterial communities? Mol Ecol 21: 5124-5137 10. Corliss JO (2004) Why the world needs protists! J Eukaryot Microbiol 51:8-22 11. D’Ambrosio U, Dolan M, Wier AM, Margulis L (1999) Devescovinid trichomonad with axostyle-based rotary motor (“Rubberneckia”): taxonomic assignment as Caduceia versatilis sp. nov. Eur J Protistol 35:327-337 12. De Bary A (1879) De la symbiose. Revue Internationale des Sciences III, pp 301-309. (Die Erscheinung der Symbiose. Vortrag gehalten auf der Versammlung Deutscher Naturforscher und Aerzte zu Cassel, K.J. Trübner, Strassburg.)

13. Desai MS, Strassert JFH, Meuser K, Hertel H, Ikeda-Ohtsubo W, Radek R, Brune A (2009) Strict cospeciation of devescovinid flagellates and

142

Int. Microbiol. Vol. 16, 2013

Bacteroidales ectosymbionts in the gut of dry-wood termites (Kaloter­ mi­­tidae). Environ Microbiol 12:2120-2132 14. Dillon RJ, Dillon VM (2004) The gut bacteria of insects: non-pathogenic interactions. Annu Rev Entomol 49:71-92 15. Dolan MF (2001) Speciation of termite gut protists: the role of bacterial symbionts. Int Microbiol 4:203-208 16. Eldredge N, Gould SJ (1977) Punctuated equilibria: the tempo and mode of evolution reconsidered. Paleobiology 3:115-151 17. Engel P, Moran NA (2013) The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev, doi: 10.1111/1574-6976. 1202 18. Gil R, Belda E, Gosalbes MJ, et al. (2008) Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae. Int Microbiol 11:41-48 19. Guerrero R (1998) Crucial crises in biology: life in the deep biosphere.

Int Microbiol 1:285-294 20. Guerrero R, Berlanga M (2006) Life’s unity and flexibility: the ecological link. Int Microbiol 9:225-235 21. Hongoh Y, Ohkuma M, Kudo T (2003) Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae). FEMS Microbiol Ecol 44:231-242 22. Hongoh Y, Deevong P, Inoue T, Moriya S, Trakulnaleamsai S, Ohkuma M, Vongkaluang C, Noparatnaraporn N, Kudo T (2005) Intra- and interspecific comparisons of bacterial diversity community structure support coevolution of gut microbiota and termite host. Appl Environ Microbiol 71:6590-6599 23. Hongoh Y, Sato T, Noda S, Ui S, Kudo T, Ohkuma M (2007) Candidatus Symbiothrix dinenymphae: bristle-like Bacteroidales ectosymbionts of termite gut protists. Environ Microbiol 9:2631-2635 24. Hongoh Y, Sharma VK, Prakash T, Noda S, Taylor TD, Kudo T, Sakaki Y, Toyoda A, Hattori M, Ohkuma M (2008) Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell. Proc Natl Acad Sci USA 105:5555-5560 25. Ikeda-Ohtsubo W, Desai M, Stingl U, Brune A (2007) Phylogenetic diversity of ‘Endomicrobia’ and their specific affiliation with termite gut flagellates. Microbiology 153:3458-3465 26. Köhler T, Dietrich C, Scheffrahn RH, Brune A (2012) High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasuti­ter­ mes spp.) Appl Environ Microbiol 78:4691-4701 27. Leadbetter JR, Schmidt TM, Graber JR, Breznak JA (1999) Acetogenesis from H2 plus CO2 by spirochetes from termite guts. Science 283:686-689 28. Lederberg J, McCray AT (2001) ’Ome Sweet ’Omics—a genealogical treasury of words. Scientist 15:8 29. Letunic I, Bork P (2011) Interactive Tree of Life v2. Outline annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39: W475-W478 30. Lewis JL, Forschler BT (2004) Protist communities from four castes and three species of Reticulitermes (Isoptera: Rhinotermitidae). Ann Ento­ mol Soc Am 97:1242-1251 31. Lilburn TG, Kim KS, Ostrom NE, Byzek KR, Leadbetter JR, Breznak JA (2001) Nitrogen fixation by symbiotic and free-living spirochetes.

Science 292:2495-2498 32. López-Sánchez MJ, Neef A, Patiño-Navarrete R, Navarro L, Jiménez R, Latorre A, Moya A (2009) Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers.

Environ Microbiol 10:3417-3422 33. Margulis L, Dolan MF, Guerrero R (2000) The chimeric eukaryote: Origin of the nucleus from the karyomastigont in amitochondriate protists. Proc Natl Acad Sci USA 97:6954-6959

guerrero et al.

34. Margulis L, Chapman M, Guerrero R, Hall J (2006) The last eukaryotic common ancestor (LECA): acquisition of cytoskeletal motility from aerotolerant spirochetes in the Proterozoic Eon. Proc Natl Acad Sci USA 103:13080-13085 35. Martinson VG, Moy J, Moran NA (2012) Establishment of characteristic gut bacteria during development of the honeybee worker. Appl Environ Microbiol 78:2830-2840 36. Maturana RH, Varela FJ (1972) Autopoiesis and cognition. In: Cohen RS, Wartofsky MW (eds) The realization of the living. Boston Studies in the Philosophy of Science. Vol 42. D. Reidel, Dordrecht, the Netherlands 37. McFall-Nagai M (2002) Unseen forces: The influence of bacteria on animal development. Develop Biol 242:1-14 38. Nakajima H, Hongoh Y, Usami R, Kudo T, Ohkuma M (2005) Spatial distribution of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium.

FEMS Microbiol Ecol 54:247-255 39. Noda S, Ohkuma M, Yamada A, Hongoh Y, Kudo T (2003) Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut. Appl Environ Microbiol 69:625-633 40. Noda S, Inoue T, Hongoh Y, et al. (2006) Identification and charac­te­ rization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach.

Environ Microbiol 8:11-20 41. Noda S, Kitade O, Inoue T, et al. (2007) Cospeciation in the triplex symbiosis of termite gut protists (Pseudotriconympha spp.), their hosts, and their bacterial endosymbionts. Molec Ecol 16:1257-1266 42. Ohkuma M (2003) Termite symbiotic systems: efficient bio-recycling of lignocellulose. Appl Microbiol Biotech 61:1-9 43. Ohkuma M (2008) Symbioses of flagellates and prokaryotes in the gut of lower termites. Trends Microbiol 16:345-352 44. Ohkuma M, Noda S, Hongoh Y, Nalepa CA, Inoue T (2009) Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus. Proc Biol Sci 276: 239-245 45. Oliver KM, Russell JA, Moran NA, Hunter MS (2003) Facultative bac­ terial symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci USA 100:1803-1807 46. Oliver KM, Degnan PH, Burke GR, Moran NA (2010) Facultative symbionts in aphids and the horizontal transfer of ecologically important traits. Annu Rev Entomol 55:247-266 47. Ruby EG (2008) Symbiotic conversations are revealed under genetic interrogation. Nat Rev Microbiol 6:752-762 48, Sabree ZL, Kambhampati S, Moran NA (2009) Nitrogen recycling and nutricional provisioning by Blattabacterium, the cockroach endosym­ biont. Proc Natl Acad Sci USA 106:19521-19526 49. Scully ED, Geib SM, Hoover K, et al. (2013) Metagenomic profiling reveals lignocellulose degrading system in a microbial community asociated with a wood-feeding beetle. PLoS ONE 8:e73827 50. Shimada K, Lo N, Kitade O, Wakui A, Maekawa K (2013) Cellulolytic protist numbers rise and fall dramatically in termite queens and kings during colony foundation. Eukaryotic Cell 12:545-550 51. Shinzato N, Muramatsu M, Matsui T, Watanabe Y (2005) Molecular phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus. Biosci Biotechnol Biochem 69:11451155 52. Stingl U, Maass A, Radek R, Brune A (2004) Symbionts of the gut flagellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: description of “Candidatus Vestibaculum illigatum”. Microbiology 150:2229-2235

Symbiogenesis and evolution

53. Stingl U, Radek R, Yang H, Brune A (2005) “Endomicrobia”: Cyto­ plas­matic symbionts of termite gut protozoa form a separate phylum of prokaryotes. Appl Environ Microbiol 71:1473-1479 54. Thong-On A, Suzuki K, Noda S, Inoue J-I, Kajiwara S, Ohkuma M (2012) Isolation and characterization of anaerobic bacteria for symbiotic recycling of uric acid nitrogen in the gut of various termites. Microbes Environ 27:186-192 55. Tokuda G, Elbourne LDH, Kinjo Y, et al. (2013) Maintenance of essential amino acid synthesis pathways in the Blattabacterium cuenoti symbiont of a wood-feeding cockroach. Biol Lett 9:20121153 56. Wallin IE (1927) Symbionticism and the origin of species. Williams & Wilkins, Baltimore, MD, USA

Int. Microbiol. Vol. 16, 2013

143

57. Warnecke F, Luginbühl P, Ivanova N, et al. (2007) Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450:560-565 58. Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6:741-751 59. Wier A, Dolan M, Grimaldi D, Guerrero R, Wagensberg J, Margulis L (2002) Spirochete and protist symbionts of a termite (Mastotermes electrodominicus) in Miocene amber. Proc Natl Acad Sci USA 99:14101413 60. Zilber-Rosenberg I, Rosenberg E (2008) Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution.

FEMS Microbiol Rev 32:723-735

Volume 16(3) SEPTEMBER 2013

Acknowledgement of Institutional Subscriptions International Microbiology staunchly supports the policy of open access (Open Access Initiative, see Int. Microbiol. 7:157161). Thus, the journal recognizes the help received from the many institutions and centers that pay for a subscription—in spite of the possibility to download complete and current issues of the journal free of charge. We would therefore like to thank those entities. Their generous contribution, together with the efforts of the many individuals involved in preparing each issue of International Microbiology, makes publication of the journal possible and plays an important role in improving and expanding the field of microbiology in the world. Some of those institutions and centers are: Area de Microbiología. Departamento de Biología Aplicada. Universidad de Almería / Biblioteca. Institut Químic de Sarrià. Universitat Ramon Llull. Barcelona / Biblioteca.

Instituto Nacional de Seguridad e Higiene en el Trabajo-Ministerio de Trabajo y Asuntos Sociales. Barcelona / Ecologia microbiana. Departament de Genètica i de Microbiologia.

Universitat Autònoma de Barcelona. Bellaterra (Barcelona) / Biblioteca. Institut de Biotecnologia i Biomedicina. Universitat Autònoma de Barcelona. Bellaterra (Barcelona) / Laboratori d’Ecogenètica. Departament de Microbiologia.

Universitat de Barcelona / Departament de Microbiologia i Parasitologia Sanitàries. Facultat de Farmàcia. Universitat de Barcelona / Societat Catalana de Biologia Institut d’Estudis Catalans. Barcelona / Departamento de Microbiologia. Universidade Federal de Minas Gerais. Belo Horizonte. Brasil / Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco, UPV-EHU. Bilbao / Biblioteca. Universidad de Buenos Aires. Argentina / Biblioteca. Facultad de Ciencias. Universidad de Burgos / Biblioteca. Departamento de Producción Animal CIAMCentro Mabegondo. Abegondo (Coruña) / Laboratorio de Microbioloxia. Universidade da Coruña. Coruña / Biblioteca.

Divisió Alimentària del IRTA-Centre de Tecnologia de la Carn. Generalitat de Catalunya. Monells (Girona) / Biblioteca Montilivi. Facultat de Ciències. Universitat de Girona / Área de Microbiología. Departamento de Ciencias de la Salud.

Universidad de Jaén / Microbiologia. Departament de Ciències Mèdiques Bàsiques. Facultat de Medicina. Universitat de Lleida / Laboratorio de Microbiología Aplicada. Centro de Biología Molecular. Universidad Autónoma de MadridCSIC. Cantoblanco (Madrid) / Laboratorio de Patógenos Bacterianos Intracelulares. Centro Nacional de Biotecnología-CSIC. Cantoblanco (Madrid) / Grupo de Investigación de Bioingeniería y Materiales (BIO-MAT). Escuela Técnica Superior de Ingenieros Industriales. Universidad Politécnica de Madrid / Biblioteca. Centro de Investigaciones Biológicas, CSIC. Madrid / Merck Sharp & Dohme de España, Madrid / Departamento de Microbiología. Facultad de Ciencias.

Universidad de Málaga / Grupo de Fisiología Microbiana.

Depto. de Genética y Microbiología. Universidad de Murcia.

Espinardo (Murcia) / Library. Department of Geosciences.

University of Massachusetts-Amherst. USA / Biblioteca de Ciencias. Universidad de Navarra. Pamplona / Grupo de Genética y Microbiología. Departamento de Producción Agraria.

Universidad Pública de Navarra. Pamplona / Microbiología Ambiental. Departamento de Biología. Universidad de Puerto Rico. Río Piedras. Puerto Rico / Biblioteca General. Universidad San Francisco de Quito. Ecuador / Biblioteca. Facultat de Medicina. Universitat Rovira Virgili. Reus / Instituto de Microbiología Bioquímica-Departamento de Microbiología y Genética. CSIC-Universidad de Salamanca / Departamento de Microbiología y Parasitología. Universidad de Santiago de Compostela. Santiago / Laboratorio de Referencia de E. coli (LREC). Facultad de Veterinaria. Universidad de Santiago de Compostela. Lugo / Departamento de Genética. Universidad de Sevilla / Tecnología de los Alimentos. Facultad de Ciencias. Universidad de Vigo / General Library. Marine Biological Laboratory. Woods Hole, Massachusetts, USA.

RESEARCH ARTICLE International Microbiology (2013) 16:145-155 doi: 10.2436/20.1501.01.189 ISSN 1139-6709 www.im.microbios.org

Non-developing ascospores in apothecia of asexually reproducing lichen-forming fungi M. Carmen Molina,1* Pradeep K. Divakar,2 Ning Zhang,3,4 Natalia González,1 Lena Struwe4,5 1 Department of Biology and Geology, ESCET, University Rey Juan Carlos, Móstoles, Madrid, Spain.

Department of Plant Biology II, Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain.

3 Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA. 4 Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ, USA. 5 Department of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ, USA 2

Received 11 April 2013 · Accepted 26 July 2013

Summary. The presence of apothecia in mixed species (vegetatively reproducing lichens, occasionally producing ascomata) has been interpreted as a mechanism to increase genetic variability in mostly clonal populations. However, spore viability from these apothecia has not been studied. We asked whether ascospores of the mixed species Physconia grisea are viable and thereby contribute to increasing the genetic diversity within populations of this species. An ontogenetic study of spores in cultures of P. grisea and a related sexual species (P. distorta), showed that although mature apothecia from both species produced and discharged meiospores capable of germination, spores from P. grisea were only rarely (0.43 %) able to continue development whereas those from P. distorta germinated and developed successfully. The strongly reduced viability of P. grisea spores suggested that they do not have a strong reproductive function, at least in the two local populations analyzed. Additionally, we show that the segregation of Physconia grisea ssp. lilacina does not have molecular support. [Int Microbiol 2013; 16(3):145155] Keywords: Physconia spp. · apothecia · sexual reproduction · germination · ontogenetic development · mixed species

Introduction Lichen fungi form obligate symbiotic relationships with their photobionts, either a green alga and/or a cyanobacterium. The largest number of lichenized fungi is found among the Asco­­

Corresponding author: M.C. Molina Departamento de Biología y Geología (Área de Biodiversidad y Conservación), ESCET Universidad Rey Juan Carlos 28933 Móstoles, Madrid, Spain Tel. +34-914887090. Fax +34-916647490 E-mail: carmen.molina@urjc.es

*

my­­­cota, in which over 40 % of the known species live in symbiosis with algae as lichens [26]. Lichen-forming fungi reproduce by sexual (e.g., apothecia, perithecia, or mazaediate ascomata) or asexual (vegetative) reproduction and develop the respective structures. When apothecia are sexually mature, meiospores are propelled upward from the asci and dispersed through several mechanisms, such as wind, water, and birds [2,41]. Sexual reproduction carries a high risk of failure since after spore germination the fungi, an obligate partner in the sym­biosis, might not be able to find a compatible photobiont, thereby failing to re-lichenize. Printzen and Ekman [49] have concluded that dispersal by ascospores over long distances is

146

Int. Microbiol. Vol. 16, 2013

rather ineffective in the fertile lichen Cladonia subcervicornis (Cladoniaceae). Lichens can potentially disperse vegetatively by symbiotic thallus fragments that have broken off [8]. Although these fragments may not necessarily regenerate a complete thallus, some lichens, such as Thamnolia subuliformis, use this mechanism exclusively [9]. Lichens can also spread through vegetative diaspores containing algal and fungal somatic cells (e.g., isidia and soredia) or with pycnidia, asexual organs that produce mitospores (conidia). Isidia are elongated, corticated outgrowths from the thallus surface that detach for mechanical dispersal. Soredia are powdery areas on the upper surface of the thallus that contain small groups of algal cells surrounded by fungal filaments. Thallus fragmentations, isidia, and soredia reduce the risk of reproductive failure since the two symbionts are dispersed together. However, the viability of vegetative diaspores after dispersion can be affected by environmental conditions, such as pollutants [21]. When the reproductive mode is vegetative, lichen species usually present either isidia or soredia, very rarely both at once. In a recent molecular study of a population of Pseudoevernia furfu­ racea (Parmeliaceae), a species that typically uses isidia as its dispersal mechanism, Ferencova et al. [18] have concluded that specimens bearing isidia and soredia at the same time should not be considered as separate species, but as a morphological variants within a polymorphic species that is simultaneously capable of executing different reproductive strategies.

It was long thought that closely related species in many groups of lichenized fungi might differ in their reproductive modes. In these “species pairs” there is a “primary” or “mother” taxon that only reproduces sexually, and a “derived” or “secondary” taxon that only reproduces vegetatively [46,55]. Sometimes, vegetative species develop apothecia together with asexual reproduction structures; in such cases, the terms “mixed reproduction” or “mixed taxa” have been used. However, molecular phylogenetic studies of species pairs have suggested that in many cases neither the sexual nor the vegetative taxa (species) form monophyletic sister clades. Rather, phylogenetic trees usually show individuals from sexual and asexual taxa to be intermixed and neither taxon forms monophyletic clades (see reviews by Crespo and Pérez-Ortega [12], Lumbsch and Leavitt [32]). Accordingly, Tehler et al. [56] have suggested that the species pair concept should be abandoned, evolutionarily and taxonomically. However, the term “mixed species” is still used to refer to populations that mostly undergo asexual reproduction but rarely bear apothecia.

The various hypotheses on the evolution of reproductive strategies and the evidence supporting them have been contradictory. While some scientists have demonstrated that soredi-

molina et al.

ate specimens can arise independently from sexual lineages at different geographical locations [30,42], others have shown the development of apotheciate (sexual) clades from rarely sexually reproducing, mostly isidiate morphotypes, i.e., mixed species [11]. Hestmark et al. [22] suggested that independent reversal to sexual reproduction from asexual stages could explain the evolution of two types of apothecia from a vegetatively reproducing lineage, which implies that the genes for sexual reproduction do not become permanently lost but only temporarily silenced. Tehler et al. [56], in their molecular phylogenetic study of Roccella from the Galapagos Islands, have emphasized that either a sorediate or a sexual ancestor of the Roccella galapagoensis aggregata species complex (Roccellaceae) can be postulated. In that particular case, either evolutionary direction, from sexual to asexual reproduction or vice versa, seems equally plausible.

Lichen-forming fungi rarely invest in both sexual and asexual reproduction on the same thallus, i.e., within the same individual, which suggests that such an energetic investment is too costly to maintain under conditions of limited resources [5]. It is unclear why certain lichen species produce ascomata infrequently. In these mixed taxa, heterothallism (obligate cross-fertilization), lack of mating partners, mutations in genes involved in gamete, gametangia or ascomata formation, and environmental factors have been invoked [23] but none has been proven. Martínez et al. [34] reported that in Lobaria pulmonaria, a lichen with a mixed reproductive strategy, the largest individuals are more likely to develop reproductive structures, initially carrying out asexual reproduction that later is replaced by sexual reproduction.

Those authors have also found indications that macro- and microclimatic variables influence the type of reproductive structures that are formed. Lawrey [28] correlates the mixed reproductive mode in lichens with temperate ecosystems, which climate-wise are seasonally more variable and unpredictable than tropical climates. It is possible that mixed taxa can “turn on” their sexual reproductive strategy as a response to environmental stress and high selection pressure (limited resources, desiccation, low relative humidity, high solar radiation, low diversity of compatible photobionts, forest fragmentation, air pollution, etc.), as an alternative means to reproduce. Populations that had some degree of sexual reproduction in the past might have been favored, through natural selection, during stressful events, since sexual reproduction increases genetic variability and therefore also the likelihood of adaptation and survival in new environments. By contrast, if conditions are stable then asexual reproduction allows population maintenance with fewer resources such that these

Non-developing spores in Physconia grisea

populations would be favored by natural selection [5].

Buschbom and Mueller [6] have also suggested that changes in the reproductive strategy of lichens are governed by tradeoffs in the success of the symbiosis, with the two modes of reproduction alternating based on selective pressures. These hypotheses presume that occasional apothecia from mixed species are functional and generate viable meiotic spores.

However, until now, there has been no effort to determine the viability of spores from such rare apothecia in mixed species.

As far as we know, ours is the first study evaluating this hypothesis.

The mixed species Physconia grisea has sorediate margins and simple white rhizines on the lower thallus side and only rarely does it produce apothecia (although when the apothe­cia are present, they can be abundant) [45]. It occurs on the nutrient-rich or eutrophicated bark of deciduous trees, often along roads, and on calcareous as well as eutrophicated non-calcareous stones. Its distribution is restricted to Europe, the Atlas Mountains of northern Africa, and the Near East [45].

Some authors have proposed that the vegetative-only morpho­ type and the rare apothecia morphotype in P. grisea populations should be distinguished as different subspecies (P. grisea ssp. lilacina, Poelt 1966), i.e., forming a subspec­ies pair, although this subspecies segregation has yet to be tested at the molecular level. Other authors (e.g., Lawrey [28]) have interpreted such morphotypes as morphological variations within a single species with mixed reproduction. Wornik and Grube [57] have proposed that Physconia distorta is the sexually reproducing species pair partner of the mixed species P. grisea.

However, phylogenetic studies have shown that the two species are not sister species or close-clade species [15], even though both belong to the monophyletic Physconia group [16].

The mixed species Physconia grisea, a primarily vegetatively reproducing species, was chosen here as a study model to determine whether the occasionally occurring apothecia produce viable sexual spores. Our hypothesis was that viable spores from these rare apothecia provide an adaptive advantage that can increase genetic variability in later generations and populations. Accordingly, we compared sporulation and germination rates as well as the growth of ascospores in various axenic cultures of P. grisea and P. distorta. The latter was selected as a comparison species since it only undergoes sexual reproduction and has already been successfully cultured in vitro [35,37]. Moreover, P. distorta and P. grisea are sympatric species that can compete for the same Trebouxia species as their photobiont [57]. Additionally, DNA sequencing of the field-collected apotheciated morphotypes of P. grisea has confirmed them as belonging to the P. grisea s. str. clade [15].

Int. Microbiol. Vol. 16, 2013

147

Materials and methods Lichen material. Physconia grisea specimens were collected from Casa de Campo on Quercus ilex (N 41º 24′ 32.88′′, W 03º 45′ 10.52′′, alt. 630 m, Madrid, Spain) by P. Divakar. Physconia distorta was collected from Montejo de la Sierra on Quercus pyrenaica (N 41º 04′ 63′′, W 03º 29′ 65′′, alt.

1293 m, Madrid, Spain) also by P. Divakar. One thallus from two local populations was used from each species, selected among 15–20 apothecia per thallus. According to Fedrowitz et al. [17], a “local population” is defined as all single thalli of the same species on a tree, and the metapopulation consists of all local populations within a certain forest landscape. The selected apothecia were of intermediate sizes (about 5 mm in diameter) to ensure sexual maturity [39]. Both localities have acceptable air quality, with pollutant concentrations lower than those that usually affect medium-sensitive lichens. Apothecia density was calculated as the number of apothecia per cm2 and three random apothecia were used. Samples were processed for spore discharge immediately after field collection (see below).

Isolation and culture. Plurisporic isolates were obtained from apothecia of both lichen species. The fungi were cultured from discharged spores following the methods of Ahmadjian [1]. The apothecia were washed for 20 min in distilled water followed by 30 min in phosphate-buffered saline (PBS) containing 0.01 % Tween 80 (v/v). Ascomata were then soaked in sterile double-distilled water for 2 h, with the water changed several times during that period. Finally, apothecia were carefully cleaned and lightly dried with absorbent paper under a magnifier glass.

Clean ascomata were attached to the inner side of Petri dish lids with petroleum jelly. The bottom halves of the Petri dishes contained either Basal Bold medium (BBM) or 1 % glucose BBM (1G-BBM; [3]) and were inverted over the lids, allowing the ascospores to discharge upwards onto the medium.

The lids with apothecia were removed after spore discharge was complete and replaced by new lids. The Petri dishes were then inverted to the normal culture position with medium at the bottom of each dish.

After germination, 36 uncontaminated multispores isolated (6 isolated on each plate and 6 plates per medium) from P. distorta and P. grisea were subcultured on 11 types of different media: 2 % glucose LBM (2G-LBM); 3 % glucose LBM (3G-LBM); 3 % sucrose LBM (3S-LBM) according to Lilly and Barnett [29]; 1 % glucose BBM (1G-BBM); 2 % glucose BBM (2GBBM); 8 % sucrose BBM (8S-BBM); malt-yeast extract (MY), in accordance with Behera and Makhija [3]; 0.2 % glucose malt-yeast extract (0.2G-MY), modified by us from Ahmadjian [1] as follows: 5 g of malt extract, 2 g of glucose, 0.25 g of yeast extract, and 15 g of agar in 1 liter of twice-distilled water; potato dextrose agar (PDA); corn meal agar (CMA); and charcoal agar (CA), following the manufacturer’s instructions (Difco).

Cultures were incubated at 18–20 °C in the dark. Periodically, mycobionts were examined using an Axioskop (Zeiss Germany) and a Nikon Eclipse 80 microscope with a magnifier glass. For photography, an automatic ring flash system was attached to the camera lens (SPOT Insight Wide-field 2MP).

Photographs used white light and Nomarsky interphase contrast. When necessary, the colonies were slightly stained with lactophenol cotton blue, especially when colony growth was compact.

The variables used to evaluate sexual reproductive fitness were: percentage of apothecia that propelled spores upward (=sexually mature); number of spores ejected by each apothecium, percentage of spores that germinated from each apothecium, globular colony growth (defined as the diameter of the central part of the colonies, where the cells are globose and very compact) at 300 days. The lengths and widths of the spores were calculated based on at least 10 randomly selected spores and using the software NIS-Elements D 3.0 included with the Nikon Eclipse 80 microscope.

148

Int. Microbiol. Vol. 16, 2013

Statistical analysis. To determine whether the germination percentages on the two types of media (BBM and 1G-BBM) and obtained with the two species (Physconia distorta and P. grisea) were statistically different, a bifactorial analysis of variance (ANOVA) was performed. Variances were checked for homogeneity using Cochran’s test. The Student-Newman-Keuls (SNK) test was used to discriminate among different treatments after a significant F-test. Data were considered significant when P < 0.05. The data of P. distor­ ta growth on the different enriched media were not strictly parametric and thus were analalyzed with the Kruskal-Wallis test. All tests were done with the software Statistica 6.0 for Windows (Statsoft, Tulsa, OK, USA).

Molecular identity. Because of the frequent occurrence of cryptic species in lichenized fungi and especially in the Physconia group (e.g., P. distorta vs. P. thorstenii, [16]), we confirmed species identities using molecular sequencing of the internal transcribed spacer (nuITS). Samples derived from field-collected specimens of P. grisea (frozen specimens) and P. distorta (mycobiont cultures) were ground into powder with sterile plastic pestles.

Total genomic DNA was extracted using the DNeasy plant mini kit (Qiagen) according to the manufacturer’s instructions, but with modifications as described in Crespo et al. [13], and then used for PCR amplification of fungal nuITS rDNA using the primers ITS1F and ITS4 (see [15]). PCR and sequencing were carried out following Crespo et al. [13]. Sequence and species identities were confirmed using the “megaBLAST” search function in GenBank [54].

Phylogenetic analysis. New ITS sequences were added to the alignment published in Divakar et al. [16]. The sequences generated for this study were deposited in GenBank under accession numbers KC559094 (P. grisea, MAF-Lich 17760) and KC559093 (P. distorta, MAF-Lich 17761).

Sequences were aligned using the program MAFFT ver. 6 [25] and the G-INS-I alignment algorithm, “1PAM/K = 2” scoring matrix, and offset value = 0.1; the remaining parameters were set to default values. The alignment was analyzed using Bayesian (B/MCMC) and maximum likelihood (ML) approaches. The program MrBAYES 3.1.2 [24] was used to analyze trees following a MCMC method. Two species of Anaptychia were selected as the outgroup, as this genus was supported as the sister group to Physconia in Cubero et al.

[15]. The analyses were performed based on the general time reversible model of nucleotide substitution [50], including estimation of invariant sites, assuming a discrete gamma distribution with six rate categories and allowing site-specific rates (GTR+I+G). The nucleotide-substitution model and parameters were selected using the Akaike information criterion as implemented in jModelTest [48]. No molecular clock was assumed. Initial runs were conducted starting with random or neighbor-joining trees to check the number of simultaneous MCMC chains necessary to avoid trapping on local optima.

Subsequently, a run with 2 million generations, starting with a random tree and employing 12 simultaneous chains each, was executed. Every 100th tree was saved into a file. The first 200,000 generations (i.e., 2000 trees) were deleted as the “burn in” of the chains. We plotted the log-likelihood scores of sample points against generation time using TRACER 1.0 to ensure that stationary phase was achieved after the first 200,000 generations by checking whether the log-likelihood values of the sample points reached a stable equilibrium value [24]. Of the remaining 18,000 trees, a majority rule consensus tree with average branch lengths was calculated using the sumt option of MrBayes.

A ML analysis was performed using the program RAxML v7.2.7, as implemented on the CIPRES Web Portal, with the GTRGAMMA model [52,53].

Support values were assessed using the “rapid bootstrapping” option with 1000 replicates. Branches with posterior probabilities (pp) ≥95 % and ML bootstrap values ≥70 % were considered strongly supported.

molina et al.

Results The sequences obtained from one specimen from each species were identified as Physconia grisea and P. distorta, respectively, through Genbank comparisons, confirming our initial species identification. The ITS PCR products from these specimens are about 600 base pairs (bp) in length. In the molecular phylogenetic tree (Fig. 1), the sequence obtained from an axenic culture of P. distorta clustered with the sequences of other non-cultured samples of P. distorta in the “distorta clade.”

This relation was strongly supported; however, the monophyly of P. distorta was not. The P. distorta clade included other species, such as P. detersa and P. subpulverulenta. Whether P. detersa and P. subpulverulenta merit species-level ranking has been questioned in other studies [15,16]. Those authors have highlighted the species-pair problems and have suggested that both P. detersa and P. subpulverulenta belong to P. distorta and are not separate lineages. Since resolving this controversy was not the goal of our study we left this question open. The sequence obtained from apotheciated P. grisea sample grouped within the “grisea clade” (pp 1.00). This clade is formed by specimens from six different populations, two of which are fertile and four of which reproduce vegetatively.

Phylogenetic clusters related to reproductive strategy (sexual vs. vegetative reproduction) were not distinguished in the “grisea clade”.

The collected specimens of P. grisea and P. distorta showed abundant apothecia (respectively, 2.07 ± 0.33 and 3.10 ± 0.31 apothecia·cm–2). All ascomata ejected spores from mature asci after 12 h and spore germination was observed after 6 days for both species. Contamination by bacteria or other fungi was observed near many spores or spore groups ejected from the apothecia. Contaminations are expected when working with field-collected specimens, although contamination levels were highly variable between apothecia (data not shown). The spores of both species were subglobular and uniseptated, with brown external ornamentation capsules (Fig. 2). The spore length and width (mean ± standard deviation) were 25.99 ± 8.66 µm × 15.32 ± 5.11 µm for P. grisea, and 28.62 ± 5.74 µm × 16.85 ± 3.75 µm for P. distorta. When a meiospore germinated, its capsule was broken open at two points (bipolar germination), allowing the development of germ tubes (Fig. 2A,B). The meiospores were attached to the media on the top plate as either isolated spores or groups of 2–3 or 6–8 spores (Fig. 2C). The number of ascospores ejected per apothecium varied considerably, with some ascomata releasing just a few dozens of spores and others releasing hun-

Int. Microbiol. Vol. 16, 2013

149

Int Microbiol

Non-developing spores in Physconia grisea

Fig. 1. The majority-rule consensus tree from the Bayesian analysis based on nuITS sequences of Physconia. Branches that received strong support in any of two analyses (pp ≥0.95 and RaxML bootstrap ≥70 %) are shown in bold. The phylogenetic position of the two new sequences of P distorta (obtained from culture) and P. grisea (field collected) are shown in bold and bold with an asterisk, respectively. Symbols: #, vegetative specimens; †, apotheciated specimens.

Int. Microbiol. Vol. 16, 2013

molina et al.

Int Microbiol

150

Int Microbiol

Fig. 3. Degeneration of Physconia grisea spores after a month on BBM (A), 0.2G-MY (B), and S-LBM (C). The arrow shows the collapsed cells and the cytoplasmic contents lost in the culture medium. Scale bar = 10 µm.

Fig. 2. Subglobular, bipolar, and uniseptate germinated ascospore from Physconia grisea. (A,ºB). First germination stage. Arrows show the break points of the spore capsule with germ tubes. (C) Eight germinated spores group from asci. Scale bar = 10 µm.

dreds of spores. Physconia grisea released an average of 62.3 ± 34.5 spores and P. distorta 73.4 ± 34.2 spores.

The germination rates of the two species did not significantly differ (P > 0.05). However, for both species the germination percentage on 1G-BBM was significantly lower than that on BBM (F1,10 = 8,25; P = 0.016). The averages and standard deviations were 79.73 ± 18.96 for P. grisea and 88.28 ± 15.2 for P. distorta on BBM and 40.1 ± 30.31 for P. grisea and 51.66 ± 34.01 for P. distorta on 1G-BBM. The standard deviations were high because of the large variations in the percentage of germination between apothecia. For example, for spores

germinated in 1G-BBM, the range was 18–86 % for P. distorta and 12–72 % for P. grisea.

Germinated ascospores of P. grisea and P. distorta showed hyphae with short and septated cells, and the cytoplasmic content could be easily seen in cultures incubated in organic (1GBBM) and inorganic (BBM) media. However, the behavior of the two species after germination was very different in each medium.

After one month, the hyphal cells of P. grisea cultured on BBM contained large vacuoles and the cells had begun to contract and shorten. Over the next 20 days, the hyphae became completely wrinkled and lost their cytoplasmic content so that they were no longer viable, excluding thus further growth (Fig. 3A). In a few rare cases, 10 % of the ascomata of P. grisea produced a small amount of ascospores (single or groups) that continued to grow after a month, but only on inorganic medium; however, they generated poor mycelia, with the hyphae growing only on the surface and at very slow rates (0.31 ± 0.1 µm per day; 100 µm in 315 days). These meiospores accounted

Int. Microbiol. Vol. 16, 2013

Non-developing spores in Physconia grisea

Table 1. Growth activity of sexual spores from Physconia grisea and P. distorta grown on inorganic media (BBM) and several enriched media. Activity is indicated by the following abbreviations: nt, not tested; –, without growth; –/+, only a 0.43 % of germinated meiospores grew, but very slowly and weakly. The number of + indicate intensity of growth according to Fig. 5 Media Basal bold medium (BBM)

P. grisea

P. distorta

–/+

+

3 % Glucose LBM (3G-LBM)

–

++++

0.2 % Glucose malt-yeast extract (0.2 G-MY)

–

+++

Corn meal agar (CMA)

–

++

2 % Glucose BBM (2G-BBM)

–

++

1 % Glucose BBM (1G-BBM)

–

+

8 % Sucrose BBM (8S-BBM)

–

nt

Malt-yeast extract (MY)

–

nt

3 % Sucrose LBM (3G-LBM)

–

nt

2 % Glucose LBM (2G-LBM)

–

nt

Potato dextrose agar (PDA)

–

nt

Charcoal agar (CA)

–

nt

Fig. 4. Developmental stages of Physconia distorta spore germination. (A) Com­pacted growth with globular cells, observed on 1G-BBM media. (B) Poor and superficial growth with elongated cells, observed on BBM media. (C) Detail of hyphae stained with lacto­ phenol. Scale bar = 100 µm.

Int Microbiol

for 0.43 % of the total germinated spores from this species.

When healthy and uncontaminated germinated spores of P. grisea were cultured on 1G-BBM, they rapidly degenerated without completing their development. To ensure that the poor development of P. grisea was not related to the choice of culture medium, eleven different media usually successful for lichen cultures were tested (Table 1); the result was always negative over the long term (Fig. 3B,C and Table 1).

After one month, all colonies of P. distorta in organic medium (1G-BBM) consisted of well-developed radial mycelia with globular cells in the center and more elongated, distally located cells. Fattened intersepta and small lipid drops were observed. During this time, pigment synthesis was considerable, leading to dark thallus-like colonies (Fig. 4A). The globular growth rate on 1G-BBM was 5.34 ± 0.26 µm per day (240 µm in 50 days). P. distorta mycelium also grew on basal media (BBM) but was more poorly developed, with long intersecta and a few lipid drops. Globular growth was slower in BBM (2.96 ± 0.57 µm per day; 133 µm in 50 days) and the hyphae were more filamentous, developing only on the surface and without generating colonies (Fig. 4B,C). The mycelia lacked pigmented areas. When germinated ascospores of P. distorta were transferred to enriched media (3G-LBM, 0.2GMY, CMA, 2G-BBM, and 1G-BBM), after 15–20 days all

151

Int. Microbiol. Vol. 16, 2013

molina et al.

Int Microbiol

152

Fig. 5. Kinetics of Physconia distorta growth on different enriched media: CMA (closed circle), 3G-LBM (open circle), 0.2GMY (closed square), 2G-BBM (open square), 1G-BBM (closed triangle), BBM (open triangle).

transferred spore groups grew, with the growth rate correlating with the type of medium (Fig. 5). After 50 days, the growth rates on the different media were statistically different ( 52 = 52; P < 0.001). Their ranking, in decreasing order, was as follows: CMA > 3G-LBM > 0.2G-MY = 2G-BBM = 1GBBM = BBM, with CMA yielding statistically higher growth rates than the other media types for initial growth. However, there was a change in the rate of development starting after 100 days. Thus, at 300 days growth was also statistically different between cultures ( 52 = 70.61; P < 0.001), but the rank order of the growth rates on the tested media were: 3GLBM > 0.2G-MY > CMA = 2G-BBM > 1G-BBM = BBM.

Discussion The morphotypes of P. grisea (“mixed species”) that have apothecia and undergo vegetative reproduction belong to the P. grisea clade s. str. described by Cubero et al. [15]. This allowed us to reject the hypothesis that these morphotypes should be considered as a separate subspecies (as suggested by Poelt [46]). All ascomata from the observed thalli of P. grisea

and P. distorta species were sexually mature. The sexual-only and mixed species produced similar amounts of spores, which indicated that occasionally occurring apothecia of P. gri­sea could produce and eject spores in a similar manner to P. distor­ ta, the sexually-only reproducing species. The amount of spore production varied widely in both species, suggesting that, although the selection of apothecia was standardized [39], the sexual age of the apothecia from different thalli was not homogeneous. This is not surprising considering the hetero­thallic life cycle of lichens (self-sterile), i.e., cross fertilization and genetic recombination among different individuals [23,55], as well as the slow growth rate of lichens in nature. Moreover, the ascospore germination rate was higher in inorganic medium (BBM) than in medium supplemented with glucose (1GBBM). This inhibition or suppression of germination by glucose had been described previously by Molina et al. [39], who found that high concentrations of sugar control fungal germination. Since algal cells in the symbiosis are responsible for carbohydrate production, this behavior could be interpreted as an algal defense mechanism, according to the putative parasitism theory [40]. Belandria et al. [4] have attributed such results to the rapid proliferation of unwanted fungi or bacteria in

Non-developing spores in Physconia grisea

enriched media, which can promote allelochemical activities inhibiting the germination of the lichen ascospores.

Even though the processes of sporulation and germination were similar in the two species, later ontogenetic development was strikingly different. When grown on enriched media, sexual-only P. distorta formed well-developed thalluslike colonies. This behavior has been described in other lichenforming fungi (e.g., [10,35]). The development of P. distorta varied significantly depending on the culture medium, with CMA being the most suitable during the first 50 days. How­ ever, after 300 days the best medium was 3G-LBM, likely because it is enriched with nutrients, vitamins, and asparagine. Colonies grown on basal medium (BBM) without a carbon source had poorly formed mycelium, with long intersepta and few lipid drops. Colonies on BBM also developed more slowly and only on the surface. Similar poor, superficial, and filamentous growth has been reported for lichenized [19,35] and phytoparasitic fungi (e.g., [38]) and is associated with fungal resource-searching in nutrient-poor media.

Germinated P. grisea spores (from the “mixed species”) were unable to complete development in any of the enriched culture media, and only a very small fraction survived in non-enriched media. It is difficult to properly assess the finding that only 0.43 % of the total germinated spores of P. grisea showed extended growth in inorganic, non-enriched medium. Since only an extremely low percentage of P. gri­ sea spores was able to grow and only at a remarkably slow growth rate (average of 100 µm growth in almost 1 year), we assumed that its sexual spores hardly contributed to the survival of mixed-species P. grisea. The probability is no doubt very low that these hyphae will find the needed compatible photobiont to establish a lichen association before the hyphae die [17,33].

Although the growing conditions of lichen-forming fungi are not genus-specific [10,36], it was surprising that none of the eleven culture media tested were adequate for the ontogenetic development of P. grisea. This suggests that 99.57 % of germinated meiospores of P. grisea would not be able to develop in any of the media, not because the spores were immature or in an unsuitable medium, but because in this species they might have been non-functional and/or non-viable over both the short and long term. Indeed, many of the media tested in this study have been used successfully in other lichen species, including Physciaceae [3,14,35].

Another reason for the lack of successful spore germination and development of P. grisea in the tested media could be that this species needs to find a suitable photobiont at a very early stage of its development and therefore does not thrive in

Int. Microbiol. Vol. 16, 2013

153

laboratory cultures. If so, this poses an obstacle to successful lichen re-synthesis, especially when P. grisea shares its habitat with other species, such as P. distorta (sympatric species).

Since these species also compete for photobionts [57], lichen re-synthesis in P. distorta could be more successful, favored by the initially strong ontogenetic development of this species to establish lichen symbiosis and by its fewer specific nutritional requirements. Regardless, the strong developmental differences between these two species are remarkable, with spores of P. distorta but not of P. grisea developing in all culture media tested.

Preliminary transplant experiments using the lichen Icma­ dophila splachnirima (Icmadophilaceae), another mixed species, have indicated that, in more exposed microhabitats, apothecial growth is reversibly arrested at an early developmental stage and always accompanied by the formation of marginal soralia. This observation suggests an environmentally triggered switch from sexual to asexual reproduction, possibly in response to adverse, more stressful growth conditions [31], in which there is a need to save resources. If the apothecia do not represent an adaptive advantage because the cost is greater than the benefit (as suggested by Ludwig et al. [31]), or if they are simply non-functional because the spores cannot complete their development, negative selection would be expected to discourage apothecia, which would then be observed only rarely or occasionally. This would account for the local populations of P. grisea. According to Honegger and Zippler [23], genetic defects in the cascade of ascomatal development are possible, although they cannot be explained without genetic studies of the mating-type pathway. Lichen forming fungi have yet to be studied in depth, but several asex­ ual non-lichenized ascomycetes are known to fail to differentiate ascomata because of defects in genes of the complex mating-type pathway [25].

Harada [20] and Ohmura et al. [43] have considered that the high genetic variability observed in the mixed species Parmotrema tinctorium (Parmeliaceae) may be due in part to genetic recombination occurring in infrequent apothecia, but they have not tested this hypothesis empirically. Moreover, other mechanisms could explain such high genetic variability, such as somatic mutations, somatic recombination (parasexuality), historical genetic variability because of ancestral sexual states, or/and cryptic sex [9]. By contrast, Otalora et al. [44] have determined high genetic diversity values for bionts in Degelia plumbea (a sexual species) and extremely low values in Degelia atlantica (a mixed species). In our study, P. grisea meio­spores produced from apothecia in in vitro conditions were almost always non-functional and therefore might not

154

Int. Microbiol. Vol. 16, 2013

have notably increased the genetic variability within the population. We suggest, following Honegger and Zipper [23], that in P. grisea the ascomatal developmental system would be defective. The defects might be isolated phenomena in a group of individuals or an established characteristic in the genetic structure of the population. To further test this hypothesis, an additional analysis of mating-type and related genes in populations of P. grisea is necessary.

It is possible that the non-viability of the spores of P. gri­ sea grown in culture was due to seasonal or population factors [51]. Therefore, further studies are needed to clarify the presence and frequency of mating-type genes in the populations and/or to analyze the genetic variability in the population based on sequence or microsatellite data. Our work emphasizes the importance of testing spore viability at the population level before concluding that sexual spores are true sources of genetic variability in mixed system reproducing species, as has often been assumed [20,43]. This study of spore viability is of particular interest considering that lichens may have other sources of genetic variability, such as somatic mutations, somatic recombination (parasexuality), and historical genetic variability reflecting ancestral sexual states. The contribution of these sources to increasing genetic diversity in populations should be determined.

Acknowledgements. The authors thank the following colleagues, who provided supplies or other input for this project at Rutgers University: James White, Ilya Raskin, Debashish Bhattacharya, Marshall Bergen, and Mónica Torres. We acknowledge the Ministerio de Ciencia e Innovación (CGL201021646) for providing the funds for this research, and Ramón y Cajal grant (RYC02007-01576) to PKD. This project was also supported by a research grant to MCM from the Universidad Rey Juan Carlos, as Visiting Scientist.

Competing interests: None declared.

References 1. Ahmadjian V (1993) The lichen symbiosis. John Wiley, New York, NY, USA 2. Bailey RH, James PW (1979) Birds and the dispersal of lichen propagules. Lichenologist 11:105-106 3. Behera BC, Makhija U (2001) Effect of various culture conditions on growth and production of salazinic acid in Bulbothrix setschwanensis (lichenized ascomycetes) in vitro. Curr Sci India 80:1424-1427 4. Belandria G, Asta J, Nurit F (1989) Effects of sulphur dioxide and fluoride on ascospore germination of several lichens. Lichenologist 21:79-86 5. Bowler PA, Rundel P W (1975) Reproductive strategies in lichens. J Lin Soc Bot 70:325-340 6. Buschbom, J, Mueller, GM (2006) Testing “species pair” hypotheses: evolutionary processes in the lichen-forming species complex Porpidia flavocoerulescens and Porpidia melinodes. Mol Biol Evol 23:574-586

molina et al.

7. Brunauer G, Stocker-Worgotter E (2005) Culture of lichen fungi for future production of biologically active compounds. Symbiosis 38:187-201 8. Büde B, Scheidegger C (2008) Thallus morphology and anatomy. In: Nash III TH (ed) Lichen Biology, 2nd edn., Cambridge University Press, Cambridge, UK, pp 40-68 9. Cassie DM, Piercey-Normore MD (2008) Dispersal in a sterile lichenforming fungus, Thamnolia subuliformis (Ascomycotina: Icmadophilaceae). Botany 86:751-762 10. Cordeiro LMC, Iacomini M, Stocker-Wörgötter E (2004) Culture studies and secondary compounds of six Ramalina species. Mycol Res 108:489-497 11. Cornejo C, Chabanenko S, Scheidegger C (2009) Phylogenetic analysis indicates transitions from vegetative to sexual reproduction in the Loba­ ria retigera group (Lecanoromycetidae, Ascomycota). Lichenologist 41: 275-284 12. Crespo A, Pérez-Ortega S (2009) Cryptic species and species pairs in lichens: a discussion on the relationship between molecular phylogenies and morphological characters. An Jardín Bot Madrid 66:71-81 13. Crespo A, Blanco O, Hawksworth DL (2001) The potential of mitochondrial DNA for establishing phylogeny and stabilising generic concepts in the parmelioid lichens. Taxon 50:807-819 14. Crittenden PD, David JC, Hawksworth DL, Campbell FS (1995) Attempted isolation and success in the culturing of a broad spectrum of lichen-forming and lichenicolous fungi. New Phytol 130:267-297 15. Cubero OF, Crespo A, Esslinger TL, Lumbsch HT (2004) Molecular phylogeny of the genus Physconia (Ascomycota, Lecanorales) inferred from a Bayesian analysis of nuclear ITS rDNA sequences. Mycol Res 108:498-505 16. Divakar PK, Amo G, del Prado R, Esslinger TL, Crespo A (2007) Upper cortex anatomy corroborates phylogenetic hypothesis in species of Physconia (Ascomycota, Lecanoromycetes). Mycol Res 11:1311-1320 17. Fedrowitz K, Kuusinen M, Snäll T (2012) Metapopulation dynamics and future persistence of epiphytic cyanolichens in a European boreal forest ecosystem. J Appl Ecol 49:493-502 18. Ferencova Z, Del Prado R, Perez-Vargas I, Hernandez-Padron C, Crespo A (2010) A discussion about reproductive modes of Pseudevernia furfu­ racea based on phylogenetic data. Lichenologist 42:1-12 19. Hamada N, Miyagawa H (1995) Secondary metabolites from isolated lichen mycobionts cultured under different osmotic conditions. Lichenologist 27:201-205 20. Harada H (2003) Does Parmotrema tinctorum reproduce by ascospores?

Lichenology 2:37-40 21. Hauck M, Paul A, Mulack C, Fritz E, Runger M (2006) Effects of manganese on the viability of vegetative diaspores of the epiphytic lichen Hypogymnia physodes. Environ Exp Botany 47:127-142 22. Hestmark G, Miadlikowska J, Kauff F, Fraker E, Molnar K, Lutzoni F (2011) Single origin and subsequent diversification of central Andean endemic Umbilicaria species. Mycologia 103:45-56 23. Honneger R, Zippler U (2007) Mating systems in representatives of Par­ meliaceae, Ramalinaceae and Physciaceae (Leconoromycetes, lichenforming ascomycetes). Mycol Res 111:424-432 24. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754-755 25. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286-298 26. Kirk PM, Cannon PF, Minter MW, Stalpers JA (2008) Dictionary of the Fungi, 10th edn, CAB International, Wallingford, UK 27. Lande R, Arnold SJ (1983) The measurement of selection on correlated characters. Evolution 37:1210-1226

Non-developing spores in Physconia grisea

28. Lawrey JD (1980) Sexual and asexual reproductive patterns in Parmo­ trema (Parmeliaceae) that correlate with latitude. Bryologist 83:344-350 29. Lilly VG, Barnett HL (1951) Physiology of the Fungi. McGraw-Hill, New York, NY, USA 30. Lohtander K, Myllys L, Sundin R, Källersjö M, Tehler A (1998) The species pair concept in the lichen Dendrographa leucophaea (Arthoniales): analyses based on ITS sequences. Bryologist 101:404-411 31. Ludwig E (2011) Marginal soralia and conidiomata in Icmadophila splachnirima (Icmadophilaceae) from southern New Zealand. Austral Lichenol 68:4-11 32. Lumbsch HT, Leavitt SD (2011) Goodbye morphology? A paradigm shift in the delimitation of species in lichenized fungi. Fungal Divers 50:59-72 33. Marshall WA (1996) Aerial dispersal of lichen soredia in the maritime Antarctic. New Phytol 134:523-530 34. Martínez I, Flores T, Otálora MAG, Belinchón R, Prieto M, Aragón G, Escudero A (2012) Multiple-scale environmental modulation of lichen reproduction. Fungal Biol 116:1192-1201 35. Molina MC, Crespo A (2000) Comparison of development of axenic cultures of five species of lichen-forming fungi. Mycol Res 104:595-602 36. Molina MC, Crespo A, Blanco O, Hladun N, Hawksworth D (2002) Mol­ ecular phylo­geny and status of Diploicia and Diplotomma, with observations on Diploicia subcanescens and Diplotomma rivas-martinezii.

Lichenologist 34:509-519 37. Molina MC, Crespo A, Vicente C, Elix JA (2003) Differences in the composition of phenolics and fatty acids of cultured mycobiont and thallus of Physconia distorta. Plant Physiol Biochem 41:175-180 38. Molina MC, Fontaniella B (2008) Yeast-mycelial dimorphism of haploide strains of Sporisorium scitamineum. In: Legaz ME, Vicente C (eds) Biochemical responses of plants to invaders. Research Signpost, Kerala, pp 97-108 39. Molina MC, Stocker-Wörgötter E, Türk R, Vicente C (1997) Axenic culture of the mycobiont of Xanthoria parietina in different nutritive media, effect of carbon source in spores germination. Endocyt Cell Res 12:103-109 40. Molina MC, Vicente C (1995) Correlationships between enzymatic activity of lectins, putrescine content and chloroplast damage in Xanthoria parieti­ na phycobionts. Cell Adh Comm 3:1-12 41. Muñoz J, Felicísimo AM, Cabezas F, Burgaz AR, Martínez I (2004) Wind as a long-distance dispersal vehicle in the Southern Hemisphere.

Science 304:1144-1147 42. Myllys L, Lohtander K, Tehler A (2001) β-Tubulin, ITS and group I intron sequences challenge the species pair concept in Physcia aipolia and P. caesia. Mycologia 93:335-343

Int. Microbiol. Vol. 16, 2013

155

43. Ohmura Y, Kawachi M, Kasai F, Watanabe M (2006) Genetic combinations of symbionts in vegetatively reproducing lichen, Parmotrema tinc­ torum, based on ITS rDNA sequences. Bryologist 109:43-59 44. Otálora MAG, Salvador C, Martínez I, Aragón G (2013) Does the reproductive strategy affect the transmission and genetic diversity of bionts in cyanolichens? A case study using two closely related species. Microb Ecol 65:517-530 45. Otte V, Esslinger TL, Litterski B (2002) Biogeographical research on European species of the lichen genus Physconia. J Biogeogr 29:11251141 46. Poelt J (1966) Zur Kenntnis der Flechtengattung Physconia in der Alten Welt und ihrer Beziehungen zur Gattung Anaptychia. Nova Hedwigia 12:107-135 47. Poelt J (1970) Das Konzept der Artenpaare bei den Flechten. Vortrage aus dem Gesamtgebiet der Botanik, N.F. 4:187-198 48. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253-1256 49. Printzen C, Ekman S (2003) Local population subdivision in the lichen Cladonia subcervicornis as revealed by mitochondrial cytochrome oxidase subunit 1 intron sequences. Mycologia 95:399-406 50. Rodríguez F, Oliver JF, Martín A, Medina JR (1990) The general stochastic model of nucleotide substitution. J Theor Biol 142:485-501 51. Sangvichien E, Hawksworth DL, Whalley AJS (2011) Ascospore discharge, germination and culture of fungal partners of tropical lichens, including the use of a novel culture technique. IMA Fungus 2:143-153 52. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688-2690 53. Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML Web Servers. Syst. Biol. 57:758-771 54. Sayers WE, Barrett T, Benson DA, et al. (2011) Data resources of the National Center for Biotechnology Information. Nucleic Acids Res 39: D38-D51 55. Tehler A (1982) The species pair concept in lichenology. Taxon 31: 708-714 56. Tehler A, Irestedt M, Bungartz F, Wedin M (2009) Evolution and reproduction modes in the Roccella galapagoensis aggregate (Roccellaceae, Arthoniales). Taxon 58:438-456 57. Wornik S, Grube M (2010) Joint dispersal does not imply maintenance of partners hips in lichen symbioses. Microb Ecol 59:150-157

RESEARCH ARTICLE International Microbiology (2013) 16:157-163 doi: 10.2436/20.1501.01.190 ISSN 1139-6709 www.im.microbios.org

Identification of rhizobial strains nodulating Egyptian grain legumes Hamdi H. Zahran,1 Rajaa Chahboune,2 Silvia Moreno,2 Eulogio J. Bedmar,2* Medhat Abdel-Fattah,1 Manal M. Yasser,1 Ahmed M. Mahmoud1 Department of Botany, Faculty of Science, University of Beni-Suef, Beni-Suef, Egypt. 2Department of Soil Microbiology and Symbiotic Systems, Experimental Station of the Zaidin, National Research Council (EEZ-CSIC), Granada, Spain

1

Received 27 July 2013 · Accepted 25 September 2013

Summary. Fifty four bacterial strains were isolated from root nodules of the grain legumes Cicer arietinum, Lens esculentus, Phaseolus vulgaris, Pisum sativum, and Vicia faba grown in cultivated lands of Beni-Suef Governorate (Egypt). Repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) clustered the strains into 15 REP-PCR groups. The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR pattern showed that the strains were closely related to members of the family Rhizobiaceae of the Alphaproteobacteria. Pairwise alignments between globally aligned sequences indicated that the strains from V. faba had 99.6 % identity with Rhizobium leguminosarum, and those from P. vulgaris 99.76 % and 100 % with sequences from R. leguminosarum and R. mesosinicum, respectively. Strains from P. sativum had 99.76 %, 99.84 %, and 99.92 % sequence identity with R. leguminosarum, R. etli, and R. pisi, respectively, and those from L. escu­­ lentus had 99.61 % identity with sequences from R. leguminosarum. Sequences of the strains from C. arietinum had 100 % identity with those of Mesorhizobium amorphae and M. robiniae, respectively. Nitrogenase activity, determined as acetylenedependent ethylene production, was detected in nodules formed after inoculation of the corresponding host plant with the representative rhizobial species. [Int Microbiol 2013; 16(3):157-163] Keywords: Rhizobium · Mesorhizobium · legumes · 16S rRNA gene · phylogenetic trees

Introduction Nitrogen is the most significant yield-limiting element in many agricultural production systems. External inputs of nitrogen to agriculture may come from mineral fertilizers, the production of which is heavily dependent on fossil fuels. Alternatively, Corresponding author: E.J. Bedmar Department of Soil Microbiology and Symbiotic Systems Estación Experimental del Zaidín, CSIC Apartado Postal 419 18080 Granada, Spain Tel. +34-958181600 E-mail: eulogio.bedmar@eez.csic.es

*

nitrogen can be obtained from symbiotic nitrogen fixation by nodule-forming legume and non-legume associated rhizobial and actinorhizal bacteria, respectively [28].

Members of the Leguminosae (Fabaceae) comprise 17,000 to 19,000 species and play an important ecological role, with representatives in nearly every terrestrial biome on Earth [17].

These plants are best characterized by their ability to establish N2-fixing symbiotic associations with Alphaproteobacteria of the genus Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Ensifer [10,24,39], collectively referred to as rhizobia. Other non-rhizobial genera have been shown to nodulate legumes [4,23,29,35,37], which can also be nodulated by Betaproteobacteria, specifically, the genera

158

Int. Microbiol. Vol. 16, 2013

Burkholderia and Cupriavidus [3,5,8,11,19,30]. During the infection process, an exchange of molecular signals occurs between the two partners, leading to the formation of root nodules, where nitrogen fixation takes place [32]. Because of this ability, legumes can grow in arid, nitrogen-deficient soils, acting as pioneer plants for soil stabilization and colonization, enhancing their fertility, and, consequently, preventing erosion and desertification. Despite potential errors in accurate calculations of N2 fixation at global scales, an overall estimate of 50–70 Tg of biologically fixed N in agricultural systems has been estimated [13].

Since the use of rhizobia as a biofertilizer is a friendly environmental alternative to mineral fertilization, inoculation of legumes is a common agricultural practice, including in Egypt, especially in the country’s newly reclaimed soils [45,46]. Nearly 95 % of Egypt’s land surface can be categorized as arid and semi-arid. In these ecosystems, abiotic stresses, such as salinity or drought, limit legume cultivation and therefore crop production [45,46]. In Egypt, the grain legumes chickpea (C. arietinum), lentil (L. esculentus), common bean (P. vulgaris), pea (P. sativum), and broad bean (V. faba) are widely cultivated all along the Nile River for human consumption. In previous studies, R. etli and R. galli­ cum were isolated from root nodules of P. vulgaris plants growing in the Ismailia desert and the Ashmun area of the Nile Valley and Nile Delta [25]. Phylogenetic analyses based on partial sequencing of the 16S rRNA gene of 34 free-living rhizobial strains directly isolated from soils taken at the same locations identified 38.2 % of the strains as E. meliloti, 29.4 % as highly related to E. medicae, 23.5 % as Agrobacterium tumefaciens, and 8.8 % as taxonomically similar to R. etli [26]. Recently, the phenotypic characteristics and nodulation capacity of more than 50 rhizobial-like strains, isolated from the root nodules of lentils, common beans, peas, chickpeas, and broad beans, have been described [47]. Here we report on the identification of those strains on the basis of their 16S rRNA gene phylogenies. Our data contribute to further characterizing endosymbiotic bacteria associated with Egyp­ tian grain legumes.

Materials and methods Isolation of bacteria from nodules and culture conditions.

Nodules (10/plant) were collected from roots of agriculturally grown, healthy C. arietinum, L. esculentus, P. vulgaris, P. sativum, and V. faba plants near the towns of Tezmant and Beni-Suef (Beni-Suef Governorate, Middle Egypt) (20 plants/location), where they are agriculturally-grown. Nodules were surface-sterilized as indicated earlier [47], placed independently in Petri dishes, and crushed in a drop of sterile water with a sterile glass rod.

Zahran et al.

The resulting suspension was streaked onto Petri dishes containing yeast extract-mannitol (YEM) medium [42] supplemented with 0.025 g Congo Red/l. After incubation of the plates at 30 ºC for 7 days, colony-forming units, which represented all of the colony types that could be distinguished by microscopic observation, were chosen. All rhizobial strains used in this study were routinely grown on YEM medium.

DNA extraction and PCR amplifications. For DNA extraction and PCR amplifications, genomic DNA was isolated from bacterial cells using the RealPure Genomic DNA extraction kit (Durviz, Spain), according to the manufacturer’s instructions. The quantity of DNA was determined using a Nanodrop spectrophotometer (NanoDrop ND1000, Thermo Fisher Scientific, USA). Repetitive extragenic palindromic (REP)-polymerase chain reactions (PCR) were performed using primers REPIR-I and REP2-I, according to de Bruijn [6]. PCR amplifications of 16S rRNA gene fragments were carried out using the two opposing primers 41f and 1488r as previously reported [12]. Amplification products were purified using the Qiagen PCR product purification system and subjected to cycle sequencing using the same primers as for PCR amplification, with ABI Prism dye chemistry. The products were analyzed with a 3130 × l automatic sequencer at the sequencing facilities of Estación Experimental del Zaidin, CSIC, Granada, Spain. The obtained sequences were compared to those in the GenBank database using the BLAST program [1] and with the sequences held in the EzTaxon-e server [15]. The sequences were aligned using Clustal W software [33]. The distances were calculated according to Kimura’s two-parameter model [16]. Phylogenetic trees were inferred based on the maximum likelihood (ML) method [9], using MEGA 5.0 software [31].

Plant nodulation tests and nitrogenase activity. Seeds of C.­ arie­ tinum, L. esculentus, P. vulgaris, P. sativum, and V. faba were surface-sterilized as above and allowed to germinate at 30 °C in the dark. Seedlings (1–4/pot) were planted in 1/2-kg pots containing sterile sand and vermiculite (1:1, v:v) and inoculated separately with each of the 15 strains. Uninoculated plants were used as a control for nodulation experiments. Plants were grown under natural daylight supplemented with artificial lighting, fed with N-free mineral solution [22], and harvested at 10 % flowering to check for nodule formation. Nitrogenase activity was determined as acetylene-dependent ethylene production, as described previously [47].

Accession numbers. Accession numbers of the nucleotide sequences of the rhizobial species used in this study are shown in the figure trees.

Results REP-PCR and 16S rRNA gene phylogenetic analysis.

Fifty-four bacterial strains, 12 from P. sativum, 11 each from C. arietinum and V. faba, and 10 each from L. escu­lentum and P. vulgaris, were isolated from extracts of nodules taken from healthy, agriculturally-grown plants in Beni-Suef Governorate (Egypt) [47].

The 54 isolates were represented by 15 different REPPCR patterns (Table 1). The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP pattern revealed that all of the isolates were members of the family Rhizobiaceae of the Alphaproteobacteria, of which 12 belonged to the genus Rhizobium and three to the

Int. Microbiol. Vol. 16, 2013

Rhizobial strains nodulating Egyptian legumes

159

Table 1. Phylogenetic classification of bacterial strains isolated in this study Strainsa

REP-PCR pattern

Closest related genusb

Family

BSPV1, BSPV2, BSPV3, BSPV4, BSPV5

I

Rhizobium

Rhizobiaceae

BSPV6, BSPV7

II

Rhizobium

Rhizobiaceae

BSPV9

III

Rhizobium

Rhizobiaceae

BSPV11, BSPV12

IV

Rhizobium

Rhizobiaceae

BSPS1, BSPS2, BSPS3, BSPS4, BSPS5, BSPS6

V

Rhizobium

Rhizobiaceae

BSPS7, BSPS8, BSPS9

VI

Rhizobium

Rhizobiaceae

BSPS10, BSPS11, BSPS12

VII

Rhizobium

Rhizobiaceae

BSCA1, BSCA2

VIII

Mesorhizobium

Rhizobiaceae

BSCA3, BSCA4, BSCA8, BSCA11

IX

Mesorhizobium

Rhizobiaceae

BSCA5, BSCA6, BSCA7, BSCA9, BSCA10

X

Mesorhizobium

Rhizobiaceae

BSVF1, BSVF2

XI

Rhizobium

Rhizobiaceae

BSVF3, BSVF4, BSVF5, BSVF6, BSVF7

XII

Rhizobium

Rhizobiaceae

BSVF8, BSVF9, BSVF10, BSVF11

XIII

Rhizobium

Rhizobiaceae

BSLE1, BSLE3, BSLE4, BSLE5, BSLE6, BSLE7, BSLE8

XIV

Rhizobium

Rhizobiaceae

BSLE9, BSLE10, BSLE11

XV

Rhizobium

Rhizobiaceae

Strains named BS to indicate Beni-Suef Governorate, followed by the letters PV, PS, CA, VF, and LE, representing P. vulgaris, P. sativum, C. arietinum, V. faba, and L. esculentum, respectively. Strains shown in bold were chosen as the representative strains of each REP-PCR group. b Based on the 16S rRNA gene. a

Mesorhizobium group (Table 1). The ML phylogenetic tree (Fig. 1) and EzTaxon-e analysis (Table 2) inferred from the 16S rRNA genes sequences indicated that strains BSPV2, BSPV7, BSPS4, BSVF2, BSVF5, BSVF9, BSLE4, and BSLE10 clustered with R. leguminosarum USDA 2370T, based on identity values > 99.6 %, and that strains BSPS7, BSPS10, and BSPV9 grouped with R. pisi DSM 30132T, R. etli CFN 42T, and R. mesosinicum CCBAU 25010T, respectively, with identity values > 99.8 % in all cases. Strain BSCA1 clustered with M. amorphae ACCC 19665T, and strains BSCA8 and BSCA9 with M. robiniae CCNWYC 115T. These three strains had 100 % identity with the 16S rRNA gene sequences of their corresponding type strain.

Plant nodulation tests. The 15 rhizobial strains identified in this study nodulated their original host plants.

Nodules fixed N2, with nitrogenase activity values, determined as acetylene-dependent ethylene production, varying from

51 nmol C2H2 plant-1 h-1 in C. arietinum nodulated by strain BSCA8 to 480 nmol C2H2 plant–1 h–1 in P. vulgaris inoculated with strain BSPV2.

Discussion In this study, rhizobial bacteria from root nodules of the grain legumes C. arietinum, L. esculentus, P. vulgaris, P. sa­ti­­vum, and V. faba growing in cultivated lands of the Beni-Suef Governorate (Egypt) were identified. REP-PCR fingerprinting was used to group the strains. This technique has been extensively used to cluster bacteria at the subspecies or strain level [6,40] and is known to be a powerful tool for studies on microbial ecology and evolution [14].

Phaseolus vulgaris is a promiscuous legume able to form symbioses with several species of Rhizobium, including R. leguminosarum, R. etli, R. gallicum, R. giardinii, and

Int. Microbiol. Vol. 16, 2013

Zahran et al.

Int Microbiol

160

Fig. 1. Maximum likelihood phylogenetic tree based on partial 16S rRNA gene sequences of strains from nodules of P. vulgaris, P. sativum, C. arietinum, V. faba, and L. esculentus and phylogenetically related species within the genera Rhizobium and Mesorhizobium. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Values lower than 70 are not shown. Bar, 1 substitution per 100 nucleotide position. The tree is rooted on Bosea thiooxidans DSM 9653.

R. tropici [10,24]. We found that common beans were nodulated by R. leguminosarum as well as by R. lusitanum and R. mesosinicum (Table 2). Nodulation of P. vulgaris

by R. leguminosarum [10, 24] and R. lusitanum [38] is well established. Our results extend those data with the finding that P. vulgaris can be nodulated by R. mesosinicum, a bacterium

first isolated from root nodules of Albizia julibrissin [18].

These results, however, do not agree with those previously published, in which R. etli and R. gallicum were isolated from nodules of P. vulgaris growing in Egyptian soils [25]. The discrepancy may reflect differences in soil characteristics, since R. etli and R. gallicum were isolated from plants grown

Int. Microbiol. Vol. 16, 2013

Rhizobial strains nodulating Egyptian legumes

161

Table 2. EzTaxon-e closest relative species of strains isolated in this study Strains

Original host

Closest related type strains

Similarity (%)

BSPV2

P. vulgaris

R. leguminosarum USDA 2370 T

99.76

BSPV7

P. vulgaris

R. leguminosarum USDA 2370 T

99.76

BSPS4

P. sativum

R. leguminosarum USDA 2370 T

99.76

BSVF2

V. faba

R. leguminosarum USDA 2370T

99.76

BSVF5

V. faba

R. leguminosarum USDA 2370 T

99.76

BSVF9

V. faba

R. leguminosarum USDA 2370 T

99.61

BSLE4

L. esculentus

R. leguminosarum USDA 2370 T

99.61

BSLE10

L. esculentus

R. leguminosarum USDA 2370 T

99.61

BSPV9

P. vulgaris

R. lusitanum P1-7 T

100.00

BSPV11

P. vulgaris

R. mesosinicum CCBAU 25010 T

99.84

BSPS7

P. sativum

R. pisi DSM 30132 T

99.92

BSPS10

P. sativum

R. etli CFN 42 T

99.84

BSCA1

C. arietinum

M. amorphae ACCC 19665 T

100.00

BSCA8

C. arietinum

M. robiniae CCNWYC 115 T

100.00

BSCA9

C. arietinum

M. robiniae CCNWYC 115 T

100.00

in desert areas [25] whereas R. leguminosarum, R. lusitanum, and R. mesosinicum were obtained from nodules of plants cultivated in agricultural, fertile areas.

Strains isolated from P. sativum were discriminated into three genotypes, R. leguminosraum, R. pisi, and R. etli (Table 2). Pisum sativum was previously shown to be nodulated by R. leguminosarum and R. pisi [10, 25]. The latter species was originally isolated from nodules of pea plants and corresponds to a reclassification of strain R. leguminosarum DSM 30132 [21]. To our knowledge, ours is the first report showing that R. etli produces effective nodules on roots of P. sativum.

Strains from C. arietinum were identified as M. amorphae and M. robiniae (Table 2). M. amorphae was isolated from Amorpha fruticosa plants grown in Chinese [43] and American soils [44], and M. robiniae is found in root nodules of Robinia pseudoacacia growing in China [48], but there were no reports that this rhizobial species nodulates C. arietinum.

The only species isolated from root nodules of V. faba was R. leguminosarum (Table 2), which is consistent with previous reports [10,24]. R. leguminosarum was shown to

form nodules in faba bean plants from Ethiopia [2], France [7], Jordan [20], China [34], and Canada [41]. It was also the predominant rhizobial species isolated from nodules of agriculturally grown faba bean plants in Egypt [27].

Rhizobial strains isolated from L. esculentus grouped into two genotypes that were identified as R. leguminosarum (Table 1). R. leguminosarum bv. viciae is the specific microsymbiont of the legumes of the tribe Vicieae, which comprises the genera Vicia, Pisum, Lens, and Lathyrus [10, 24]; accordingly, its isolation from Egyptian lentils is not surprising.

Because inoculation of legumes is a common practice in Egypt [45, 46], the identification, selection, and maintenance of superior rhizobial strains for each host plant are critical.

Collectively, based on 16S rRNA gene sequences, our results show that Egyptian agriculturally grown members of the genus Lens as well as broad beans, peas, common beans, and chickpeas can be nodulated by different species of Rhizobium and Mesorhizobium. The recognition of this diversity is essential to improve our knowledge of endosymbiotic bacterial populations and thus to study the activities and applications

162

Int. Microbiol. Vol. 16, 2013

of rhizobial strains important in agriculture, environmental protection, and biotechnology. In this context, we isolated a number of rhizobial strains that could be assayed for increased productivity of agriculturally grown legumes in Egypt. The 15 selected strains identified in this study are true symbionts of their corresponding host plant as, after nodule isolation, they were able to establish new and effective N2-fixing symbioses with them. Although the acetylene reduction technique cannot be used as a quantitative assay of N2 fixation [36], in this study it allowed us to determine whether the nodulated legume roots actively fixed N2 but not the effectiveness of each symbiotic association.

Acknowledgements. This study was supported by ERDF-cofinanced grant RNM4746 from Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain). A.M. Mahmoud thanks the Egyptian Government for a Partnership and Ownership Initiative (PAROWN) grant to support his stay in the Department of Microbiology and Symbiotic Systems, Estación Expe­ ri­mental del Zaidín (EEZ), Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.

Competing interests. None declared

References 1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410 2. Amanuel G, Kuhne RF, Tanner DG, Vlek PLG (2000) Biological nitrogen fixation in faba bean (Vicia faba L.) in the Ethiopian highlands as affected by P fertilization and inoculation. Biol Fert Soils 32:353-359 3. Angus AA, Hirsch AM (2010) Insights into the history of the legumesbetaproteobacterial symbiosis. Mol Ecol 19:28-30 4. Ardley JK, Parker MA, De Meyer SE, Trengove RD, O’Hara GW, Reeve WG, Yates RJ, Dilworth MJ, Willems A, Howieson JG (2011) Microvirga lupini sp. nov., Microvirga lotononidis sp. nov., and Micro­ virga zambiensis sp. nov. are Alphaproteobacterial root nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxo­no­ mically separate legume hosts. Int J Syst Evol Microbiol. 62:2579-2588 5. Bontemps C, Elliott GN, Simon MF, Dos Reis Junior FB, Gross E, Lawton R, Neto NE, Loureiro MF, Faria SM, Sprent JI, James EK, Young JPW (2010) Burkholderia species are ancient symbionts of legumes. Mol Ecol 19:44-52 6. De Bruijn FJ (1992) Use of repetitive (Repetitive Extragenic Palindromic and Enterobacterial Repetitive Intergenic Consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol 58:2180-2187 7. Depret G, Houot S, Allard MR, Breuil MC, Nouaim R, Laguerre G (2004) Long-term effects of crop management on Rhizobium leguminosarum biovar viciae populations. FEMS Microbiol Ecol 51:87-97 8. Elliott GN, Chou JH, Chen WM, Bloemberg G, Bontemps C, MartínezRomero E, Velázquez E, Young JPW, Sprent JI, James EK (2009) Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions. Environ Microbiol 11:762-778 9. Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368-376

Zahran et al.

10. Graham PH (2008) Ecology of the root nodule bacteria of legumes. In: Dilworth MJ, James EK, Sprent JI, Newton WE (eds) Nitrogen-fixing leguminous symbioses. Springer Science + Business Media B.V. pp. 23-58 11. Gyaneshwar P, Hirsch AM, Moulin L, Chen WM, Elliott GN, Bontemps C, Estrada-de los Santos P, Gross E, Dos Reis Junior FB, Sprent JI, Young JPW, James EKW (2011) Legume-nodulating betaproteobacteria: diversity, host range, and future prospects. Mol Plant Microbe Interact 24:1276-1288 12. Herrera-Cervera JA, Caballero-Mellado J, Laguerre G, Tichy HW, Requena N, Amarger N, Martínez-Romero E, Olivares J, Sanjuan J (1999) At least five rhizobial species nodulate Phaseolus vulgaris in a Spanish soil. FEMS Microbiol Ecol 30:87-97 13. Herridge DF, Peoples MB, Boddey RM (2008) Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 311:1-18 14. Ishii S, Sadowsky MJ (2009) Applications of the REP-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution. Environ Microbiol 11:733-740 15. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JK, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716-721 16. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.

J Mol Evol 16:111-120 17. Lewis G, Schrire B, Mackinder B, Lock M (2005) Legumes of the world.

Royal Botanic Gardens, Kew. 18. Lin DX, Chen WF, Wang FQ, Hu D, Lin DX, Chen WF, Wang FQ Hu D, Wang ET, Sui XH, Chen WX (2009) Rhizobium mesosinicum sp. nov., isolated from root nodules of three different legumes. Int J Syst Evol Microbiol 59:1919-1923 19. Mishra RPN, Tisseyre P, Melkonian R, Chaintreuil C, Miché L, Klonowska A, González S, Bena G, Laguerre G, Moulin L (2012) Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and biodiversity of Burkholderia phymatum and other betaproteobacteria. FEMS Microbiol Ecol 79:487-503 20. Mutch LA, Tamimi SM, Young JPW (2003) Genotypic characterization of rhizobia nodulating Vicia faba from soils of Jordan: a comparison with UK isolates. Soil Biol Biochem 35:709-714 21. Ramírez-Bahena MH, García-Fraile P, Peix A, Valverde A, Rivas R, Igual JM, Mateos PF, Martínez-Molina E, Velázquez E (2008) Revision of the taxonomic status of the species Rhizobium leguminosarum (Frank 1879) Frank 1889AL, Rhizobium phaseoli Dangeard 1926AL and Rhizobium trifolii Dangeard 1926AL. R. trifolii is a later synonym of R. leguminosarum. Reclassification of the strain R. leguminosarum DSM 30132 (=NCIMB 11478) as Rhizobium pisi sp. nov. Int J Syst Evol Microbiol 58:2484-2490 22. Rigaud J, Puppo A (1975) Indol-3-acetic catabolism by soybean bacteroids. J Gen Microbiol 88: 223-228 23. Rivas R, Velázquez E, Willems A, Vizcaíno N, Subba-Rao NS, Mateos PF, Gillis M, Dazzo FB, Martínez-Molina E (2002) A new species of Devosia that forms a unique nitrogen-fixing root nodule symbiosis with the aquatic legume Neptunia natans (L.F.) Druce. Appl Environ Microbiol 68:5217-5222 24. Rivas R, García-Fraile P, Velázquez E (2009) Taxonomy of bacteria nodulating legumes. Rev Microbiol Insight 2:251-269 25. Shamseldin AAY, Vinuesa P, Thierfelder H, Werner D (2005) Rhizobium etli and Rhizobium gallicum nodulate Phaseolus vulgaris in Egyptian soils and display cultivar-dependent symbiotic efficiency. Symbiosis 38:145-161 26. Shamseldin AAY, Sadowsky MJ, El-Saadani M, An CS (2008) Molecular biodiversity and identification of free living Rhizobium strains from diverse Egyptian soils as assessed by direct isolation without trap hosts.

Amer-Euras J Agric Environ Sci 4:541-549

Rhizobial strains nodulating Egyptian legumes

27. Shamseldin AAY, El-Saadani M, Sadowsky MJ, An CS (2009) Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodul­ ating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis. Soil Biol Biochem 41:45-53 28. Sprent JI (2009) Legume nodulation: a global perspective. Oxford, Wiley-Blackwell 29. Sy A, Giraud E, Jourand P, Garcia N, Willems A, de Lajudie P, Prin Y, Neyra M, Gillis M, Boivin-Masson C, Dreyfus B (2001) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. J Bacteriol 183:214-220 30. Talbi C, Delgado MJ, Girard L, Ramírez-Trujillo A, CaballeroMellado J, Bedmar EJ (2010) Burkholderia phymatum strains capable of nodulating Phaseolus vulgaris are present in Moroccan soils. Appl Environ Microbiol 76:4587-4591 31. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony. Mol Biol Evol 28:2731-2739 32. Terpolilli JJ, Hood GA, Poole PS (2012) What determines the efficiency of N2-fixing Rhizobium-legume symbioses?. Adv Microbiol Physiol 60:325-389 33. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 24:4876-4882 34. Tian CF, Wang ET, Han TX, Sui XH, Chen WX (2007) Genetic diversity of rhizobia associated with Vicia faba in three ecological regions of China. Arch Microbiol 188: 273-282 35. Trujillo ME, Willems A, Abril A, Planchuelo AM, Rivas R, Ludeña D, Mateos PF, Martínez-Molina E, Velázquez E (2005) Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov. Appl Environ Microbiol 71:1318-1327 36. Unkovich M, Herridge D, Peoples M, Cadisch G, Boddey R, Giller K, Alves B, Chalk P (2008) Measuring plant-associated nitrogen fixation in agricultural systems. ACIAR, Canberra, Australia 37. Valverde A, Velázquez E, Fernández-Santos F, Vizcaíno N, Rivas R, Mateos PF, Martínez-Molina E, Igual JM, Willems A (2005) Phyllobacterium trifolii spp. nov. nodulating Trifolium lupini and Lupinus in Spanish soils. Int J Sys Evol Microbiol 55:1985-1989

Int. Microbiol. Vol. 16, 2013

163

38. Valverde A, Igual JM, Peix A, Cervantes E, Velázquez E (2006) Rhizobium lusitanum sp. nov., a bacterium that nodulates Phaseolus vulgaris. Int J Syst Evol Microbiol. 56:2631-2637 39. Velázquez E, García-Fraile P, Ramírez-Bahena MH, Peix A, Rivas R (2010) Proteobacteria forming nitrogen fixing symbiosis with higher plants. In: Sezenna ML (ed) Proteobacteria: Phylogeny, metabolic diversity and ecological effects. Nova Science Pub., New York, NY, USA, pp. 37-56 40. Versalovic J, Lupski JR (1998) Interspersed repetitive sequence in bacterial genomes. In: de Bruijn FJ, Lupski JR, Weinstock GM (eds) Bacterial genomes: Physical structure and analysis. Chapmann and Hall, New York, NY, USA, pp. 437-454 41. Vessey JK, Chemining’wa GN (2006) The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie. Soil Biol Biochem 38:153-163 42. Vincent JM (1970) A manual for the practical study of the rootnodule bacteria. IBP15. Blackwell Scientific Publications, Oxford and Edinburgh, UK 43. Wang ET, van Berkum P, Sui XH, Beyene D, Chen WX, MartínezRomero E (1999) Diversity of rhizobia associated with Amorpha fruti­ cosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov. Int J Syst Bacteriol 49:51-65 44. Wang ET, Rogel MA, Sui XH, Chen WX, Martínez-Romero E, van Berkum P (2002) Mesorhizobium amorphae, a rhizobial species that nodulates Amorpha fructicosa, is native to American soils. Arch Microbiol 178:301-305 45. Zahran HH (1999) Rhizobium–legume symbiosis and nitrogen fixation under severe conditions and in arid climate. Microbiol Mol Biol Rev 63:968-989 46. Zahran HH (2009) Enhancement of rhizobia-legumes symbioses and nitrogen fixation for crops productivity improvement. In: Khan MS, Zaidi A, Musarrat J (eds) Microbial strategies for crop improvement.

Springer-Verlag, Berlin, pp. 227-254 47. Zahran HH, Abdel-Fattah M, Yasser MM Mahmoud AM, Bedmar EJ (2012) Diversity and environmental stress responses of rhizobial bacteria from Egyptian grain legumes. Aust J Basic Appl Sci 6:571-583 48. Zhou PF, Chen WM, Wei GH (2010) Mesorhizobium robiniae sp. nov., isolated from root nodules of Robinia pseudoacacia. Int J Syst Evol Microbiol 60:2552-255

RESEARCH ARTICLE International Microbiology (2013) 16:165-176 doi: 10.2436/20.1501.01.191 ISSN 1139-6709 www.im.microbios.org

Characterization of the gene cluster involved in allantoate catabolism and its transcriptional regulation by the RpiR-type repressor HpxU in Klebsiella pneumoniae Karla Guzmán, Evangelina Campos, Laura Aguilera, Lorena Toloza, Rosa Giménez, Juan Aguilar, Laura Baldoma, Josefa Badia* Department of Biochemistry and Molecular Biology, Institute of Biomedicine, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain Received 30 July 2013 · Accepted 25 September 2013

Summary. Bacteria, fungi, and plants have metabolic pathways for the utilization of nitrogen present in purine bases. In Klebsiella pneumoniae, the genes responsible for the assimilation of purine ring nitrogen are distributed in three separated clusters. We characterized the gene cluster involved in the metabolism of allantoate (genes KPN_01761 to KPN_01771). The functional assignments of HpxK, as an allantoate amidohydrolase, and of HpxU, as a regulator involved in the control of allantoate metabolism, were assessed experimentally. Gene hpxU encodes a repressor of the RpiR family that mediates the regulation of this system by allantoate. In this study, the binding of HpxU to the hpxF promoter and to the hpxU-hpxW intergenic region containing the divergent promoter for these genes was evidenced by electrophoretic mobility shift assays. Allantoate released the HpxU repressor from its target operators whereas other purine intermediate metabolites, such as allantoin and oxamate, failed to induce complex dissociation. Sequence alignment of the four HpxU identified operators identified TGAA-N8-TTCA as the consensus motif recognized by the HpxU repressor. [Int Microbiol 2013; 16(3):165-176] Keywords: Klebsiella pneumoniae · allantoate metabolism · allantoate amidohydrolase · purine catabolism · RpiR-type repressor

Introduction Bacteria, fungi, and plants have metabolic pathways for the utilization of nitrogen present in pyrimidine and purine bases [40,41].

When ammonia is limiting, many microorganisms obtain nitrogen from these organic compounds. Deamination of the purine Corresponding author: J. Badia Departament de Bioquímica i Biologia Molecular Facultat de Farmàcia, Universitat de Barcelona Av. Diagonal, 643 08028 Barcelona, Spain Tel. +34-934034496. Fax +34-934024520 E.mail: josefabadia@ub.edu *

bases adenine and guanine yields one molecule of ammonia, and hypoxanthine and xanthine, respectively. The catabolic pathway for hypoxanthine and xanthine assimilation occurs in two stages.

In the first, both compounds are oxidized to uric acid, which is then converted to allantoate via allantoin by two sequential ringopening steps. In the second, allantoate is degraded to carbon dioxide and ammonia through alternative metabolic routes depending on the organism [10,35,40,41].

In enterobacteria, purine catabolism has been well characterized in Escherichia coli and some species of Klebsiella. In the presence of oxygen, the adenine catabolic pathway is incomplete in E. coli [10,32,44] whereas Klebsiella can assimilate all adenine nitrogens under these conditions [28,33].

166

Int. Microbiol. Vol. 16, 2013

In K. oxytoca M5a1, a cluster of 23 genes responsible for the utilization of purines as sole nitrogen source was identified [28]. Functional assignments of some genes of this cluster were based on growth, complementation tests, and sequence similarity. Comparison with the K. pneumoniae MGH78578 genome revealed that, at least in this strain, these genes are organized in three separate clusters [28], two of which have been studied in detail in K. pneumoniae strain KC2653 [15,33].

The first cluster (KPN_01661 to KPN_01666), in­volved in the oxidation of hypoxanthine to allantoin, is formed by seven genes organized in four transcriptional units, hpxDE, hpxR, hpxO, and hpxPQT [33]. Expression of this system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. The induction of hpxPQT requires uric acid formation, whereas the expression of hpxDE is induced by hypoxanthine through the HpxR regulatory protein encoded by hpxR [33].

The second cluster (KPN_01787 to KPN_01791) is organized in three transcriptional units, hpxSAB, hpxC, and guaD. It contains genes involved in the metabolism of allantoin to allantoate and in guanine deamination. Gene hpxS encodes a regulatory protein that mediates regulation of the hpxSAB operon by allantoin, although full induction of hpxSAB by allantoin requires both HpxS and the nitrogen assimilation control protein (NAC), which regulates a subset of genes that are dependent on RNA polymerase bearing s70 for their transcription [2]. The expression of guaD is mainly regulated by nitrogen availability through the action of NtrC [15].

Regarding the third gene cluster (KPN_01761 to KPN_01771) presumably involved in allantoate catabolism, there have been no studies dealing with its transcriptional regulation, and functional assignment has only been reported for gene KPN_01762 (hpxJ). This gene encodes a protein that belongs to the pyridoxal 5′-phosphate (PLP)-dependent aspartate aminotransferase superfamily, specifically, an aminotransferase that preferentially converts ureidoglycine plus an α-keto acid into oxalurate plus the corresponding amino acid [12]. The gene encoding this aminotransferase is also related to the allantoate amidohydrolase genes in several organisms [42,43]. For instance, a gene related to alanine-glyoxylate aminotransferase (pucG) has been reported in the purine degradation cluster of Bacillus subtilis. Bacillus species are known to have an efficient metabolic system for the use of oxidized purines. This activity seems to enable soil and gut bacteria to use animal-produced purine waste as a source of carbon and nitrogen [30].

In this study, we were able to functionally identify the hpxK gene product in K. pneumoniae strain KC2653 as an al-

guzmán et al.

lantoate amidohydrolase. This gene belongs to the hpxFGHI­ JK operon (KPN_01766 to KPN_01761), which is located next to the regulatory gene hpxU (KPN_01767) and genes predicted to be involved in oxalurate metabolism (KPN_01768 -KPN_01771; hpxWYXZ). Analysis of the transcriptional regulation of this cluster by HpxU is also presented.

Materials and methods Bacterial strains and plasmids. The genotypes and sources of the bacterial strains, plasmids, and promoter fusions are given in Table 1. All K. pneumoniae strains were derived from strain W70 [23].

Growth conditions and preparation of cell extracts. Cultures were grown at 30 ºC with aeration in Luria broth (LB) [6] or in W4 minimal medium [38] supplemented with glucose at 0.4 % as the sole carbon source.

For nitrogen-limiting conditions, freshly prepared glutamine was used at 0.04­­ %. Ammonium sulfate and glutamine, both at 0.2 %, were used to achieve nitrogen excess [4,5]. Allantoin, allantoate, ureidoglycolate, and oxamate were usually used at 0.05 %. When required, the following antibiotics were added to the indicated final concentrations: ampicillin (Ap), 100 mg/ml; kanamycin (Km), 50 mg/ml; streptomycin (Sm), 50 mg/ml; chloramphenicol (Cm), 20 mg/ml; rifampicin (Rf), 50 mg/ml; and tetracycline (Tet), 12.5 mg/ml.

5-Bromo-4-chloro-3-indolyl b-d-galactoside (X-Gal) and isopropyl-b-dthio­galactoside (IPTG) were used at 30 and 10 mg/ml, respectively.

Cell extracts were obtained by sonic disruption of bacterial cells collected by centrifugation at the end of the exponential phase and resuspended in the appropriate buffer.

Enzyme activities. For b-galactosidase assays, the cultures were grown to an OD600 of 0.5. The cells were collected by centrifugation, washed in 1 % KCl, and suspended at a concentration that contained 1–1.5 mg of protein per ml [1]. b-Galactosidase activity was assayed in detergent-treated whole cells using o-nitrophenyl-b-d-galactopyranoside (ONPG) as the substrate and expressed as U/mg of cell protein [25]. One unit of b-galactosidase activity corresponds to the amount of enzyme that hydrolyzes 1 nmol of ONPG per min. The data reported are the averages of at least four separate experiments performed in triplicate.

Allantoate amidohydrolase activity was assayed spectrophotometrically as described by Muratsubaki et al. [26]. The reaction mixture consisted of 100 mM Tris–HCl buffer (pH 8.5), 0.1 mM MnCl2, 0.3 mM NADPH, 0.25 mM α-ketoglutarate, 2.5 mM allantoate, and yeast glutamate dehydrogenase (1 kU/ml). The reaction was started by the addition of the enzyme and activity was assayed by monitoring the decrease in absorbance at 340 nm induced by NADPH oxidation. One unit of enzyme activity was defined as the amount of enzyme that transforms 1 μmol of ammonia per min. Protein concentration was quantified by the method of Lowry et al. [22], with bovine serum albumin as the standard.

DNA manipulation and site-directed mutagenesis. Bacterial genomic DNA was obtained using the Wizard Genomic DNA purification kit (Promega). Plasmid DNA was prepared using the Wizard Plus SV Midipreps DNA purification system (Promega). DNA manipulations were performed essentially as described by Sambrook and Russell [34]. DNA fragments were PCR-amplified using chromosomal DNA as template. When necessary, specific restriction sites were incorporated at the 5′-ends of the primers to facilitate cloning of the fragments into the appropriate vector. PCRs were performed with pfu DNA polymerase under standard conditions. DNA was se-

Int. Microbiol. Vol. 16, 2013

Repressor HpxU in K. pneumoniae

167

Table 1. Strains, fusions and plasmids used in this study Strain or plasmid

Relevant characteristics

Source/reference

K. pneumoniae strains KC2653

hutC515 Δ[bla]-2 dadA1str-6

[21]

KC2738

hutC515 ntrC2::Tn5-131

[3]

JB101

KC2653 hpxK::cat

This study

DH5aF′

f80d lacZΔM15 recA1 endA1 λ– gyrA96 thi-1 hsdR17 (rK– mK+) phoA supE44 relA1 deoR Δ(lacZYA-argF) U169

Gibco BRL

XL1-Blue

recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacIqZΔM15 Tn10(Tcr)]

Stratagene

S17 λ pir

Tpr Smr recA thi pro hsdR- M+RP4::2-Tc::Mu::Km Tn7 λ

Biomedal

EB6193

RP4-2 tet::Mu -1 Kan::Tn7 integrant leu-63::IS10 recA1 creC510 hsdR17 endA1 zbf-5 uidA(ΔMuI):pir+ thi SpR/SmR

RA Bender

E. coli strains

Fusionsa f(hpxF-lacZ)

hpxF (–180 to +140) fused to lacZ

This study

f(hpxU-lacZ)

hpxR (–195 to +105) fused to lacZ

This study

f(hpxW-lacZ)

The same fragment as for hpxU but cloned in the opposite direction

This study

Plasmids pGEMT

Apr; cloning vector for PCR products

Promega

pCAT19

Apr Tn9-CAT(Cmr)

[14]

pUC18Not

Apr; identical to pUC18 but with NotI in the multiple-cloning site

Biomedal

pRS415

Ap ; promoterless lacZYA reporter for operon fusions with replication origin of pBR322

[37]

pUTmini-Tn5 Tc

Apr oriR6K mobRP4 tnp* mini-Tn5 Tc

Biomedal

pCB1583

Apr Kmr; promoterless lacZ reporter for integration of operon fusions on host genome with oriR6K replication origin; rpsL

RA Bander

pMAL-c2x

Apr; vector for cytoplasmic expression of MBP fusion proteins

r

N.E. BioLabs

a Nucleotide sequences are given in the 5′- to 3′- direction for the coding strand of each gene and numbered relative to the gene transcription initiation nucleotide at position +1.

quenced using an automated ABI377 DNA sequencer and fluorescent dye termination methods. The primers used in this work are available upon request.

To construct an HpxK-defective mutant, a fragment encompassing hpxK was PCR-amplified and cloned into pUC18Not. The resultant recombinant plasmid was then digested with PsyI (with a single restriction site in hpxK) and ligated with a Cm cassette amplified from plasmid pCAT19 [14]. The recombinant plasmid, pUC18-hpxK::cat, was then digested with NotI to obtain the hpxK::cat insert. This fragment was cloned into the NotI restriction site of pUTmini-Tn5Tc and introduced into E. coli S17-1(pir) by electroporation. To introduce the hpxK::cat mutation into the K. pneumoniae chromosome, conjugation was performed with a Rf-resistant derivative of strain KC2653 as a recipient. Transconjugants displaying the Rfr Cmr Tcs phenotype were selected. Chromosomal insertion was confirmed by PCR. Mapping of the 5´-end of the transcripts. The 5′-end of the transcripts was determined by the rapid amplification of cDNA 5′-ends (5′-RACE) [34] using a commercial 5′-RACE kit (Roche Diagnostics). Total RNA was isolated from KC2653 cells grown aerobically to an OD600 between 0.5 and 1 in glucose minimal medium with Gln 0.04 % as nitrogen source (nitrogen-

limiting conditions) using the Qiagen RNeasy total RNA kit and then treated with RNAse-free DNase (Ambion). The cDNAs were transcribed from RNA with specific hpxU, hpxFGHIJK, or hpxWXYZ antisense oligonucleotides. A homopolymeric(dA) tail was added (via terminal transferase) to the 3′-termini of the corresponding cDNAs. The reverse transcription products were amplified with nested gene-specific primers and an oligo(dT) anchor primer.

The double-stranded cDNAs obtained were cloned into pGEMT vectors for sequencing.

Construction of lacZ transcriptional fusions. Transcriptional fusions were constructed by inserting the promoter fragments into plasmid pRS415 [37], which carries a cryptic lacZ operon and confers resistance to ampicillin. To construct the hpxF-lacZ fusion, a 320-bp fragment encompassing the hpxF 5′-region (positions –180 to +140) was PCR-amplified. The hpxW-lacZ fusion was constructed by PCR-amplifying a 300-bp fragment encompassing the hpxU-hpxW intergenic region (positions –195 to +105 with respect to the hpxU transcriptional start site). The same fragment was cloned in the opposite direction to obtain the hpxU-lacZ fusion. In all cases, primers bearing BamHI or EcoRI sites at their 5′-ends were used to facilitate cloning

168

Int. Microbiol. Vol. 16, 2013

of the fragment into the EcoRI–BamHI sites of plasmid pRS415. For all constructs, plasmid DNA was sequenced to ensure that the fragment was inserted in the correct orientation and that no mutations were introduced during the amplification.

To transfer the lacZ fusions into K. pneumoniae chromosome as a single copy, the recombinant plasmid was first digested with EcoRI and SacI, and the fragment containing the promoter fusion was subcloned into plasmid pCB1583 [21]. In this λpir-dependent plasmid the lacZ gene is flanked by genes of the K. pneumoniae d-ribose operon, thus allowing integration of the cloned fusion by homologous recombination into the d-ribose operon of the recipient strain. After the transformation of strain EB6193, recombinant plasmids containing f(hpxF-lacZ), f(hpxU-lacZ), and f(hpxW-lacZ) were selected as blue bacterial colonies on LB-Xgal-Km plates and then introduced into strain KC2653 by electroporation. After several rounds of selection in different growth media, stable recombinants were isolated as those displaying a KmS SmR d-ribose negative phenotype.

achieved in strain XL1-Blue incubated at 37 ºC for 16 h, in the presence of 0.3 mM IPTG. The fusion proteins were then affinity-purified chromatographed with amylose resin (New England BioLabs) as described previously [15]. The MalE-HpxK fusion protein was eluted with column buffer containing 10 mM maltose and then digested with factor Xa by incubation at room temperature for 12 h. However, MalE-HpxU folding hindered cleavage of the fusion protein in solution. Thus, instead, recombinant MalE-HpxU was used in gel shift experiments, since the MalE protein was unable to bind to the analyzed promoter regions (not shown). Purified proteins were analyzed by SDS–PAGE, performed according to the standard procedure [19], and then either used immediately or stored at –20 °C in 20 % glycerol.

DNA binding studies. The non-radioactive digoxigenin (DIG) gel shift kit for the 3′-end labeling of DNA fragments (Roche Applied Science, Indianapolis, IN) was used for protein-DNA binding assays. The fragments obtained by PCR were labeled with terminal transferase and digoxigeninddUTP according to the manufacturer’s instructions. Labeled DNA fragments were incubated with purified HpxU in 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 10 mM MgCl2, 10 % glycerol, and 2 mM dithiothreitol in a total volume of 20 ml. All mixtures contained 500-fold molar excesses of poly(dI-dC) as non-specific competitor. HpxU binding mixtures were incubated at 37 ºC for

Int Microbiol

Expression and purification of recombinant proteins. HpxU and HpxK were purified using the malE gene fusion system. For this purpose, the corresponding genes (hpxU and hpxK) were PCR-amplified and cloned into plasmid pMal-c2x. The overproduction of MalE-fused proteins was

guzmán et al.

Fig. 1. Gene organization and metabolic map for allantoate catabolism in K. pneumoniae strain KC2653. (A) Scheme of the gene organization in cluster KPN_01761 to KPN_01771. The arrows indicate the extent and direction of the transcription of these genes. The predicted function of the hpx-encoded proteins is indicated below each gene. (B) Metabolic pathway for allantoate metabolism. Metabolites and proteins with putative functions are in gray.

Repressor HpxU in K. pneumoniae

20 min, and loaded onto a pre-run gel of 5 % native polyacrylamide, containing 10 % glycerol in 1× TBE (Tris-borate-EDTA buffer). Blotting was performed using a Biorad electro-blotting system (model Trans blot) according to the manufacturer’s instructions. The chemiluminescence of DIG-labeled DNA-protein complexes on the nylon membranes was detected using Hyperfilm ECL (Amersham Pharmacia).

Results and Discussion Physical organization of the hpxFGHIJK-hpxUhpxWYXZ cluster. The gene cluster encompassing genes KPN_01761 to KPN_1771 in K. pneumoniae has been proposed to encode proteins involved in allantoate metabolism [28], namely, genes KPN_01761 to KPN_01766 (hpxFGHI­ JK) in the conversion of allantoate to oxalurate, genes KPN_01768 to KPN_01771 (hpxWXYZ) in oxalurate metabolism, and gene KPN_01769 (hpxU) in the regulation of this cluster (Fig. 1). To determine whether these genes were also present in strain KC2653, the corresponding region was PCRamplified using primers designed according to the genome sequence of K. pneu­moniae strain MGH78578. This analysis confirmed the presence of these genes (KPN_01768 to KPN_01771) in the KC2653 genome.

Information available in databases suggested that this gene cluster is present in different strains of the genus Klebsi­ ella. However, no experimental evidence for the transcriptional regulation of these genes is currently available. We therefore performed an in silico analysis of the hpxF 5′-flanking region and the hpxU-hpxW intergenic region using the Footprint and Promscan programs [http://www.promscan.uklinux. net]. Putative s70-promoters were identified upstream of hpxF, hpxU, and hpxW. The 5′-ends of the hpxFGHIJK, hpxU, and hpxWXYZ trans­cripts were experimentally determined by the 5′-RACE method. Total RNA was obtained from aerobic cultures of strain KC2653 on glucose-glutamine (nitrogen-limiting conditions). For hpxFGHIJK, the transcriptional start site (tss) was identified at 89 nucleotides upstream of the hpxF start codon. Inspection of the sequences upstream of nucleotide +1 revealed the presence of –10 (tttaAt) and –35 (TTGTCC) sequences, similar to the s70 consensus sequence, separated by 15 bp (positions matching the consensus are underlined) (Fig. 2). For hpxU, the tss was identified 50 nucleotides upstream of the ATG codon. Inspection of the sequences upstream of nucleotide +1 revealed the presence of –10 (TAAGGC) and –35 (TTCCCA) sequences similar to the s70 consensus and separated by 15 bp (Fig. 2). Although these spacers do not fit

Int. Microbiol. Vol. 16, 2013

169

the optimal 17 ± 1 nucleotides, functional spacers ranging in size from 15 to 21 bp have been reported previously [16,17].

For hpxWXYZ, the tss was identified 20 bp upstream of the start codon for hpxW. In this case, sequences similar to σ70 RNA polymerase consensus were found at –10 (TGTTCT) and at –35 (TTCAGA), separated by 18 bp (Fig. 2).

Functional assignment of HpxK as an allantoate amidohydrolase. Genes involved in the conversion of allantoate to oxalurate are clustered together, forming the hpxFGHIJK operon. Sequence analysis by PBLAST showed that HpxK displays similarity to allantoate amidohydrolases.

HpxK is 34 % identical and 49 % similar to E. coli AllC [10,36], and 40 % identical and 55 % similar to Pseudomonas fluorescens PuuE [29]. In AllC, residues involved in allantoate binding are conserved in the K. pneumoniae HpxK sequence (His219, Asn268, Arg281, and His379). This finding suggested that HpxK catalyzes the conversion of allantoate to ureidoglycine (Fig. 1B). The involvement of HpxK in allantoate metabolism was assessed by phenotypic characterization of strain JB101, a KC2653 derivative hpxK::cat knockout mutant (Table 1). This strain was unable to grow on allantoate as a nitrogen source but displayed normal growth on ureidoglycollate or oxamate.

To confirm HpxK function, the hpxK gene of strain KC2653 was cloned into plasmid pMal-c2X, and the protein was purified by affinity chromatography. Enzyme activity of the purified enzyme was assayed using allantoate as substrate.

Since allantoate amidohydrolase from various sources is a Mn2+-dependent enzyme [43], we measured the allantoate amidohydrolase activity of HpxK in the absence or presence of the divalent cations Mn2+, Ca2+, Mg2+, Cu2+, Zn2+, and Co2+ (at final concentrations of 0–0.2 mM). Maximum activity was obtained with Mn2+ at 0.1 mM (100 % activity). At this concentration, the other divalent cations yielded significantly lower activity: Co2+ (31 %), Ca2+ (9 %), Mg2+, and Cu2+ (<1 %), with no activity occurring in the presence of Zn2+. In the absence of divalent cations, the activity was around 10 % of that obtained with Mn2+. Therefore, Mg2+, Cu2+, and Zn2+ inhibited enzyme activity. The kinetic parameters for allantoate were determined in the presence of 0.1 mM MnCl2 from the double reciprocal Lineweaver-Burk plot. This analysis yielded a Km of 0.105 mM and a Vmax of 0.042 μmol/min/mg.

The ureidoglycine produced by the action of HpxK on allantoate is further metabolized by HpxJ. The functional assignment of K. pneumoniae HpxJ was previously reported by French and Ealick [12]. The protein acts as an aminotransferase catalyzing the conversion of ureidoglycine to oxalurate,

Int. Microbiol. Vol. 16, 2013

guzmán et al.

Int Microbiol

170

Fig. 2. Promoter sequences of hpxF, hpxU and hpxW genes. For each gene, the ATG initiation codon is boxed, the consensus sequence for RNA polymerase (–10 and –35 sequences) is indicated, and the transcriptional start site (tss) is shown by a black arrowhead labeled +1. Binding sites recognized by HpxU are boxed in gray. IRF, IRF1, IRF2, IRU, and IRW inverted repeats are indicated by arrows below the corresponding sequence.

coupled to the conversion of an α-keto acid to the correspond­ ing amino acid (Fig. 1B). Aminotransferases are rather promiscuous with respect to their amino donors and acceptors.

For example, HpxJ displays specificity for ureidoglycine as a donor but can use different substrates as acceptors, including pyruvate, glyoxylate, and oxaloacetate [12]. Glyoxylate could be provided by the action of enzymes encoded by genes belonging to the hpxWXYZ operon. PBLAST analysis showed that HpxY shared 85 % similarity with amidohydrolases belonging to the AtzE family [7]. The best protein characterized in the Atz family was biuret hydrolase from Pseudomonas sp.

[24], which catalyzes the hydrolysis of biuret to allophanate plus ammonium in the atrazine catabolic pathway [9]. Since biuret is structurally related to oxalurate, we hypothesize that

HpxY would be involved in the conversion of oxalurate to ammonium plus 2-carboxyamino-2-oxoacetate. In fact, K. oxy­toca HpxY has been classified as a putative oxalurate amidohydrolase (information available from GenBank, reference EU884423).

Information provided in this entry suggest that K. oxy­toca HpxW is a putative oxamate amidohydrolase able to catalyze the hydrolysis of oxamate to ammonium plus glyoxylate. This reaction would provide the glyoxylate used in the transamination reaction catalyzed by HpxJ [12], thereby avoiding the toxicity due to glyoxylate accumulation [27]. A similar mech­ anism of detoxification was proposed for the catabolism of purines in Bacillus subtilis [30].

As discussed above, allantoate is endogenously generated from purine metabolism. The presence of genes encoding

Repressor HpxU in K. pneumoniae

transport proteins in the hpxFGHIJK operon suggested that allantoate is also used as an extracellular substrate. An in silico analysis of the hpxFGHI gene products evidenced the simi­ larity of these proteins to proteins of the histidine and glutamine ABC transport systems [8], which suggests that they are components of the allantoate uptake system.

171

promoter constructs in strain KC2738 vs. the wild-type strain (not shown). These results ruled out the involvement of the Ntr system in the transcriptional regulation of this gene cluster.

Moreover, chromatin immnoprecipitation studies have not identified these genes as NAC targets in K. pneumoniae [13].

The expressions of f(hpxF-lacZ), f(hpxW-lacZ), and f(hpxUlacZ) were also analyzed in nitrogen-limiting medium in the presence of either allantoin or downstream pathway metabolites such as allantoate or oxamate. The expression of f-(hpxF-lacZ) was induced 13-fold by allantoin or allantoate, whereas no induction was obtained with oxamate (Fig. 3). Since allantoin metabolism generates allantoate, this latter metabolite may be the effector molecule needed for hpxFGHIJK induction, in agreement with the proposed role of this operon in allantoate dissimilation. Regarding hpxU expression, a 40–50 % increase in b-galactosidase activity in the presence of allantoin or allantoate but not with oxamate was observed, indicating that this transcriptional unit was also regulated by the presence of allantoate (Fig. 3). Assays of hpxW showed that the addition of allantoin or allantoate to nitrogen- limiting cultures did not significantly modify b-galactosidase expression, but a 50 % increase in b-galactosidase activity was achieved in the presence of oxamate (Fig. 3). Induction by oxamate is compatible with the involvement of hpxWXYZ in the metabolism of this compound.

Int Microbiol

Transcriptional regulation of the hpxFGHIJKhpxU-hpxWYXZ unit by nitrogen availability and pathway metabolites. To determine whether the expression of this gene cluster is regulated by nitrogen availability, the expression of the promoter fusions f(hpxF-lacZ), f(hpxW-lacZ), and f(hpxU-lacZ) was analyzed against the genetic background of strain KC2653 grown in glucose-minimal medium with nitrogen excess or nitrogen limitation. As shown in Fig. 3, nitrogen limitation resulted in weak repression of HpxU expression and weak activation of HpxW expression, suggesting that the transcription of these genes is somewhat regulated by nitrogen. To study this regulation, the expression of the three promoter fusions was analyzed in the genetic background of the ntrC mutant strain KC2738 [3]. NtrC activates the transcription of another transcriptional regulator, NAC, described in Introduction [2]. No changes in the b-galactosidase expression pattern were observed for any of these

Int. Microbiol. Vol. 16, 2013

Fig. 3. Expression analysis of f(hpxF-lacZ), f(hpxU-lacZ), and f(hpxW-lacZ). Cells of strain KC2653 bearing the indicated promoter fusions were grown in medium containing excess nitrogen (indicated as NH4+), with 0.4 % glucose and 0.2 % each of ammonium sulfate and glutamine, or in a nitrogen-limiting medium (indicated by a minus sign) supplemented with 0.4 % glucose and 0.04 % glutamine. Where indicated, 0.05 % allantoin (All), allantoate (Alt), or oxamate (Oxm) was added to the nitrogen-limiting medium.

Int. Microbiol. Vol. 16, 2013

guzmĂĄn et al.

Int Microbiol

172

Fig. 4. Binding of HpxU to promoter fragments of the hpxF promoter region. (A) Diagram of the hpxF promoter region showing the tss. The promoter fragments used as probes and their end terminus positions with respect to position +1 of hpxF are shown below. The HpxU binding site is indicated by a gray box and the corresponding inverted repeat sequence (IRF) is shown above the diagram. The underlined bases indicate the nucleotides conserved in both halves of the palindromic sequence. The two palindromic sequences, IRF1 and IRF2, identified inside the IRF are also shown and delimited by arrows. (B) EMSA performed with probe P320 in the absence or presence of allantoin (All), allantoate (Alt), or oxamate (Oxm) as a putative effector molecule (EM). (C) EMSA performed with probe P320 in the presence of increasing concentrations of allantoate. (D) EMSA performed with the indicated probes, corresponding to the hpxF promoter fragments, in the absence or presence of 300 ÂľM allantoate.

Analysis of HpxU binding to promoter regions of hpxFGHIJK-hpxU-hpxWXYZ cluster. PBLAST analysis of HpxU showed that this protein has high sequence identity with transcriptional regulators of the RpiR/AlsA family.

Analysis of the 279 amino acid residues of HpxU revealed that it had the helix-turn-helix domain of the RpiR family between

residues 2 and 115. In addition, proteins of the RpiR family have a C-terminal SIS domain (sugar isomerase domain, INTERPRO:IPR001347). In HpxU, the SIS domain is located between residues 123 and 250. Some members of this family (RpiR, IolR, and GlvR) control sugar phosphate metabolic pathways [11,18,39,45], although genes encoding Rpi-type

Repressor HpxU in K. pneumoniae

173

is consistent with the proposed role for HpxU in the regula­ tion of the hpxFGHIJK operon. Binding experiments performed in the presence of allantoin, allantoate, or oxamate showed that only allantoate abolished HpxU binding to probe P320 (Fig. 4B). We next incubated HpxU (0.3 mg) with probe P320 in the presence of increasing concentrations of allan­ toate. The amount of free probe increased with the allantoate

Int Microbiol

regulators have also been identified in gene clusters involved in the metabolism of nitrogenous compounds, such as agmatine [20]. Our analysis suggested that HpxU acts as a transcriptional regulator of genes of the allantoate metabolic pathway.

Binding of HpxU to the 5′-region of hpxFGHIJK was analyzed by electrophoretic mobility shift assays (EMSA). As seen in Fig. 4B, the binding of HpxU to probe P320 (Fig. 4A)

Int. Microbiol. Vol. 16, 2013

Fig. 5. Binding of HpxU to promoter fragments of the hpxU-hpxW intergenic region. (A) Diagram of the hpxU-hpxW intergenic region showing the tss for each gene. The promoter fragments used as probes and their end terminus positions with respect to position +1 of hpxU are shown below. The sequence of the two inverted repeats, IRU and IRW, are indicated above the diagram. The conserved residues in each inverted repeat are underlined. (B) EMSA performed with probe P300 in the absence or presence of allantoin (All), allantoate (Alt), and oxamate (Oxm) as putative effector molecules (EM). (C) EMSA performed with the indicated probes in the absence or presence of 300 mM allantoate. (D) Alignment of the palindromic sequences recognized by HpxU, identified in this study. The derived consensus sequence is indicated below. Nucleotides conserved in at least three of these sequences are indicated by capital letters, and those conserved in all four operators by asterisks.

174

Int. Microbiol. Vol. 16, 2013

concentration and complete dissociation of the HpxU-DNA complexes was obtained at 0.3 mM allantoate (Fig. 4C).

Over­all, these results suggested that allantoate was the effector molecule required to release the HpxU-mediated repression of the hpxFGHIJK operon.

To precisely locate the HpxU binding site, the hpxF promoter region was split into several fragments (Fig. 4A) that were tested as probes in subsequent experiments. HpxU-DNA complexes were detected with probes P149 and P70 but not with probe P150 or P140 (Fig. 4D). These studies allowed us to locate the HpxU binding site between positions +15 and +84 with respect to the hpxF tss. An in silico analysis of this region showed the presence of an inverted repeat (5′-TGAAACAAAAGATTCTTTGAATCTGTTTTTGCA-3′) between positions +35 and +67, which we refer to as the IRF (inverted repeat for hpxF) (Figs. 2 and 4A). To analyze whether IRF was the recognition motif for HpxU, two additional probes, P92 and P249, were designed, each containing only half of the IRF palindromic sequence. EMSA studies showed HpxU binding to both probes although only one protein-DNA complex was visible (Fig. 4D). More detailed inspection of these sequences revealed that each half of the IRF palindrome was itself an inverted repeat (IRF1 and IRF2), which in turn suggested that both IRF1 and IRF2 acted as HpxU binding sites.

Analysis of the IRF1 and IRF2 operator sequences identified TGAA-N8-TTCT and TGAA-N8-TGCA as the putative sites recognized by HpxU in the hpxF promoter. These sequences display similarity with the operator sites recognized by other RpiR-like regulators [18].

Binding analysis of HpxU to the hpxU-hpxW intergenic region was also undertaken. Computational analysis of this region allowed us to identify two palindromic sequences as putative HpxU binding sites, IRU (5′-TGAAACGAAA GTTACA-3′), located at positions +43 to +28 with respect to the hpxU tss, and IRW (5′-TGAACGTTTCGTTTCA-3′), at positions -51 to -35 with respect to the the putative hpxW tss (Fig. 2). These positions are in agreement with the repressor func­ tion proposed for HpxU. EMSA experiments performed with different probes were consistent with the binding of HpxU to probe P180, encompassing IRU and IRW, but not to probes P135 and P120, which lacked these sequences (Fig. 5). HpxU binding studies to P150 (containing only IRW) and P165 (containing only IRU) confirmed the functionality of both proposed HpxU binding sites. As expected, allantoate abolished HpxU binding to IRW and IRU sites. These sites were separated by 84 nucleotides, corresponding to approximately eight turns in the DNA helix. Interactions between HpxU proteins simultaneously bound to IRU and IRW might lead to the

guzmán et al.

formation of a DNA loop responsible for the transcriptional repression of each transcriptional unit.

Sequence alignment of the four proposed HpxU operators (IRF1, IRF2, IRW, IRU) revealed a high degree of identity and a common inverted repeat consensus sequence, TGAAN8-TTCA (Fig. 5D), which most likely represented the HpxU binding motif (underlined nucleotides are conserved in all four operators). The right arm of this motif was less conserved and might determine the HpxU binding affinity of these sites.

Model for the coordinated regulation of the three clusters involved in the purine catabolic pathway. As noted above, the genes required for purine nitrogen assimilation are distributed in three separate clusters in the K. pneumoniae genome. Two clusters had been previously studied by our group [15,33]. In those studies, genes of the purine pathway are shown to be co-ordinately regulated through sequential transcriptional activation of gene subsets by upstream metabolic pathway intermediates. The first cluster (KPN_01661 to KPN_01666) comprises genes involved in hypoxanthine oxidation to allantoin. Genes encoding the hypoxanthine dehydrogenase subunits (hpxDE) are induced by hypoxanthine through the activator protein HpxR, encoded in the same cluster. Uric acid, the product of hypoxanthine oxidation, is required for induction of the hpxPQT operon encoding the enzymes involved in the conversion of uric acid to allantoin [33]. Allantoin itself is in turn required for the induction of the hpxSAB operon, located in the second cluster (KPN_01787 to KPN_01791), in which the gene hpxB encodes allantoinase. Full induction of hpxSAB by allantoin requires the specific regulator HpxS and the global nitrogen regulatory protein NAC [15]. Lastly, the results presented here show that allantoate, the product of allantoinase activity, is required for the induction of genes in the third cluster (KPN_01761 to KPN_01771), which in turn triggers the induction of the hpxFGHIJK operon, involved in the conversion of allantoate to oxalurate. The finding that the addition of allantoate to glucose-glutamine cultures of strain KC2653 bear­ing f(hpxW-lacZ) did not yield any increase in b-galactosidase activity (Fig. 3) suggested that additional signals were needed for hpxWXYZ expression. Since each gene cluster encodes specific transport systems and regulatory proteins that recognize the cognate substrates, our findings were consistent with an integrated genetic system for the uptake and assimilation of purine-derived compounds present in the environment.

The assimilation pathway of purines as a nitrogen source is also transcriptionally regulated by nitrogen availability through different mechanisms. Genes hpxPQT (in the first

Repressor HpxU in K. pneumoniae

cluster) and guaD (in the second cluster), both with a s54-dependent promoter, are activated by the global regulatory protein NtrC. In genes with s70-dependent promoters, only hpxDE (in the first cluster) and hpxSAB (in the second cluster) are activated under nitrogen-limiting conditions. This nitrogen regulation is mediated by NAC in the case of the hpxSAB operon, whereas for hpxDE an as yet unidentified repression mechanism that is involved under nitrogen excess conditions has been proposed [15,33]. Transcriptional control by nitrogen is expected for genes involved in the catabolism of compounds that can be used as nitrogen sources [13,31]. Our results suggest that the genes involved in allantoate catabolism (third cluster) would not be significantly regulated by nitrogen availability, in accordance with additional roles for allantoate or its derived metabolites besides facilitating the supply of nitrogen. In the metabolic context of this bacterial species, this cluster might contribute to detoxifying intracellular glyoxy­late or other α-ketoacids through HpxJ aminotransferase activity.

Acknowledgements. This research was supported by grant BFU 201022260-C02-01 from the Ministerio de Educación y Ciencia, Spain, to L. Baldoma. K. Guzmán received a predoctoral fellowship from the Generalitat de Catalunya, Spain. We thank Robert A. Bender for providing plasmid pCB1583.

Competing interests. None declared.

References 1. Baldauf SL, Cardani MA, Bender RA (1988) Regulation of the galactose-inducible lac operon and the histidine utilization operons in pts mutants of Klebsiella aerogenes. J Bacteriol 170:5588-5593 2. Bender RA (2010) A NAC for regulating metabolism: the nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae. J Bacteriol 192:4801-4811 3. Bender RA, Friederich B (1990) Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70. J Bacteriol 172: 7256-7259 4. Bender RA, Janssen KA, Resnick AD, Bluemenberg M, Foor F, Magasanik B (1977) Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol 129:1001-1009 5. Bender RA, Snyder PM, Bueno R, Quinto M, Magasanik B (1983) Nitrogen regulation system of Klebsiella aerogenes: the nac gene. J Bacteriol 156:444-446 6. Bertani G (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186:595-600 7. Cameron SM, Durchschein K, Richman JE, Sadowsky MJ, Wackett LP (2011) A new family of biuret hydrolases involved in s-triazine ring metabolism. ACS Catal 2011:1075-1082 8. Campbell JD, Deol SS, Ashcroft FM, Kerr ID, Sansom MS (2004) Nucleotide-dependent conformational changes in HisP: molecular dynamics simulations of an ABC transporter nucleotide-binding domain. Biophys J 87:3703-3715

Int. Microbiol. Vol. 16, 2013

175

9. Cheng G, Shapir N, Sadowsky MJ, Wackett LP (2005) Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism.

Appl Environ Microbiol 71:4437-4445 10. Cusa E, Obradors N, Baldomà L, Badía J, Aguilar J (1999) Genetic analysis of a chromosomal region containing genes required for assimilation of allantoin nitrogen and linked glyoxylate metabolism in Escherichia coli. J Bacteriol 181:7479-7484 11. Daddaoua A, Krell T, Ramos JL (2009) Regulation of glucose metabolism in Pseudomonas. The phosphorilative branch and Entner–Doudoroff enzymes are regulated by a repressor containing a sugar isomerase domain. J Biol Chem 284:21360-21368 12. French JB, Ealick SE (2010) Biochemical and structural characterization of an ureidoglycine aminotransferase in the Klebsiella pneumoniae uric acid catabolic pathway. Biochemistry 49:5975-5977 13. Frisch RL, Bender RA (2010) Expanded role for the nitrogen assimilation control protein (NAC) in the response of Klebsiella pneumonia to nitrogen stress. J Bacteriol 192:4812-4820 14. Fuqua WC (1992) An improved chloramphenicol resistance gene cassette for site-directed marker replacement mutagenesis. BioTechniques 12: 223-225 15. Guzmán K, Badia J, Giménez R, Aguilar J, Baldoma L (2011) Transcriptional regulation of the gene cluster encoding allantoinase and guanine deaminase in Klebsiella pneumoniae. J Bacteriol 193:2197-2207 16. Hidalgo E, Demple B (1997) Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor. EMBO J 16:1056-1065 17. Huerta AM, Francino MP, Morett E, Collado-Vides J (2006) Selection for unequal densities of σ70 promoter-like signals in different regions of large bacterial genomes. PLOS Genetics 2:1740-1749 18. Kohler PRA, Choong EL, Rossbach S (2011) The RpiR-like repressor IolR regulates inositol catabolism in Sinorhizobium meliloti. J Bacteriol 193: 5155-5163 19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 222:680-685 20. Landette JM, Arena ME, Pardo I, Manca de Nadra MC, Ferrer S (2010) The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase. Int Microbiol 13:169-177 21. Liu Q, Bender RA (2007) Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae. J Bacteriol 189:7593-7599 22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-273 23. MacPhee DG, Sutherland IW, Wilkinson JF (1969) Transduction in Klebsiella. Nature 221:475-476 24. Martinez B, Tomkins J, Wackett LP, Wing R, Sadowsky MJ (2001) Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. J Bacteriol 183: 5684-5697 25. Miller JH (1992) A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 26. Muratsubaki H, Satake K, Enomoto K (2006) Enzymatic assay of allantoin in serum using allantoinase and allantoate amidohydrolase. Anal Biochem 359:161-166 27. Pellicer MT, Fernández C, Badía J, Aguilar J, Lin ECC, Baldomà L (1999) Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection. Characterization of the glc promoter.

J Biol Chem 274:1745-1752 28. Pope SC, Chen LL, Stewart V (2009) Purine utilization by Klebsiella oxy­­toca M5a1: Genes for ring-oxidizing and -opening enzymes. J Bacteriol 191:1006-1017 29. Ramazzina I, Cendron L, Folli C, Berni R, Monteverdi D, Zanotti G, Percudani R (2008) Logical identification of an allantoinase analog

176

Int. Microbiol. Vol. 16, 2013

(puuE) recruited from polysaccharide deacetylases. J Biol Chem 283: 23295-23304 30. Ramazzina I, Costa R, Cendron L, Berni R, Peracchi A, Zanotti G, Percudani R (2010) An aminotransferase branch point connects purine catabolism to amino acid recycling. Nat Chem Biol 6:801-806 31. Reitzer L (2003) Nitrogen assimilation and global regulation in Esche­ richia coli. Annu Rev Microbiol 57:155-176 32. Rintoul MR, Cusa E, Baldomà L, Badia J, Reitzer L, Aguilar J (2002) Regulation of the Escherichia coli allantoin regulon: coordinated function of the repressor AllR and activator AllS. J Mol Biol 324:599-610 33. de la Riva L, Badia J, Aguilar J, Bender RA, Baldoma L (2008) The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: Gene organization and transcriptional regulation. J Bacteriol 190:7892-7903 34. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 35. Schultz AC, Nygaard P, Saxild HH (2001) Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J Bacteriol 183:3293-3302 36. Serventi F, Ramazzina I, Lamberto I, Puggioni V, Gatti R, Percudani R (2010) Chemical basis of nitrogen recovery through the ureide pathway: formation and hydrolysis of S-ureidoglycine in plants and bacteria. ACS Chem Biol 5:203-214 37. Simons RW, Houmanand F (1987) Improved single and multicopy lacbased cloning vectors for protein and operon fusions. Gene 53:85-96

guzmán et al.

38. Smith GR, Halpern YS, Magasanik B (1971) Genetic and metabolic control of enzymes responsible for histidine degradation in Salmonella typhi­murium. J Biol Chem 246:3320-3329 39. Sfrensen KI, Hove-Jensen B (1996) Ribose catabolism of Escherichia coli characterization of the rpiB gene encoding ribose phosphate iso­ merase B and of the rpiR gene, which is involved in regulation of rpiB expression. J Bacteriol 178:1003-1011 40. Todd CD, Tipton PA, Blevins DG, Piedras P, Pineda M, Polacco JC (2006) Update on ureide degradation in legumes. J Exp Bot 57:5-12 41. Vogels GD, Van der Drift C (1976) Degradation of purines and pyri­mi­ dines by microorganisms. Bacteriol Rev 40:403-468 42. Werner AK, Romeis T, Witte CP (2010) Ureide catabolism in Arabidopsis thaliana and Escherichia coli. Nat Chem Biol 6:19-21 43. Werner AK, Sparkes IA, Romeis T, Witte CP (2008) Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean. Plant Physiol 146:418-430 44. Xi H, Schneider BL, Reitzer L (2000) Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage. J Bac­ teriol 182:5332-5341 45. Yamamoto H, Serizawa M, Thompson J, Sekiguchi J (2001) Regulation of the glv operon in Bacillus subtilis: Yfia (GlvR) is a positive regulator of the operon that is repressed trough CcpA and cre. J Bacteriol 183:5110-5121

RESEARCH ARTICLE International Microbiology (2013) 16:177-189 doi: 10.2436/20.1501.01.192 ISSN 1139-6709 www.im.microbios.org

Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation Anna Plasencia,1 Frederic Gich,1* Mireia Fillol,1 Carles M. Borrego1,2 Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Girona, Spain. 2Water Quality and Microbial Diversity, Catalan Institute for Water Research (ICRA), Girona, Spain

1

Received 30 July 2013 · Accepted 30 September 2013 Summary. A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35 % of the SybrGold-stained cells, which corresponded to 6.61 × 106 cells/ml and 1.76 ± 0.09 × 106 archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1.1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = –1.058, P = 0.308 and ANOVA t14 = 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001). [Int Microbiol 2013; 16(3):177-189] Keywords: ammonia-oxidizing archaea and bacteria · ammonia monooxygenase alpha subunit (amoA) · Lake Kivu · microcosm · multi-color CARD-FISH

Introduction Following the pioneering work of Könneke and co-workers with Nitrosopumilus maritimus [24], the ability to grow ammonia-oxidizing archaea (AOA) under laboratory conditions Corresponding author: F. Gich Institute of Aquatic Ecology University of Girona Montilivi, s/n 17071 Girona, Spain Tel. +34-972418261. Fax +34-972418150 E-mail: frederic.gich@udg.cat *

has made rapid progress. The first isolation of an autotrophic nitrifying archaeon not only was considered a milestone in microbial ecology but it also provided clues to the enrichment or isolation of several new mesophilic nitrifying Candidatus (Ca.) species from both aquatic and terrestrial habitats [14,20,25,30,44] and one thermophilic species from a terrestrial hot spring [10,18]. Nitrosopumilus maritimus was initially identified as the first mesophilic member of the kingdom Crenarchaeota to be isolated in pure culture, but further genome-based studies of that species and its nitrifying relatives, i.e., Crenarchaeum symbiosum and “Ca. Nitrososphaera gargensis,” provided robust genetic data to accommodate

178

Int. Microbiol. Vol. 16, 2013

these archaeal nitrifiers within a new archaeal kingdom, the Thaumarchaeota [41]. Thus far, marine species are affiliated with Marine Group 1.1a cluster (with the exception of “Ca. Nitrosoarchaeum koreensis” isolated from soil) whereas archaeal soil nitrifiers belong to either Marine Group 1.1a-associated or to Soil Group 1.1b. The unique thermophilic Thaumarchaeota described to date have been assigned to group ThAOA [17]. Since all cultured members of this new kingdom rely on the oxidation of ammonia for their energy metabolism, this metabolic trait has been considered as a defining characteristic of Thaumarchaeota.

Despite these advances in our knowledge, the cultivation of AOA remains a difficult, time-consuming task and the final isolation of the targeted species in a pure culture is only rarely achieved. Among the alternative strategies to study AOA that have been tested thus far are state-of-the-art genomic techniques [7] and experimentation with highly enriched cultures containing satellite bacterial communities [14,20]. These culture-dependent methods provide highly valuable information allowing the proper interpretation of data obtained by cultureindependent techniques in ecological studies focused on AOA [42]. Physiological characterization and determination of the growth requirements of recently described species are also useful to refine cultivation strategies and thereby improve cultivability. For instance, differences in the ammonia oxidation kinetics of AOA and ammonia-oxidizing bacteria (AOB) not only provide an elegant explanation for their niche segregation but can also be used to selectively enrich AOA in samples containing a heterogeneous bacterial and archaeal nitrifying community [29]. Data from such investigations indicate that archaeal nitrifiers from aquatic habitats are similar in their tolerances of oxygen, ammonia concentrations, and pH range whereas soil representatives have greater variability in relation to optimal pH and their tolerance of high ammonia concentrations (e.g., 15 mM for “Ca. Nitrosoarchaeum koreensis” [20]). These differences are consistent with the higher ammonia concentrations found in soils than in aquatic environments and they suggest specific adaptations for planktonic and terrestrial archaeal nitrifiers [47]. For example, the capacity to use simple organic compounds including pyruvate has been described for “Ca. Nitrososphaera viennensis” [44], and genes involved in organic carbon utilization have been identified in AOA genomes ([7] and references therein).

Recently, we reported the accumulation of AOA in the oxycline of Lake Kivu, as part of a heterogeneous community consisting of members of Marine Group 1.1a and Soil Group 1.1b [28]. The presence of the latter group in the water column is generally thought to be a consequence of water inputs

plasencia et al.

by surface runoff, which are also responsible for the large nutrient loads entering the lake [33,34]. Assuming different growth requirements and physiological constraints for soil and planktonic AOA, the presence of the former in the water column has raised the question whether they are capable of active growth or simply passively accumulate in the lake’s rare biosphere. To answer this question and to identify active members of the archaeal community we established an experimental microcosm using water collected at the oxycline of Lake Kivu. Ammonia concentrations and incubation conditions were manipulated to stimulate AOA growth, which was monitored for almost a year using multi-color catalyzed reporter deposition fluorescent in situ hybridization (CARDFISH) and qPCR [16S rRNA and the alpha subunit of ammonia monooxygenase (amoA) for both AOA and AOB]. The former technique was chosen to avoid potential overestimation of archaeal abundance following the non-specific hybridization of archaeal probe ARC915 to members of the phylum Bacteroidetes. Simultaneous hybridization of the probes ARC915 and CF319a allowed us to identify non-specific targets and to correctly quantify archaeal cells throughout the incubation. Archaeal members of the microbial community growing in the microcosm were identified by means of denaturing gradient gel electrophoresis (DGGE) fingerprinting and clone libraries. In addition, the effect of different organic compounds on archaeal growth was monitored using 96-deep well microtiter plates to elucidate whether some organic compounds stimulate archaeal nitrifiers, as recently demonstrated [44].

Materials and methods Set-up of the experimental microcosm. Lake Kivu is located between Rwanda and the Democratic Republic of the Congo, 1,463 m above sea level. The lake is a deep (maximum depth: 489 m) meromictic and oligotrophic body of water with steeply increasing temperature and salinity gradients. Water samples were collected during a sampling campaign conducted during the rainy season in March 2007. In a previous study, the maximal abundance of AOA in Lake Kivu was detected at the oxic–anoxic interface (30–50 m depth) of the main basins [28]. Accordingly, in this study we initiated an enrichment culture using water collected at a depth of 50 m from the Lake Kiwu Northern Basin (NB50). The experimental set-up consisted of a sterile 1-l Erlenmeyer flask filled with 500 ml of lake water and capped with a cotton wool stopper to ensure oxic conditions. At day 0, the water was supplemented with NH4Cl (1 mM), NaHCO3 (2.7 µM), and KH2PO4 (1.8 mM) to stimulate the growth of the autochthonous nitrifying community. The enrichment culture was maintained for 246 days at 22 ºC in the dark without shaking. Samples were periodically collected to quantify archaeal and bacterial abundances and to monitor changes in microbial community composition (see below). The samples were coded using a standard label (M50) followed by indicating the time in days at which each sample was collected (e.g., M50_246 was collected on day 246).

Enrichment culture cultivation in microtiter plates. To determine whether the AOA present in the enrichment could be further stimulated by the addition of organic compounds, we used a multi-enrichment system consisting of 96-deep-well polystyrene microtiter plates (Corning Inc., Corning, NY, USA) containing different media (Fig. 1). The plate wells were filled with synthetic freshwater mineral medium for nitrifying Crenarchaeota [24] but with the following modifications: NaCl (17.11 mM, final concentration), NH4Cl (1 mM), MgCl2·6H2O (1.97 mM), CaCl2·2H2O (0.68 mM), and KCl (6.71 mM). Once autoclaved, the basal medium was amended with KH2PO4 (0.03 mM), NaHCO3 (17 mM), trace elements solution, and vitamin V7 solution [5]. The medium was supplemented with the following substrate combinations: (i) NH4Cl (15 mM, final concentration), (ii) a mixture of organic acids (acetate, citrate, formate, α-ketoglutarate, propionate, pyruvate and succinate, 200 mM each), (iii) yeast extract (0.001 % w/v [44]), (iv) mineral medium, and (v) the original microcosm enrichment. The last two treatments were considered as controls. Each medium combination (900 ml total volume) was dispensed into six wells in order to analyze several replicates for each treatment (Fig. 1).

For treatments 2, 3, and 4, an additional row of six wells was filled with the same medium amended with an antibiotic mixture (carbenicillin, an inhibitor of cell wall synthesis [100 mg/l]; streptomycin, an inhibitor of ribosomal protein synthesis [100 mg/l]; polymyxin B, a disruptor of the lipid bilayer [20 mg/l]; and triclosan, an inhibitor of fatty acid synthesis [10 mg/l]) to inhibit bacterial growth, with the aim of enriching archaeal cells. The inhibitors were selected among a larger group of diverse antimicrobial agents after several inhibition tests previously carried out against Sulfolobus solfataricus DSM1617, Bacillus subtilis CECT 39, Escherichia coli CECT 831, and Pseudomonas aeruginosa CECT 532 (data not shown). The medium-containing wells were then manually inoculated with 100 µl (10 % inoculum) of the enrichment suspension (M50_246), resulting in a final concentration of 6.6 × 105 cells/ml. Peripheral wells were not inoculated but were instead used as controls for potential contamination during handling. Finally, the plates were covered with a sterile porous adhesive film (Kisker, Steinfurt, Germany) and then incubated at 18 ºC in the dark for two months. The activity of the inhibitor mixture in the enrichment plates throughout incubation was assessed after 7, 30, and 60 days of incubation by susceptibility diffusion tests using 10-µl aliquots withdrawn from the wells and transferred to nutrient agar lawn growth cultures of B. subtilis CECT 39 and P. aeruginosa CECT 532. No substantial decrease in the antimicrobial activity of the mixture against these strains was evident in any of the performed tests (data not shown).

Multi-color CARD-FISH. Archaeal abundance was periodically monitored in the microcosm and measured at the end of the incubation period in microtiter plate cultures using CARD-FISH [36] and the archaea specific probe ARC915 [28]. Samples of 0.5 and 9 ml were taken from three replicate wells of the microtiter plate cultures and from the microcosm, respectively, fixed with paraformaldehyde (final concentration, 2 % [w/v]) overnight at 4 ºC, and filtered onto 25-mm diameter, white, 0.22-µm-pore-size polycarbonate filters (Millipore, Eschborn, Germany). To overcome non-specific binding of the ARC915 probe to members of the Bacteroidetes (see the Discussion), the multi-color CARD-FISH protocol was optimized to evaluate the extent of the probe bias by the simultaneous hybridization of probe ARC915 and the Bacteroidetes-specific probe CF319a [2]. As positive controls, fixed cells of Sulfolobus solfataricus DSM 1617 and Cytophaga fermentans DSM 9555 were included as independent samples in every set of hybridizations. Immediately after cell permeabilization, a first hybridization step using probe ARC915 (0.85 ng/µl, final concentration) was followed by a washing step and then a signal amplification step with Alexa-Fluor 488 tyramide conjugate as described [28], except that 55 % (v/v) formamide was used. A portion of the filter was cut, air-dried, embedded in antifading solution (Citifluor, London, UK), and then examined using an Axioskop epifluorescence microscope (Zeiss, Jena, Germany) to confirm the correct fluorescence signal of ARC915.

Int. Microbiol. Vol. 16, 2013

179

Int Microbiol

Ammonia oxidizers from Lake Kivu

Fig. 1. Schematic view of the microcosm and the microtiter plates used as cultivation systems. Mineral medium (MM) and MM+NH 4Cl contained 1 and 15 mM ammonium chloride, respectively.

Next, the filters were prepared for the second hybridization by inactivating horseradish peroxidases as previously done with cellular peroxidases but in this case extending the incubation time in 1 M HCl to 25 min. The air-dried filters were then submitted to the second hybridization using probe CF319a ( 0.28 ng/µl, final concentration) followed, after a wash, by a signal amplification step with Alexa-Fluor 546 tyramide conjugate exactly as described for probe ARC915. Finally, the filter sections were air-dried, DAPI (4′,6-diamidino-2-phenylindole) stained [28], embedded in antifading solution (Citifluor, London, UK) and examined using an Axioskop epifluorescence microscope (Zeiss) equipped with a 50-W Hg bulb and appropriate filter sets for Alexa-Fluor 488, Alexa-Fluor 546, and DAPI. To avoid overestimation of archaeal abundance because of the non-specific hybridization of probe ARC915 to members of Bacteroidetes, archaeal cells were defined as those showing positive ARC915 and negative CF319a hybridization signals and positive DAPI staining. For total cell counts, the filters were immersed in SybrGold (diluted 1:10,000 [v/v]) in the dark at room temperature for 20 min and then washed into 30 ml of double-distilled particle-free water and in 30 ml of 70 % cold ethanol. Between 250 and 600 SybrGold-stained cells were counted in ten randomly selected microscopic fields from one filter section using an Axioskop epifluorescence microscope (Zeiss). The results are expressed as the mean cell counts from one filter section; hence, a standard deviation does not apply.

Molecular characterization of the microcosm. DNA from the autochthonous community in Lake Kivu was extracted from water samples collected during the sample campaign using a combination of enzymatic cell lysis and cetyltrimethyl ammonium bromide (CTAB) as previously described [28]. The same protocol was applied to extract DNA from enrichment culture samples (10 ml) collected at different times throughout the 246-day incubation. The DNA concentration was determined spectrophotometrically using NanoDrop (Thermo Scientific, Wilmington, DE, USA) and immediately

180

Int. Microbiol. Vol. 16, 2013

stored at –80 °C until use. Amplification of partial archaeal 16S rRNA and amoA gene sequences, DGGE fingerprinting, further acrylamide band excision containing DNA melting types, and PCR reamplification of the selected melting types were carried out as previously described [28]. Additionally, the composition of both the autochthonous prokaryotic planktonic community from Lake Kivu and that enriched in the experimental microcosm was assessed by clone libraries using amplicons of the archaeal and bacterial 16S rRNA genes and the primer pairs 21f/958r and 28f/1492r, respectively [11,49]. PCR products were purified using the QIAquick spin kit (QIAGEN, Valencia, CA, USA) and polyadenylated to improve cloning efficiency by adding 26 µl of the clean PCR product to 4.5 µl of a PCR mix containing 0.98× PCR buffer (Invitrogen, Paisley, UK), 0.16 mM of MgCl2 (QIAGEN), 0.26 mM of dATP (Promega, Madison, WI, USA) and 0.61 units of Taq polymerase (QIAGEN).

The mix was then incubated for 10 min at 72 ºC in a GeneAmp 2700 thermocycler (Applied Biosystems). The polyadenylated products were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Randomly selected clones were grown overnight in LB liquid medium containing ampicillin and then amplified using the M13 primers. Clones showing the correct size insert were sequenced in both directions. Sequencing of the clones and phylotypes recovered from the DGGE bands corresponding to the 16S rRNA and amoA genes was carried out externally (Macrogen, Seoul, South Korea).

Quantitative PCR (qPCR). Copy numbers of the 16S rRNA gene of Bacteria, Archaea, and Marine Group 1.1a Thaumarchaeota and of the amoA gene from AOA and AOB were quantified by qPCR as previously described, with minor modifications [45]. All qPCR assays were performed in duplicate in a 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using MicroAmp optical 96-well reaction plates covered with optical caps (Applied Biosystems). The reaction mixtures (20 µl) contained 10 µl of SsoFast EvaGreen supermix (Bio-Rad, Richmond, CA, USA), 2 µl of template DNA (5 ng), 1 and 0.5 µM of each 16S rRNA and amoA primer pair, respectively, 2 µl of bovine serum albumin (10 mg/ml) (Sigma-Aldrich, Steinheim, Germany), and 4 µl of molecular biology-grade water (Sigma). The standard curves were obtained from serial dilutions of linearized plasmids (pGEM-T Easy; Promega) containing standard sequences. The PCR efficiency was 80–90 %, with R2 values >0.99. The results are expressed as the mean and standard deviation of duplicate qPCR assays. Negative controls resulted in undetectable values in all cases.

Phylogenetic analyses. Consensus sequences from clones or DGGE bands were aligned using the NAST algorithm [12] and analyzed to detect chimeras using the Bellerophon tool available at the GreenGenes website (http://greengenes.lbl.gov). High quality, chimera-free sequences were then aligned in Mothur v1.20.0 [39] using the SILVA archaeal database as the reference alignment. Aligned sequences were imported into ARB [26] loaded with the SILVA 16S rRNA ARB-compatible database (SSURef-104, October 2010) and manually checked to refine the alignment. Clone sequences were then exported and loaded into Mothur to assign operational taxonomic units (OTU, defined at a 97 % cutoff) and their representative sequences after calculation of a neighbor-joining (NJ) distance matrix using the Jukes-Cantor algorithm. Rarefaction curves, Good’s coverage, and Shannon diversity index were also determined in Mothur. OTU assignment for short DGGE sequences (ca. 500 bp) were analyzed separately following the same procedure but using the alignment containing all of the 16S rRNA gene sequences retrieved in this study. A phylogenetic tree was constructed using the representative sequences for each OTU and the reference sequences from the ARB database of at least 900 bp using the maximum likelihood (ML) algorithm with 100 bootstrap replicates. Shorter sequences from DGGE fingerprints were then added to this tree using the ARB “parsimony (quick add marked)” tool and termini filter, thereby maintaining the overall tree topology. A NJ tree with JukesCantor correction with 1000 bootstrap replicates was also generated using the

plasencia et al.

ARB software and resulted in a similar tree topology.

Environmental sequences for archaeal amoA genes were obtained from public databases and aligned with those retrieved from DGGE gels using the MAFFT on-line alignment tool and applying the recommended parameters [http://mafft.cbrc.jp/alignment/server/]. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 and manually checked. The ML method was used to generate 1000 bootstrap replicates. The resulting tree topology was compared with the one obtained using the NJ method, applying the Jukes-Cantor algorithm in both cases to calculate phylogenetic distances.

Statistical analyses. Data on archaeal abundance were used to calculate a 5 % trimmed mean for each combination of three wells from the microtiter plate cultures. Data on the archaeal numbers from the microtiter plate cultures were evaluated using Levene’s test for homogeneity of variances and Shapiro–Wilk’s test for normality. The data were normally distributed and homoscedastic, although a logarithmic transformation was needed in some cases. Archaeal numbers obtained from all seven treatments were compared using analysis of variance (ANOVA) and ANOVA linear contrasts. All tests were performed using SPSS v15.0 (SPSS, Chicago, IL, USA).

Nucleotide sequence accession numbers. The 16S rRNA and amoA gene sequences obtained in this study were deposited in the GenBank database under accession numbers KF418394 to KF418540 and KF418541 to KF418542.

Results Archaeal growth during incubation. The abundance of total archaeal cells increased from 2 × 104 cells/ml in the autochthonous lake community at 50 m depth to 6.61 × 106 cells/ml after 246 days of incubation (Fig. 2A). The percentage of false positives (i.e., cells hybridizing with probes ARC915 and CF319a) rapidly decreased from 63.81 % at the beginning of the incubation period to 0.76 % at the end, suggesting that the enrichment conditions were not optimal for members of Bacteroidetes (Fig. 2B). False-positives comprised only a minor fraction of the SybrGold-stained cells, ranging from 2.87 % at the beginning of the incubation period to less than 0.5 % at the end, thus confirming a specific cell identification. Archaeal growth was further confirmed uing qPCR, by monitoring archaeal 16S rRNA gene copies, which increased from 1.13 ± 0.08 × 105 to 1.76 ± 0.09 × 106 cells/ml at days 64 and 211, respectively.

Crenarchaeal 16S rRNA gene copies were determined in order to precisely quantify the AOA present in the microcosm.

The abundance of Crenarchaeota increased from 6.62 ± 0.36 × 104 to 6.68 ± 0.52 × 105 gene copies/ml for the first 133 days. Afterwards, archaeal 16S rRNA gene abundances remained almost constant until the end of the incubation period (5.20 ± 0.62 × 105 gene copies/ml at 246 days, Fig. 2C). Overall, Crenarchaeota accounted for between 22.72 ± 0.03 % and

Int. Microbiol. Vol. 16, 2013

181

Int Microbiol

Ammonia oxidizers from Lake Kivu

Fig. 2. (A) Total archaeal cell numbers and relative abundances of Archaea in the enrichment microcosm throughout the incubation period, as determined with multi-color CARD-FISH. (B) Reduction of false-positives for the ARC915 probe in samples collected during the incubation. (C) 16S rRNA gene copy numbers for Bacteria, Archaea, and Crenarchaeota and Marine Group 1.1a Thaumarchaeota as determined in the microcosm during the 246-day incubation. (D) Gene copy numbers for archaeal and bacterial amoA in the enrichment microcosm throughout the incubation.

46.86 ± 17.10 % of the total archaeal 16S rRNA gene copies.

Assuming that mesophilic Crenarchaeota harbor a single genomic copy of the 16S rRNA gene and that Euryarchaeota contain at most two to four genomic copies of the 16S rRNA gene [http://rrndb.umms.med.umich.edu], the former would represent a significant fraction of the total archaeal community quantified by qPCR, thus suggesting the positive stimulation of AOA by the enrichment conditions. Nonetheless, bacterial abundances always exceeded those of archaea by one and two orders of magnitude at the beginning and end of the microcosm experiment, respectively, confirming that archaea were a minor component of the microbial community in the microcosm. In fact, bacterial 16S rRNA genes increased during the first 133 days, from 3.73 ± 0.01 × 106 copies/ml to 2.96 ± 0.05 × 108 copies/ml (Fig. 2C).

Since the cultivation conditions were designed to promote ammonia oxidation, archaeal and bacterial amoA gene copy numbers were also monitored to determine the effect of medium conditions on both groups of ammonia-oxidizers. Archaeal amoA gene copies rapidly increased during the first

133 days of cultivation, from less than 10±0.17 copies/ml to 6.24 ± 0.08 × 103 copies/ml, whereas during the same period the bacterial amoA gene was not detected (Fig. 2B). Instead, bacterial amoA was detected only later and the copy number consistently increased until the end of the incubation period, when it outnumbered that of the archaeal amoA gene by three orders of magnitude (Fig. 2D).

Phylogenetic composition of bacterial and archaeal communities. Two clone libraries were generated from samples obtained at the initial phase of enrichment (64 days) and at the end of the experiment (246 days). The bacterial community, which was initially dominated by Betaproteobacteria (81.7 % of the total clones in the 64-day library), became a progressively more diverse community mainly composed of Alpha- and Betaproteobacteria (65.2 % and 25 % of total clones in the 246-day library, respectively).

The major representatives of these proteobacterial classes were members of the Rhizobiales and Burkholderiales, respec­tively, most of which are well-known nitrogen-fixing

Int. Microbiol. Vol. 16, 2013

plasencia et al.

Int Microbiol

182

Fig. 3. Negative image of a SybrGold-stained DGGE gel showing: (A) archaeal 16S rRNA gene fingerprints obtained from the natural archaeal assemblage used as inoculum (Lake Kivu, Northern Basin, 50 m depth, NB50) and the archaeal community enriched in the experimental microcosm at different sampling intervals (from day 64 to 246), and (B) fingerprints for the archaeal amoA gene. Band labels correspond to codes used in the phylogenetic tree (DGGE1, DGGE2, Fig. 4; M50_DGGE1 and M50_DGGE2, Fig. 5). L stands for DGGE ladder.

soil bacteria. Note that no sequences of known AOB were identified in the clone library (355 clones in total) although the presence of these bacteria had been deduced by qPCR targeting their amoA genes (Fig. 2D). The composition of the archaeal community was assessed by DGGE fingerprinting and by clone libraries. 16S rRNA gene fingerprints obtained at selected dates showed two bands that persisted throughout the incubation and coincided with the band pattern obtained from the planktonic community in Lake Kivu (Fig. 3A). The sequence recovered from band DGGE1 clustered within uncultured crenarchaeota from Marine Group 1.1a, mainly represented by marine AOA. Specifically, DGGE1 exactly matched phylotype aG2 (FJ536698) and was highly similar (>99 %) to bG4 (Fig. 3A); these two phylotypes were previously identified in the Northern Basin of Lake Kivu [28]. In addition, the sequence similarity of DGGE1 to two previously characterized ammonia oxidizers, “Ca. Nitrosoarchaeum koreensis” MY1 (HQ331116) [20] and “Ca. Nitrosoarchaeum limnia” SFB1 (NZ_CM001158) [7], was 100 % and 99.3 %, respectively (Fig. 4).

The phylotype DGGE1 also showed a sequence similarity of 92.2 % to previously enriched AOA-DW (JQ669391) and AOA-AC5 (JQ669390), two archaeal sequences from freshwater sediment samples of Acton Lake and Delaware Lake (OH, USA) [14]. Phylotype DGGE2 clustered within the Miscellaneous Crenarchaeotic Group (MCG), showing 96 % and

94 % similarity to uncultured MCG from Griffy Lake (IN, USA) and Lake Pavin (France), respectively, and was only distantly related (88.3 %) to aG7, a previously identified MCG phylotype occurring in the same basin of Lake Kivu.

Further DGGE analysis targeting archaeal amoA gene fragments showed two melting types that agreed with the results obtained with the 16S rRNA genes (Fig. 3B). Only one of these bands was very similar to that from the autochthonous community and it clearly persisted in the enrichment throughout the incubation. The recovered amoA sequences grouped in two subclusters, Kivu1 (melting type M50_DGGE1: M50_0, M50_133 and M50_246) and Kivu2 (melting type M50_ DGGE2: M50_0) (Fig. 5). The first amoA variant was identical to amoA sequences previously retrieved from the Lake Kivu oxycline [28] and it grouped within a freshwater clade showing high similarities (>99 %) to the amoA sequence from “Ca. Nitrosoarchaeum koreensis” MY1 (HQ331117). The second variant occurred as a faint band and was not detected after 133 days (Fig. 3B). This phylotype had not been previously found in Lake Kivu and showed high pairwise similarity (99.72 %) to an amoA clone sequence (FJ951737) retrieved from the surface sediments of Lake Taihu (China). Overall, the DGGE fingerprints for the archaeal 16S rRNA and amoA genes indicated the persistence of AOA throughout the incubation period and therefore their presence as a significant fraction of the archaeal community in the microcosm.

Int. Microbiol. Vol. 16, 2013

183

Int Microbiol

Ammonia oxidizers from Lake Kivu

Fig. 4. Maximum-likelihood phylogenetic tree calculated for the full and partial 16S rRNA gene sequences retrieved from Lake Kivu and microcosm samples (in bold). Sequences obtained from the natural community (NB50) after inoculation of the enrichment culture (M50_0) and at the end of the 246-day incubation (M50_246) are specified for each OTU. DGGE band sequences are coded according to the band numbers in Fig. 3. Sequences identical to those of the cultured isolates are shaded in gray. Significant bootstrap values (≥50 %) from 100 replicates are shown at branch nodes. Scale bar indicates an estimated sequence divergence of 10 %.

We analyzed 146 clones, which grouped into six OTUs (defined at 97 % cutoff). Three of these OTUs were affiliated with Marine Group 1.1a Thaumarchaeota and grouped together 130 sequences (89 % of the total). From these AOA sequences, 126 could be assigned to OTU-2 (86 % of the clones), which exactly matched a phylotype previously identified in Lake Kivu (aG2, FJ536698, [28] and showed a 99 % pairwise similarity to “Ca. Nitrosoarchaeum limnia” SFB1 (NZ_CM001158) and “Ca. Nitrosoarchaeum koreensis” MY1 (HQ331116) [20]. In addition, the representative clone sequence from OTU-2 had a 99 % pairwise similarity with phylotype DGGE-1, previously identified by 16S rRNA DGGE fingerprinting. Phylotype DGGE-2 was not identified in our clone libraries.

Only clone sequences affiliated with OTU-2 were identified in the clone libraries constructed from the natural lake community (NB50) and from the microcosm enrichment (M50_0 and M50_246). Sequences affiliated with OTU-2 were prevalent in all clone libraries analyzed, representing 84 %, 93.5 %, and 85 % of the total sequences in the NB50, M50_0, and M50_246 libraries, respectively). Variations in the relative abundance of OTU-2 sequences caused changes in diversity, as estimated by the Shannon index (data not shown). The remaining OTUs comprised OTU-5 and OTU-6, which contained sequences only identified at the beginning (M50_0) and at the end (M50_246) of the cultivation period, OTU-3 and OTU-4, which grouped together sequences from the autochthonous community in the lake (NB50), and OTU-

Int. Microbiol. Vol. 16, 2013

plasencia et al.

Int Microbiol

184

Fig. 5. Maximum-likelihood phylogenetic tree for archaeal amoA partial gene sequences retrieved from microcosm samples (in bold). Significant bootstrap values (â&#x2030;Ľ50 %) from 1,000 replicates are shown at branch nodes; those corresponding to maximum likelihood and neighbor joining analyses are indicated above and below the line, respectively. Scale bar indicates an estimated sequence divergence of 5 %.

1, which included sequences recovered from the lake community and from the microcosm at the end of the incubation.

Substrate tests in microtiter plates. The analyses of non-inoculated control wells in the microtiter plates showed no contamination in any of the experiments. The addition of antibiotics to the medium was expected to inhibit the growth

of bacteria present in the microcosm, thereby enhancing the growth of archaea. However, a decrease in archaeal abundances was determined by CARD-FISH in all antiobioticsupplemented media (Fig. 6); therefore, the effect of antibiotic addition on archaeal growth was evaluated. Indeed, the statistical analyses revealed significant differences (ANOVA t14 = 12.12, P < 0.001) in archaeal cell counts from deep-well

Int. Microbiol. Vol. 16, 2013

185

Int Microbiol

Ammonia oxidizers from Lake Kivu

Fig. 6. Total cell numbers of archaea determined in microtiter plate enrichments by means of multi-color CARD-FISH in the first and the second microtiter plate cultivation systems (for details, see Materials and methods).

microtiter plate enrichments differing only in the presence or absence of antibiotics. The addition of a high concentration of ammonia (15 mM) did not significantly increase archaeal cell numbers compared to deep-wells supplemented with low ammonia concentrations (1 mM) (ANOVA t14 = –0.042, P = 0.967).

By contrast, amendment of the basal mineral medium with yeast extract yielded the highest increase in archaeal abundance, although the differences were not significant compared to the other supplements (Fig. 6). Additional tests confirmed this observation as they failed to show significant differences in archaeal cell numbers in enrichments consisting of mineral medium with or without added organic compounds (ANOVA t14 = –1.058, P = 0.308 and ANOVA t14= 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). A second transfer of the enrichment samples from individual plate wells to a new microtiter plate containing fresh medium and incubated under the same conditions also did not result in increased archaeal growth under any of the assayed conditions (Fig. 6).

Discussion Multi-color CARD-FISH. This technique is the only nucleic acid probing approach that simultaneously allows cell identification and quantitative determination of cell numbers. Although the probe ARC915 is theoretically optimal to specifically identify and count all Archaea [2] by CARD-FISH ([3,27] and references therein), several authors have reported non-specific probe binding to members of the Bacteroidetes (formerly known as the Cytophaga–Flavobacteria–Bacteroides cluster), causing overestimations of archaeal counts in natural samples [6,22,27,32,37]. To avoid this problem and thereby more precisely estimate archaeal cell numbers, we used multi-color CARD-FISH together with probes ARC915 and CF319a. To our knowledge, this is the first study in which this technique has been used to correct potential overestimations of archaeal cell counts be-

186

Int. Microbiol. Vol. 16, 2013

cause of non-specific hybridization of the ARC915 probe to members of the Bacteroidetes.

Archaeal cells were correctly quantified by subtracting cells that hybridized with both probes from the total number of ARC915-positive cells. With this method, we detected a decrease in the former over the course of the incubation, such that, finally, only those cells that hybridized with probe ARC915 alone were used to estimate archaeal abundances.

Our results therefore question whether previous estimates of archaeal cell numbers based exclusively on probe ARC915 served as reliable indicators of archaeal abundance.

CARD-FISH vs. qPCR. Quantification techniques based on cell counts (FISH or CARD-FISH) or gene copy numbers (qPCR) are different approaches to determine bacterial abundances. In microcosm experiments, CARD-FISH is a valuable and reliable monitoring tool for identifying and quantifying slow-growing cells, such as AOA, with an inherently low ribosome content that may not be sufficient to yield a fluorescence signal visible by FISH alone [17,50]. Although qPCR is a very sensitive and specific technique, its use for purposes of quantification is known to have several biases that can result in differences in archaeal abundances compared to the results obtained with CARD-FISH. First, the chosen DNA extraction method strongly affects qPCR-based quantification, as demonstrated in cultures of Dehalococcoides ethenogenes ([13,43] and references therein).

Similarly, ten-fold difference between 16S rRNA gene copies and DAPI counts have been reported for pure cultures of Bacillus cereus and Bacillus subtilis and were attributed to deficient cell lysis [4]. Since the cell wall and membrane structures of archaeal cells differ from those of bacteria, the use of a common extraction method will not assure an equivalent degree of lysis of the two cell types.

Recently, Urakawa and co-workers showed that an improved phenol-chloroform DNA extraction method (similar to our CTAB protocol) enhanced DNA recovery from Nitros­ opumilus maritimus cells by four-fold and resulted in a similar increase in the qPCR-based quantification of the amoA gene of that species [46]. We therefore assume that the limitations of this study included differences in DNA recovery, which in turn would have influenced the qPCR results. Similarly, cell-wall permeabilization by different chemical treatments implies differential effects on cell walls and therefore differences in probe hybridization. Second, mismatches between PCR primers and target DNA can affect duplex stability for PCR amplification. For example, the presence and position of a single internal primer-template mismatch reduce the

plasencia et al.

efficiency of PCR amplification, in turn resulting in an underestimation of gene copy numbers by up to 1,000-fold [8].

Moreover, the authors of a comparative study reported that cell numbers determined by CARD-FISH were always at least one order of magnitude higher than gene copy numbers determined by qPCR, because of the limitations posed by DNA extraction and primer biases [31]. Accordingly, the discrepancies found in this study between the archaeal abundances determined by CARD-FISH vs. by qPCR would reflect differences in DNA recovery and/or primer-template mismatches for some archaeal groups and, therefore, an underestimation of 16S rRNA gene copy numbers. Thus, overall, to properly quantify archaeal abundance CARD-FISH is preferred over qPCR. Qualitative and quantitative identification of target sequences by PCR-based methods. DGGE is a suitable technique for the rapid comparison of band fingerprinting between samples even though it only detects dominant members of the community (>9 %, [16]) whereas the construction of a clone library may allow the detection of phylotypes with low abundances. Moreover, DGGE allows the direct comparison of band patterns at different dates, thus providing a method to rapidly detect changes in the dominant archaeal phylotypes throughout an incubation and to compare these phylotypes with those in the autochthonous community [28]. Yet, although DGGE fingerprinting is routinously used as a screening technique to detect dominant phylotypes in enrichment cultures [15,16,23], it clearly fails in the proper iden­ tification of rare taxa (≤9 % of abundance, [16]).

To overcome this limitation, we constructed clone libraries of archaeal 16S rRNA gene from samples obtained at different dates throughout the incubation period and from the autochthonous planktonic community. The high coverage values calculated for the three clone libraries (>97 %) indicated that most, if not all, of the archaeal richness present in the enrichment was recovered.

In the present study, no AOB members were identified by either DGGE or clone library construction, but the bacterial amoA gene was quantified by qPCR. Although biases must be taken into consideration when PCR is used, the absence of any AOB clones in our libraries was surprising. Considering that AOB harbor a mean 2.5 amoA gene copies per AOB genome [45] we estimated that the AOB members in our microcosm accounted for 2.70 × 103 and 1.71 × 106 cells/ml at days 154 and 246, respectively. Since it is generally assumed that AOB carry a single 16S rRNA copy per genome [1], the number of 16S rRNA gene copies corresponding to AOB can be inferred

Ammonia oxidizers from Lake Kivu

for these dates. Accordingly, at day 246, AOB represented 0.57 % of the total bacterial 16S rRNA gene copies/ml. Consequently, the use of conventional eubacterial primers in PCR would strongly bias amplicon replication in favor of the most abundant target sequences, making it very unlikely that AOB 16S rRNA gene sequences warranting further detection in clone libraries would be amplified. Moreover, the relative abundance of AOB in the first sample used in library construction (M_64) would have been far below 0.57 % of the total 16S rRNA gene copies, since amoA was not detected at that date.

Significance of archaeal ammonia oxidizers in Lake Kivu. In a previous study carried out in Lake Kivu, we showed that AOA accumulate at the oxycline (30–50 m depth), forming a community mainly dominated by a single OTU affiliated with Marine Group 1.1a Thaumarchaeota [28].

The same study identified 11 OTUs containing fewer sequences belonging to Soil Group 1.1b Thaumarchaeota, raising the question whether these OTUs were truly planktonic or were introduced into the lake by surface runoff from surrounding crop fields [33,34]. By comparing the autochthonous archaeal community in Lake Kivu with that grown in the enrichment culture, we confirmed the dominance of a single OTU (OTU-2) affiliated with Marine Group 1.1a. In addition, the results from the clone libraries supported those obtained

by DGGE fingerprinting and clearly demonstrated that members of Thaumarchaeota Marine Group 1.1a were the major component of the archaeal community that developed in the experimental microcosm. Although this group mainly comprises AOA from aquatic habitats, either marine or freshwater, it also includes species isolated from soils (i.e., “Ca. Nitrosoarchaeum koreensis” [20]).

Culture-dependent studies have shown that different species differ in their tolerance of ammonia, consistent with the ammonia concentrations usually found in their respective habitats. For instance, Nitrosopumilus maritimus strain SCM1 and probably other marine species are able to cope with the very low ammonia concentrations usually found in the world oceans [29], whereas several AOA from soils can oxidize ammonia at concentrations up to 15–20 mM [20,44]. The optimal and maximum ammonia concentrations for the growth of a new strain of Nitrosopumilus maritimus NM25, enriched from sand in an eelgrass zone, are 15 mM and 26 mM, respectively [30]. In Lake Kivu, the average concentration of ammonia at the oxycline is below 0.5 mM [34], at which point planktonic AOA face extreme nutrient limitation. Note that in our study the OTU that contained the most sequences (OTU-2) had a high 16S rRNA gene sequence similarity to “Ca. Nitro­

Int. Microbiol. Vol. 16, 2013

187

soarchaeum koreensis”, an archaeal nitrifier isolated from an agricultural soil and able to tolerate ammonium concentrations up to 10 mM [20]. Similarly, several AOA strains enriched from Acton Lake and Delaware Lake (Ohio, USA) were shown to differ in their ammonium tolerances (1–5 mM).

One of these strains has 99.6 % 16S rRNA identity to “Ca. Nitrosoarchaeaum koreensis” [14]. Consequently, the AOA prevalent in Lake Kivu might have colonized the lake from nearby crop fields and then gradually adapted to a range of ammonia concentrations wider than those in marine environments. This hypothesis merits future investigations considering the increase in nutrient inputs by water runoff over the last several decades [33-35]. However, the identification of AOA prevalent in Lake Kivu leads to the question whether these microorganisms are ecologically relevant given the low concentrations of ammonia present in the lake. It seems more plausible to consider that the AOA were not in an ecological niche appropriate for their growth. The absence of AOB during the first 154 days of the microcosm incubation in the laboratory was in agreement with the low abundances of bacterial amoA (<102 amoA gene copies/ml; Llirós M, personal comm.) measured at several sampling sites of Lake Kivu. Remarkably, the rapid growth of AOA in the experimental microcosm (Fig. 2D) was in accordance with the higher ammonia affinities of AOA than AOB [20,29]; however, this trend was reversed during the final stages of the incubation, when AOB clearly outnumbered AOA. Stimulation of AOA growth by organic compounds and the effect of antibiotics. By using microtiter plates, we were able to examine the effect of different organic compounds on the growth of AOA in a minimal space, thereby optimizing resources and screening efforts.

The ability off AOA to grow mixotrophically in oligotrophic habitats, as determined in metagenomic analyses and isotopic studies, could provide them with a selective advantage over their strict autotrophic counterparts [7,44,48]. Findings from experimental studies and metagenomic analysis support this view, by showing that AOA can use simple organic compounds as carbon sources [7,19,48,51]. By contrast, according to our results, the AOA community was neither stimulated nor inhibited by the addition of simple organic compounds to the growth medium (Fig. 6). This finding is in agreement with the high similarity of the most abundant OTU2 to “Ca. Nitrosoarchaeum koreensis,” which is not stimulated by organic compounds [20], and with the oligotrophic conditions prevalent in Lake Kivu [38]. Previous studies on the enrichment of archaea from rhizospheres of Licopersinon esculentum (to-

188

Int. Microbiol. Vol. 16, 2013

mato plant) reported a slight stimulation of AOA growth by the addition of root extracts [40,51]. Some AOA might have a mixotrophic and/or heterotrophic metabolism, which would explain their adaptation to eutrophic environments, although AOA species differ in their responses to organic matter, which inhibits the growth of, e.g., Nitrosopumilus maritimus [24] while stimulating that of “Ca. Nitrososphaera viennensis” [44]. Thus, extreme caution is warranted when drawing conclusions from our stimulation assays, given the relatively few substrates tested and the dominance of the accompanying bacterial community, which probably was able to use the tested organic compounds more efficiently.

The inhibition of archaeal growth by medium containing antibiotics could have been caused either by direct toxicity to AOA or by an indirect effect on the satellite bacterial community. The impossibility to carry out toxicity studies on pure cultures of mesophilic AOA serious limited our ability to properly interpret the results, although our preliminary findings obtained with Sulfolobus solfataricus DSM 1617 did not indicate any effects of the inhibitor mixture on growth (data not shown).

In addition, direct effects of antibiotics on archaeal growth were reported for Sulfolobus acidocaldarius—and especially for methanogens that are pathogenic in humans [9,21]—while studies on the enrichment of mesophilic archaea describe an inhibitory effect of some antibiotics [40]. We were unable to eliminate co-cultured bacteria in the microcosm despite the antibiotic treatment used. Other authors reported the same problem in their attempts to isolate AOA species from enrichment cultures [10,18,20,25]. This difficulty has led to the conclusion that archaea may obtain some benefit from the co-cultured bacteria, either commensally or symbiotically [25]. Thus, the detection in our clone libraries of many bacterial sequences affiliated with potential N2-fixers (Rhizobiales and Burkholderiales) agrees with previous results obtained following the cultivation of acidophilic AOA from an acid soil. Further work will be needed to determine the possible metabolic implications of a relationship between potential N2-fixers and AOA in natural environments and in co-cultures.

Aknowledgements. This work was founded by the Spanish Government through projects CRENYC (CGL2006-12058-C02-01), ARKI (CGL200729823-E) and ARCANOX (CGL2009-13318-C02-02). A.P.C. and M.F.H. are recipients of pre-doctoral fellowships from the Catalan (2006FI-109) and the Spanish (FPI BES-2010-035225) Governments, respectively. Authors are indebted to Ingrid Cuesta for helping on qPCR quantification of total archaea and Marc Llirós for fruitful discussions along manuscript preparation.

Competing interests. None declared.

plasencia et al.

References 1. Aakra Å, Utåker JB, Nes IF (1999) RFLP of rRNA genes and sequencing of the 16S-23S rDNA intergenic spacer region of ammonia-oxidizing bacteria: a phylogenetic approach. Int J Syst Bacteriol 49:123-130 2. Amann R, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Micro 6:339-348 3. Auguet J-C, Nomokonova N, Camarero L, Casamayor EO (2011) Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes. Appl Environ Microbiol 77:1937-1945 4. Bach HJ, Tomanova J, Schloter M, Munch JC (2002) Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification. J Microbiol Methods 49:235-245 5. Bañeras L, Gich F, Martinez-Medina M, Miller M, Abella CA, Borrego CM (2009) New phylotypes of mesophilic filamentous anoxygenic phototrophic bacteria enriched from sulfide-containing environments. Environ Microbiol Rep 1:86-93 6. Battin TJ, Wille A, Sattler B, Psenner R (2001) Phylogenetic and functional heterogeneity of sediment biofilms along environmental gradients in a glacial stream. Appl Environ Microbiol 67:799-807 7. Blainey PC, Mosier AC, Potanina A, Francis CA, Quake SR (2011) Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis. PLoS ONE 6(2):e16626 8. Bru D, Martin-Laurent F, Philippot L (2008) Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appl Environ Microbiol 74: 1660-1663 9. Cammarano P, Teichner A, Londei P, Acca M, Nicolaus B, Sanz JL, Amils R (1985) Insensitivity of archaebacterial ribosomes to protein synthesis antibiotics. Evolutionary implications. EMBO J 4:811-816 10. de la Torre JR, Walker CB, Ingalls AE, Könneke M, Stahl DA (2008) Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ Microbiol 10:810-818 11. DeLong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci USA 89:5685-5689 12. DeSantis TZ, Jr., Hugenholtz P, Keller K, Brodie EL, Larsen N, Piceno YM, Phan R, Andersen GL (2006) NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucl Acids Res 34: 394-399 13. Duhamel M, Mo K, Edwards EA (2004) Characterization of a highly enriched Dehalococcoides-containing culture that grows on vinyl chloride and trichloroethene. Appl Environ Microbiol 70:5538-5545 14. French E, Kozlowski JA, Mukherjee M, Bullerjahn G, Bollmann A (2012) Ecophysiological characterization of ammonia-oxidizing Archaea and Bacteria from freshwater. Appl Environ Microbiol 78: 5773-5780 15. Gich F, Janys MA, König M, Overmann J (2012) Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces. Environ Microbiol 14:2984-2997 16. Gich F, Schubert K, Bruns A, Hoffelner H, Overmann J (2005) Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community. Appl Environ Microbiol 71:5908-5919 17. Hatzenpichler R (2012) Diversity, physiology, and niche differentiation of ammonia-oxidizing archaea. Appl Environ Microbiol 78:7501-7510

Ammonia oxidizers from Lake Kivu

18. Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, Daims H, Wagner M (2008) A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. Proc Natl Acad Sci USA 105: 2134-2139 19. Ingalls AE, Shah SR, Hansman RL, Aluwihare LI, Santos GM, Druffel ERM, Pearson A (2006) Quantifying archaeal community autotrophy in the mesopelagic ocean using natural radiocarbon. Proc Natl Acad Sci USA 103:6442-6447 20. Jung M-Y, Park S-J, Min D, et al. (2011) Enrichment and characterization of an autotrophic ammonia-oxidizing archaeon of mesophilic crenarchaeal Group I.1a from an agricultural soil. Appl Environ Microbiol 77:8635-8647 21. Khelaifia S, Drancourt M (2012) Susceptibility of archaea to antimicrobial agents: applications to clinical microbiology. Clin Microbiol Inf 18:841-848 22. Klammer S, Posch T, Sonntag B, Griebler C, Mindl B, Psenner R (2002) Dynamics of bacterial abundance, biomass, activity, and community composition in the oligotrophic Traunsee and the Traun River (Austria).

Water, air, and soil pollution: Focus 2:137-163 23. Koch IH, Gich F, Dunfield PF, Overmann J (2008) Edaphobacter modes­ tus gen. nov., sp. nov., and Edaphobacter aggregans sp. nov., acidobacteria isolated from alpine and forest soils. Int J Syst Evol Microbiol 58:1114-1122 24. Könneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl DA (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437:543-546 25. Lehtovirta-Morley LE, Stoecker K, Vilcinskas A, Prosser JI, Nicol GW (2011) Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil. Proc Natl Acad Sci USA 108:15892-15897 26. Ludwig W, Strunk O, Westram R, et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363-1371 27. Llirós M, Alonso-Sáez L, Gich F, Plasencia A, Auguet O, Casamayor EO, Borrego CM (2011) Active bacteria and archaea cells fixing bicarbonate in the dark along the water column of a stratified eutrophic lagoon.

FEMS Microbiol Ecol 77:370-384 28. Llirós M, Gich F, Plasencia A, Auguet J-C, Darchambeau F, Casamayor EO, Descy J-P, Borrego C (2010) Vertical istribution of ammonia-oxidizing Crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo). Appl Environ Micro­ biol 76:6853-6863 29. Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA (2009) Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461:976-979 30. Matsutani N, Nakagawa T, Nakamura K, Takahashi R, Yoshihara K, Tokuyama T (2011) Enrichment of a novel marine ammonia-oxidizing archaeon obtained from sand of an eelgrass zone. Microbes Environ 26:23-29 31. Matturro B, Heavner GL, Richardson RE, Rossetti S (2013) Quantitative estimation of Dehalococcoides mccartyi at laboratory and field scale: Comparative study between CARD-FISH and Real Time PCR. J Microbiol Methods 93:127-133 32. Moissl C, Rudolph C, Rachel R, Koch M, Huber R (2003) In situ growth of the novel SM1 euryarchaeon from a string-of-pearls-like microbial community in its cold biotope, its physical separation and insights into its structure and physiology. Arch Microbiol 180:211-217 33. Muvundja FA, Pasche N, Bugenyi FWB, et al. (2009) Balancing nutrient inputs to Lake Kivu. J Great Lakes Res 35:406-418

Int. Microbiol. Vol. 16, 2013

189

34. Pasche N, Dinkel C, Müller B, Schmid M, Wüest A, Wehrli, B (2009) Physical and biogeochemical limits to internal nutrient loading of meromictic Lake Kivu. Limnol Oceanogr 54:1863-1873 35. Pasche N, Schmid M, Vazquez F, et al. (2011) Methane sources and sinks in Lake Kivu. J Geophys Res Biogeosci 116:G03006 36. Pernthaler A, Pernthaler J, Amann R (2004) Sensitive multi-color fluorescence in situ hybridization for the identification of environmental microorganisms. In: Kowalchuk GA, et al. (eds) Molecular microbial ecology manual, 2nd ed. Kluwer, Dordrecht, the Netherlands, pp 711-726 37. Pernthaler A, Preston CM, Pernthaler J, DeLong EF, Amann R (2002) Comparison of fluorescently labeled oligonucleotide and polynucleotide probes for the detection of pelagic marine bacteria and archaea. Appl Environ Microbiol 68:661-667 38. Sarmento H, Isumbisho M, Descy J-P (2006) Phytoplankton ecology of Lake Kivu (eastern Africa). J Plankton Res 28:815-829 39. Schloss PD, Westcott SL, Ryabin T, et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537-7541 40. Simon HM, Jahn CE, Bergerud LT, Sliwinski MK, Weimer PJ, Willis DK, Goodman RM (2005) Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots. Appl Environ Microbiol 71: 4751-4760 41. Spang A, Hatzenpichler R, Brochier-Armanet C, et al. (2010) Distinct gene set in two different lineages of ammonia-oxidizing archaea supports the phylum Thaumarchaeota. Trends in Microbiol 18:331-340 42. Stahl DA, de la Torre JR (2012) Physiology and diversity of ammoniaoxidizing Archaea. Annu Rev Microbiol 66:83-101 43. Sung Y, Ritalahti KM, Apkarian RP, Löffler FE (2006) Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring dehalococcoides isolate. Appl Environ Microbiol 72:1980-1987 44. Tourna M, Stieglmeier M, Spang A, et al. (2011) Nitrososphaera vien­ nensis, an ammonia oxidizing archaeon from soil. Proc Natl Acad Sci USA 108:8420-8425 45. Trias R, García-Lledó A, Sánchez N, López-Jurado JL, Hallin S, Bañeras L (2012) Abundance and composition of epiphytic bacterial and archaeal ammonia oxidizers of marine red and brown macroalgae. Appl Environ Microbiol 78:318-325 46. Urakawa H, Martens-Habbena W, Stahl DA (2010) High abundance of ammonia-oxidizing archaea in coastal waters determined using a modified DNA extraction method. Appl Environ Microbiol 76:2129-2135 47. Verhamme DT, Prosser JI, Nicol GW (2011) Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms. ISME J 5:1067-1071 48. Walker CB, de la Torre JR, Klotz MG, et al. (2010) Nitrosopumilus ma­ ritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc Natl Acad Sci USA 107:8818-8823 49. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) SSU ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697-703 50. Wuchter C, Abbas B, Coolen MJL, et al. (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci USA 103:12317-12322 51. Xu M, Schnorr J, Keibler B, Simon HM (2012) Comparative analysis of 16S rRNA and amoA genes from archaea selected with organic and inorganic amendments in enrichment culture. Appl Environ Microbiol 78: 2137-2146

RESEARCH ARTICLE International Microbiology (2013) 16:191-198 doi: 10.2436/20.1501.01.193 ISSN 1139-6709 www.im.microbios.org

A modular reactor to simulate biofilm development in orthopedic materials Joana Barros,1,2,4* Liliana Grenho,1,2 Cândida M. Manuel,4,5 Carla Ferreira,4 Luís F. Melo,4 Olga C. Nunes,4 Fernando J. Monteiro,1,2 Maria P. Ferraz1,2,3 INEB-Instituto de Engenharia Biomédica, Portugal. 2FEUP-Faculdade de Engenharia, Universidade do Porto, Departamento de Engenharia Metalúrgica e Materiais, Portugal. 3CEBIMED-Centro de Estudos em Biomedicina, Universidade Fernando Pessoa, Portugal. 4LEPABE–Laboratory for Process Engineering, Environment, Biotechnology and Energy, Dept. Chemical Engineering, University of Porto, Portugal. 5ULP-Universidade Lusófona do Porto, Portugal

1

Received 17 August 2013 · Accepted 1 October 2013

Summary. Surfaces of medical implants are generally designed to encourage soft- and/or hard-tissue adherence, eventually leading to tissue- or osseo-integration. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation.

To understand the mechanisms of bone tissue infection associated with contaminated biomaterials, a detailed understanding of bacterial adhesion and subsequent biofilm formation on biomaterial surfaces is needed. In this study, a continuous-flow modular reactor composed of several modular units placed in parallel was designed to evaluate the activity of circulating bacterial suspensions and thus their predilection for biofilm formation during 72 h of incubation. Hydroxyapatite discs were placed in each modular unit and then removed at fixed times to quantify biofilm accumulation. Biofilm formation on each replicate of material, unchanged in structure, morphology, or cell density, was reproducibly observed. The modular reactor therefore proved to be a useful tool for following mature biofilm formation on different surfaces and under conditions similar to those prevailing near human-bone implants. [Int Microbiol 2013; 16(3):191-198] Keywords: orthopedic materials · orthopedic conditions · modular reactors · continuous flow · biomaterials · biofilm formation

Introduction Biofilm-related infections associated with indwelling medical devices, such as orthopedic implants and prostheses, have become a major clinical concern and reflect the failed attempts to prevent their formation and to treat affected patients [31].

In fact, bone-tissue and prosthetic-joint infections are among the worst complications in orthopedic surgery and traumatolCorresponding author: J. Barros INEB-Instituto de Engenharia Biomédica Universidade do Porto Rua do Campo Alegre, 823 4150-180 Porto, Portugal Phone: +351-226074900 E-mail: joanabarros@fe.up.pt

*

ogy and may lead to the complete failure of the arthroplasty, amputation, prolonged hospitalization, and even death [4,9,27].

Bacterial attachment to biomaterial surfaces is an important step in the pathogenesis of these infections. Their exact mechanism remains unclear but several studies have been directed at better understanding the development, structure, and impact of bacterial biofilms associated with indwelling medical devices. A large proportion of implant-related infections is caused by Staphylococcus aureus, S. epidermidis, and Esch­ erichia coli [1,18,27]. The pathogenicity of biofilm-dwelling S. epidermidis has at least in part been attributed to the development of extracellular polymeric substances (EPS) [18,27, 30] that protect the bacterial population against host defense mechanisms and antimicrobial agents [1,27].

192

Int. Microbiol. Vol. 16, 2013

Hydroxyapatite (HA) has exceptional biocom­patibility and bioactivity with respect to bone cells and tissues, probably because of its similarity to the hard tissues of the body [11]. It has therefore been extensively used as a coating for orthopedic implants or as a bone substitute. Bone contains natural HA crystals with needle-like and rod-likes shapes well-arranged within a polymeric matrix of collagen type I. Nanophased HA is able to bind bone and to interact with macromolecules that participate in the preliminary events leading to bone bonding and tissue regeneration [10]. However, the introduction of nanophased HA materials into the body is always associated with the risk of microbial infection, particularly in the fixation of open-fractures and in joint-revision surgeries [27]. Consequently, these implanted materials represent sites of weakness for host defenses such that they allow the attachment even of bacteria with a low level of virulence [16].

The most promising strategies for preventing orthopedic infections seek to inhibit bacterial adhesion prior to biofilm formation, especially during the initial 6 h following im­ plantation [9,10], the critical phase in the ocurrence of deviceassociated infections [9]. To achieve this goal requires a detailed understanding and quantification of the events that occur during initial bacterial adhesion and subsequent biofilm formation on biomaterial surfaces.

One way to reproducibly study and visualize biofilms and cellular attachment is to use biofilm reactors [6]. During the last several decades, attempts have been made to develop laboratory biofilm reactors that minimize the heterogeneity of experimental conditions in order to simplify the analysis and validation of biofilm data and to enable direct and real-time assessment of the bacterial colonization of submerged surfaces [6,13,14,22,23,30]. Biofilm reactors present several advantages with respect to the definition and control of hydrodynamic parameters such as flow velocity, Reynolds number, and shear stress [22]. Moreover, properly designed biofilm reactors contribute to minimizing experimental problems associated with inconsistent and ill-defined rinsing of “reversibly” bound cells, the variability of culture media, radiolabeled substrates, or vital stains, and the exposure of biofilm to medium-air interfacial forces [22]. Two of the best known types of reactors for the open continuous culture of biofilms are annular reactors (Rototorque) and the Robbins device [14,23]. However, while both operate as continuous-flow systems and contain fixed biofilm supports that can be easily removed for sampling, removal is only possible after the flow has been stopped, with the flow restarted by again closing the system [15,21]. Thus, the hydrodynamics are, in general, not similar to the conditions found near human bones in the body.

barros et al.

Additionally, in the Rototorque reactor, fluid dynamics are not uniform throughout the system, leading to non-ideal mixing and non-uniform biofilm formation [17].

In this work, an in vitro model modular reactor was designed that replicates the conditions in the vicinity of living bone, including low flow rates, physiological body temperature, darkness, and low oxygen and nutrient levels. It also allows visual surveillance of bacterial adhesion and biofilm formation on biomaterial surfaces, easy manipulation and control of the environmental conditions, and periodic sampling and analysis with minimal disturbance of the biofilm samples.

Materials and methods Preparation of nanohydroxyapatite and microhy­ droxy­ apatite samples. Commercial nanohydroxyapatite (nanoHA) and microhydroxyapatite (microHA) provided as powders were kindly supplied by Fluidinova SA-Portugal (nanoXIM_HAp202) and Plasma Biotal-UK (P218), respectively. The samples consisted of cylindrical nanoHA and microHA discs 10 mm in diameter that were prepared from 0.150 g of dry powder under a uniaxial compression stress of 8 MPa (Mestra Snow P3). All experimental conditions related to the compression and sintering procedure were previously published [2,20,24]. Briefly, three different sintering temperatures were used according to the material: 830 °C (nanoHA830) and 1000 °C (nanoHA1000), with a 15-min plateau and applying a heating rate of 20 °C/min, and 1300 °C (microHA1300), with a 1-h plateau and applying a heating rate of 20 °C/min followed by cooling to room temperature inside the oven. The samples were sterilized by two passages in 70 % ethanol during 15 min followed by a double washing in sterile physiological saline (0.9 % NaCl).

Modular reactor set-up. A transparent Perspex (polymethyl­methacrylate), modular reactor containing 27 sampling discs (nanoHA830, nanoHA1000, and microHA1300) was randomly placed in each well (Figs. 1,2). This reactor was connected to a closed Pyrex vessel containing the bacterial culture, which was supplied to the reactor at a continuous flow rate of 1.54 × 10–8 m3/s and an internal velocity of 2.19 × 10–5 m/s by means of a peristaltic pump (RS Amidata) working at 8 rpm. The complete experimental set-up was composed of modular units, a bacterial suspension vessel, a stir plate and magnetic stirrer, a waste vessel, and the circulation tubes (Fig. 1). Three modular units were used in parallel, one for each incubation time (24, 48, and 72 h), and the same conditions were maintained in all of the modular units. This system was operated as “once-through”, i.e., discarding the effluent. The entire reactor was placed inside an incubator to achieve and maintain a temperature of approximately 37 °C, with agitation of the suspension vessel throughout the experiment. All components of the modular reactor were sterilized in an autoclave except for the modular reactor itself, which was sterilized in a 15 % sodium hypochlorite solution and then rinsed with sterilized water under aseptic conditions.

Bacterial strain and culture conditions. Staphylococcus epider­ midis strain RP62A (ATCC 35984), a slime producer [3,6], was used to produce a monospecies biofilm in all experiments in this report. A plate count agar culture of the test strain not older than 2 days was incubated in 15 ml of tryptic soy broth (TSB) for 24 (±2) h at 37 ºC with agitation at 150 rpm by an orbital shaker (Certomat HK, B. Braun Biotech, Göttingen, Germany). An aliquot of 200 μl was transferred to 600 ml of fresh TSB, and the cells were

Biofilms in orthopedic materials

Int. Microbiol. Vol. 16, 2013

193

Fig. 1. Scheme of the experimental system used to obtain biofilm formation in the modular reactors. (A) Bacterial suspension vessel (5 l).

(B) Magnetic stirrer. (C) Peristaltic pumps, 8 rpm. (D) Modular reactors to collect samples at 24, 48, and 72 h. A scheme of the HA discs randomly positioned in the reactor is also given; each color represents a different surface biomaterial. (E) Waste vessel.

allowed to grow for 18 (±2) h, at 37 °C and 150 rpm, until they reached the exponential phase of growth. The inoculum was then transferred to the reactor suspension vessel in a volume of 10 % of the reactor’s useful volume (bacterial suspension containing approximately 1 × 108 cells/ml).

(data not shown). The total numbers of metabolically active and cultivable cells were determined to assess biofilm formation. The structure of the biofilm was visualized by scanning electron microscopy (SEM). Nine discs of each biomaterial were used and all experiments were performed in triplicate.

Biofilm formation on nanohydroxyapatite and microhydro­ xyapatite discs. Staphylococcus epidermidis RP62A biofilm formation on the biomaterial discs was assessed over time. The sterile material samples were placed inside the modular reactor, which was operated under the abovedescribed conditions. At 24, 48, and 72 h of incubation, the respective modular reactor was closed and the discs were collected, gently washed with sterile physiological saline (0.9 % NaCl), immersed in a flask containing 25 ml of sterile 0.9 % NaCl, and sonicated for 45 min in an ultrasonic bath (70 W, 35 kHz, Transsonic 420 ELMA) to release the attached bacteria into the suspension. The sonication time had been properly optimized in a preliminary study

Total cell numbers. The total number of cells in the diluted biofilm suspensions was determined by staining with 4′,6-diamidino-2-phenylindole (DAPI, Merck, D9542), closely following a previously reported method [2].

The averages and standard deviations of the density of the biofilm samples were adjusted to the disc area.

Metabolically active cell numbers. The redox dye 5-cyano-2,3ditolyl tetrazolium chloride (CTC) was used for direct epifluorescence microscopy counting of metabolically active bacteria [7] in dispersed biofilm samples. A 50 mM stock solution of CTC was prepared, filtered through a

Fig. 2. Scheme and image of the modular reactor. (L) length (0.127 m); (H) height (0.018 m), (W) width (0.039 m).

194

Int. Microbiol. Vol. 16, 2013

barros et al.

0.22-mm membrane, and stored in the dark at 4 ºC. For each sample, 200 µl of the biofilm suspension was collected and incubated in 4 mM CTC for 2 h in the dark at 37 ºC with shaking at 130 rpm. The stained suspension was then filtered through a 0.22-μm black polycarbonate membrane and the metabolically active bacteria were examined using an epifluorescence microscope with filter cube N2.1, since CTC excitation and emission occur at 450 and 630 nm, respectively. The averages and standard deviations of the biofilm density were calculated per unit surface area of the disc.

Cultivable cell numbers. The heterotrophic plate count is a procedure for estimating the number of colony-forming units (CFU) corresponding to cultivable bacteria. The method used in this study closely followed one previously reported [2]. The averages and standard deviations of the biofilm samples density were adjusted to the disc area.

Scanning electron microscopy (SEM). The methods for SEM observation and sample preparation closely followed those previously reported [2]. Five fields for each sample were randomly chosen to eliminate the possible uneven distribution of bacteria. Magnification was between 1000 and 15,000×; when required, higher magnifications were used to assess bacterial biofilm morphology and the interactions between the bacteria and the material surfaces.

Statistical analysis. The results of all the biofilm assays were compared using one-way analysis of variance (ANOVA), followed by post-hoc comparisons for all possible combinations of group means by applying the Tukey HSD multiple comparison test using SPSSV Statistics (vs.19.0, Chicago). In all cases, P < 0.05 denoted significance.

The modular reactor and experimental set up described in Materials and methods (Figs. 1,2) was tested in several experiments to confirm use of this system to monitor changes in the growth and accumulation of a biofilm under conditions of a laminar flow rate (1.54 × 10–8 m3/s), low shear stress (2.26 × 10–1 N/m2), and low velocity (2.19 × 10-5 m/s). The reactor internal dimensions were L = 0.127 m, H = 0.018 m, and W = 0.039 m (Fig. 2). The hydrodynamic variables were the hydraulic diameter (2.46 × 10–2 m) and the cross-sectional area of the reactor (7.02 × 10–4 m2). The hydrodynamic flow near the biofilm samplings was positioned after the inlet stabilization zone (2.0 × 10–2 m).

To verify whether the data obtained with the modular reactor were reproducible and therefore suitable for biofilm formation assays, the ability of S. epidermidis RP62A to form biofilms on different ceramic biomaterial discs (nanoHA830, nanoHA1000, and microHA1300) during up to 72 h of incubation was assessed. The results are shown in terms of total cell density (Fig. 3A), metabolically active cell density (Fig. 3B), and cultivable cell density (Fig. 3C). Biofilm structure and morphology were assessed by SEM at 72 h of incubation (Fig. 4).

Int Microbiol

Results

Fig. 3. Attached cells per unit surface area: total (A), metabolically active (B), and cultivable cells (C). The biofilms were grown on nanoHA and microHA discs in the modular reactor operated for 72 h. Different lowercase letters indicate significant differences (P < 0.05) according to a Tukey HSD test. In black, 24 h; light grey, 48 h; and dark grey, 72 h

Biofilms in orthopedic materials

Int. Microbiol. Vol. 16, 2013

195

Fig. 4. SEM micrographs of biofilm growth on nanoHA and microHA discs at 72 h of incubation. The circles indicate the water channels.

Replicates of each biomaterial were placed in randomly chosen locations inside the reactor at 24, 48, and 72 h in order to check reproducibility. Since no significant differences were found among replicates of each biomaterial (P > 0.07), it was concluded that the location of the sample inside the modular

unit, for each incubation time, did not affect the structure or the morphology of the biofilm nor the cell density.

In the S. epidermidis biofilms, similar profiles were obtained for total (Fig. 3A) and cultivable (Fig. 3C) cell density, with an increasing number of bacteria attaching to the bioma-

196

Int. Microbiol. Vol. 16, 2013

terials for up to 48 h. After this time point, the cell density of the biofilm decreased (Fig. 3A,B) except on microHA1300 discs, where the number of total adhered bacteria remained similar between 48 and 72 h of incubation (P > 0.05) (Fig. 3A). The nanoHA830 discs had the highest and the microHA1300 discs the lowest total cell density up to 48 h [(3.01 ± 0.65) × 107 total cells/mm2 and (1.33 ± 0.15) × 107 total cells/mm2, respectively] (Fig. 3A). After 48 h, the biofilms that had formed on the three biomaterials were similar (P > 0.05) (Fig. 3A).

The data obtained for cultivable Staphyloccus epidermidis were also similar, nor were there significant differences (P > 0.05) between the three biomaterials at 48 h (Fig. 3C). As with total cell numbers, after 48 h the highest number of cultivable cells occurred on the nanoHA830 discs [(1.61 ± 0.22) × 106 CFU/mm2] and the lowest number on the microHA1300 discs [(1.06 ± 0.13) × 106 CFU/mm2] (Fig. 3C).

The number of metabolically active cells (CTC-positive) decreased during 72 h of incubation (Fig. 2B). Again, the highest number of metabolically active cells was found on the nanoHA830 discs and the lowest number of on the microHA1300 discs (Fig. 3B).

As seen in the SEM images, mature biofilms had formed on the biomaterial surfaces after 72 h (Fig. 4) and their morphology and structure evidenced the production of extracellular polymeric substance (EPS) (Fig. 4A), three dimensional mushroom-like or pillar-like structures (Fig. 4C,D), and possibly water channels (Fig. 4B). In addition, the biofilms were seen to include multiple layers of bacterial cells (Fig. 4C,D) embedded in EPS (Fig. 4A).

Discussion A variety of laboratory-based model systems are available for the cultivation and study of biofilm communities. An important prerequisite of these systems is that they should simulate both the architecture and the spatial heterogeneity of the microbial community [19]. If the hydrodynamic pattern around the biofilm is overlooked, interpretations of the attachment and growth of microbial layers may be biased, because hydrodynamics directly affect shear stress and substrate mass transfer, and thus, in turn, biofilm development and architecture [28]. In this work, conditions closely mimicking those surrounding human bone, including hydrodynamic parameters such as flow rate and shear stress, were established to grow biofilms in the laboratory. Several studies [6,14,15,28] have shown that the hydrodynamic conditions determine the rate of bacterial transport as well as oxygen and nutrient diffusion to

barros et al.

the surface of the biofilm, thereby determining its structure.

While developing the modular reactor, particular attention was paid to the hydrodynamic entry length, which is an important determinant in stabilizing the hydrodynamic flow and allowed comparisons between data obtained from different locations in the reactor. For rectangular ducts of aspect ratio (width/height) > 2, the entry length in laminar flow can be estimated by the following expression, adapted from Schetz and Fuhs [25]: Le = Dh (0.25 + 0.015 Re) where Le is the entry length, Dh is the hydraulic diameter of the duct, and Re the Reynolds number based on the hydraulic diameter [25]. In the present work, the entry length given by the above equation was around 0.001 m. Since the inlet conditions of the modular reactor did not exactly replicate those indicated by Schetz and Fuhs [25], the real entry length may have been somewhat higher but it was always less than a few millimeters, clearly within the reactor’s inlet stabilization zone (0.02 m). In addition, others aspects were taken into consideration in the reactor’s design, such as easy removal of the colonized substrata (the discs) without disturbing the biofilm formed in other zones of the modular reactors; the use of highquality materials with high corrosion resistance, easy cleaning and sterilization; and the possibility of continuous macro- and microscopic monitoring. Given that one modular reactor was used for each period of incubation (24, 48, and 72 h), the colonization substrata were easily collected without disturbing the biofilm formed on the biomaterial surfaces of the other modular reactors. This kind of system is particularly suited for low flow rate experiments, namely, laminar flow (uniform and rectilinear stream lines), which better simulate orthopedic situations. Moreover it substantially reduces the cost of culture media preparation [29].

The results obtained with this reactor system are comparable to those already described by different authors using other experimental set ups. For example, similar total cell densities on different materials after 48 h were reported by Huang et al. [17], who grew E. coli biofilms in a parallel-plate flow cell reactor. The experiments of Shapiro et al. [26] were aimed at obtaining reproducible S. epidermidis RP62A biofilms on glass slides with the DFR system (Drip Flow Biofilm) and they established very low flow velocities such as in the present work. Those authors found that the mean number of viable bacteria in the biofilms increased until 48 h, with no significant differences occurring after this time among the different conditions. In another study, by Pereira et al. [23], in

Int. Microbiol. Vol. 16, 2013

Biofilms in orthopedic materials

which a flow cell reactor was used, the bacterial density on Perspex plates increased over time and the cultivable and total cell densities followed a similar pattern as in our system. Thus, although different morphological and chemical surface properties may affect the initial attachment, in the long term they do not seem to greatly affect the final build-up of the biofilm.

A decrease over time in the number of metabolically active cells (assessed in this study by CTC) was also recorded

by Créach et al. [7], in their study of the growth of E. coli on M63 + 0.01 % d-glucose. One possible explanation for this reduction in cell numbers is the effect of EPS (see below), which in significant amounts tends to limit substrate penetration through the biological matrix.

Figure 4 shows the architecture of the relatively mature biofilm and substantial EPS production. The same indicators of a mature biofilm were noted by Williams et al. [31], who used a modified CDC (Center for Disease Control and Prevention, USA) biofilm reactor to develop mature biofilms of S. aureus on the surface of polyetheretherketone (PEEK).

Well-established, mature biofilms reinforce bacterial resistance to antibiotics and influence the rate of genetic material exchange between microorganisms organized in these structures [5,8]. Thus, the reproduction of these biofilms in in vitro reactor models is clinically relevant in studying biofilm-related infections [15].

Molecular biology has allowed many new insights into biofilm development. Thus, it is now known that the icaADBC operon in S. epidermidis controls the production of polysaccharide intercellular adhesin (PIA) [12] and that this operon may in turn be controlled by oxygen levels [6]. Cotter et al.

[6] reported that higher oxygen levels reduce biofilm formation via repression of the icaADBC operon and consequently reduce the production of PIA. In our study, the low oxygen levels may have contributed to the high production of PIA in the biofilms formed on all of the biomaterial surfaces. In the micrographs, the cracks observed in the biofilms may have formed because of shrinkage during dehydration processing.

Similar artifacts were observed by Williams et al. [30] in the biofilm matrix of S. epidermidis ATCC 35984 grown using the CDC biofilm reactor.

The prevention of medical device contamination by biofilm formation remains a challenge. A promising strategy is the development of new surfaces containing two or more antibacterial agents differentially targeting the different microrganisms present in biofilms. The screening and assessment of this and other technical solutions require reliable laboratorybased systems that closely simulate physiological conditions and are easy to operate.

197

The modular reactor developed for this study proved to be useful in monitoring reproducible biofilm development under laboratory-controlled conditions. It allowed for periodic sampling by the removal of colonized discs without the need to stop the flow, thus minimizing the contamination risk and the disturbance of the biofilms forming on the other discs. In addition, together with off-line SEM observations, our reactor system provided information on the build-up of a mature biofilm (EPS production and the formation of three dimensional mushroom-like or pillar-like structures as well as water channels) on different biomaterials, under conditions similar to those that prevail in the vicinity of human-bone implants.

Acknowledgements. The authors acknowledge financial support for this work by a research grant to J. Barros from the FEDER funds through COMPETE and by FCT–Fundação para a Ciência e a Tecnologia in the framework of the project NanoBiofilm (PTDC/SAU-BMA/111233/2009).

Also, the provision of nanoHA (nanoXIM) by FLUIDINOVA, SA (MaiaPortugal) is greatly acknowledged.

Competing interest. None declared.

References 1. An YH, Friedman RJ (1997) Laboratory methods for studies of bacterial adhesion. J Microbiol Methods 30:141-152 2. Barros J, Grenho L, Manuel C, Ferreira C, Melo L, Nunes O, Monteiro F, Ferraz M (2013) Influence of nanohydroxyapatite surface properties on Staphylococcus epidermidis biofilm formation. J Biomat Appl. doi: 10.1177/0885328213507300 3. Cerca N, Pier GB, Vilanova M, Oliveira R, Azeredo J (2005) Quantitative analysis of adhesion and biofilm formation on hydrophilic and hydrophobic surfaces of clinical isolates of Staphylococcus epidermidis.

Res Microbiol 156:506-514 4. Cheatle MD (1991) The effect of chronic orthopedic infection on quality-of-life. Orthop Clin North Am 22:539-547 5. Costerton W, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G (2007) The application of biofilm science to the study and control of chronic bacterial infections. J Clin Invest 11:1466-1477 6. Cotter JJ, O’Gara JP, Stewart PS, Pitts B, Casey E (2010) Characterization of a modified rotating disk reactor for the cultivation of Staphylococ­ cus epidermidis biofilm. J Appl Microbiol 109:2105-2117 7. Creach V, Baudoux AC, Bertru G, Rouzic BL (2003) Direct estimate of active bacteria: CTC use and limitations. J Microbiol Methods 52:19-28 8. Cvitkovitch DG (2001) Genetic competence and transformation in oral streptococci. Crit Rev Oral Biol Med 12:217-243 9. Darley ESR, MacGowan AP (2004) Antibiotic treatment of Gram-positive bone and joint infections. J Antimicrob Chemother 53:928-935 10. Ferraz MP, Mateus AY, Sousa JC, Monteiro FJ (2007) Nanohydroxyapatite microspheres as delivery system for antibiotics: Release kinetics, antimicrobial activity, and interaction with osteoblasts. J Biomed Mater Res A 81:994-1004 11. Ferraz MP, Monteiro FJ, Manuel CM (2004) Hydroxyapatite nanoparticles: A review of preparation methodologies. J Appl Biomater Biomech 2:74-80

198

Int. Microbiol. Vol. 16, 2013

12. Fey PD, Olson ME (2010) Current concepts in biofilm formation of Staphylococcus epidermidis. Future Microbiol 5:917-933 13. Gattlen J, Zinn M, Guimond S, Korner E, Amberg C, Mauclaire L (2011) Biofilm formation by the yeast Rhodotorula mucilaginosa: process, repeatability and cell attachment in a continuous biofilm reactor. Biofouling 27:979-991 14. Gilmore BF, Hamill TM, Jones DS, Gorman SP (2010) Validation of the CDC biofilm reactor as a dynamic model for assessment of encrustation formation on urological device materials. J Biomed Mater Res B Appl Biomater 93:128-140 15. Goeres DM, Loetterle LR, Hamilton MA, Murga R, Kirby DW, Donlan RM (2005) Statistical assessment of a laboratory method for growing biofilms. Microbiology 151:757-762 16. Grenho L, Manso MC, Monteiro FJ, Ferraz MP (2012) Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa onto nanohydroxyapatite as a bone regeneration material. J Biomed Mater Res A 100:1823-1830 17. Huang CT, Peretti SW, Bryers JD (1992) Use of flow cell reactors to quantify biofilm formation kinetics. Biotechnol Tech 6:193-198 18. Kajiyama S, Tsurumoto T, Osaki M, Yanagihara K, Shindo H (2009) Quantitative analysis of Staphylococcus epidermidis biofilm on the surface of biomaterial. J Orthop Sci 14:769-775 19. Lawrence JR, Swerhone GDW, Neu TR (2000) A simple rotating annular reactor for replicated biofilm studies. J Microbiol Methods 42:215-224 20. Lopes MA, Monteiro FJ, Santos JD, Serro AP, Saramago B (1999) Hydrophobicity, surface tension, and zeta potential measurements of glassreinforced hydroxyapatite composites. Biomed Mater Res A 45:370-375 21. Manuel CM, Nunes OC, Melo LF (2007) Dynamics of drinking water biofilm in flow/non-flow conditions Water Res 41:551-562 22. Mittelman MW, Kohring LL, White DC (1992) Multipurpose laminar flow adhesion cells for the study of bacterial colonization and biofilm formation. Biofouling 6:39-51

barros et al.

23. Pereira MO, Morin P, Vieira MJ, Melo LF (2002) A versatile reactor for continuous monitoring of biofilm properties in laboratory and industrial conditions. Letters Appl Microbiol 34:22-26 24. Santos JD, Knowles JC, Reis RL, Monteiro FJ, Hastings GW (1994) Microstructural characterization of glass-reinforced hydroxyapatite composites. Biomaterials 15:5-10 25. Schetz JA, Fuhs AE (1999) Fundamentals of fluid mechanics. In: Schetz JA, Fuhs AE (eds) John Wiley, NY, USA. 26. Shapiro JA, Nguyen VL, Chamberlain NR (2011) Evidence for persisters in Staphylococcus epidermidis RP62A planktonic cultures and biofilms.

J Med Microbiol 60:950-960 27. Simchi A, Tamjid E, Pishbin F, Boccaccini AR (2011) Recent progress in inorganic and composite coatings with bactericidal capability for orthopaedic applications. Nanomedicine: NBM 7:22-39 28. Simoes M, Pereira MO, Vieira MJ (2007) The role of hydrodynamic stress on the phenotypic characteristics of single and binary biofilms of Pseudomonas fluorescens. Water Sci Technol 55:437-445 29. Stoodley P, Hall-Stoodley L, Costerton B, et al. (2012) Biofilms, biomaterials, and device-related infections. In: Ratner BD, et al. (eds) Biomaterials science: an introduction to materials in medicine. 3rd ed. Elsevier, p. 565-583 30. Williams DL, Bloebaum RD (2010) Observing the biofilm matrix of Staphylococcus epidermidis ATCC 35984 grown using the CDC biofilm reactor. Microsc Microanal 16:143-152 31. Williams DL, Woodbury KL, Haymond BS, Parker AE, Bloebaum RD (2011) A modified CDC biofilm reactor to produce mature biofilms on the surface of PEEK membranes for an in vivo animal model application.

Curr Microbiol 62:1657-1663

PERSPECTIVES International Microbiology (2013) 16:199-203 doi: 10.2436/20.1501.01.194 ISSN 1139-6709 www.im.microbios.org

Gold or green: the debate on Open Access policies Ernest Abadal Faculty of Library and Information Science, University of Barcelona, Barcelona, Spain Received 16 August 2013 · Accepted 20 September 2013

Summary. The movement for open access to science seeks to achieve unrestricted and free access to academic publications on the Internet. To this end, two mechanisms have been established: the gold road, in which scientific journals are openly accessible, and the green road, in which publications are self-archived in repositories. The publication of the Finch Report in 2012, advocating exclusively the adoption of the gold road, generated a debate as to whether either of the two options should be prioritized. The recommendations of the Finch Report stirred controversy among academicians specialized in open access issues, who felt that the role played by repositories was not adequately considered and because the green road places the burden of publishing costs basically on authors. The Finch Report’s conclusions are compatible with the characteristics of science communication in the UK and they could surely also be applied to the (few) countries with a powerful publishing industry and substantial research funding. In Spain, both the current national legislation and the existing rules at universities largely advocate the green road. This is directly related to the structure of scientific communication in Spain, where many journals have little commercial significance, the system of charging a fee to authors has not been adopted, and there is a good repository infrastructure. As for open access policies, the performance of the scientific communication system in each country should be carefully analyzed to determine the most suitable open access strategy. [Int Microbiol 2013; 16(3):199-203] Keywords: open access · scientific communication ∙ scientific journals ∙ repositories ∙ open access policies

Introduction The earliest public demonstrations in favor of open access go back some 12 years, with the letter of the Public Library of Science (2001) and the Budapest Open Access Initiative

Corresponding author: E. Abadal Faculty of Library and Information Science University of Barcelona Melcior de Palau, 140 08014 Barcelona, Spain Tel. +34-934035787. Fax +34-934035772.

E-mail: abadal@ub.edu *

(2002). Both advocated a change in the model of science communication and essentially proposed unrestricted, free access to academic content. Now, more than 10 years later, the open access movement has matured, in the sense that it is widely known by all agents of science communication— whether they be authors, publishers or librarians. Moreover, it has acquired remarkable institutional support from universities, research funding agencies, and the European Union, among others.

This maturity is also confirmed by the many studies on open access published in the intervening years and focusing on scientific journals, repositories, authors, legal aspects, etc. These have been partially compiled by Bailey in two

200

Int. Microbiol. Vol. 16, 2013

bibliographies [2,3]. In addition, this topic has been dealt with in texts of wider dissemination. For example, STM Reports [15,16]—published by the International Association of Scientific, Technical & Medical Publishers (STM), the leading global trade association for academic and professional publishers whose members are responsible for the publication of 66 % of all journal articles—analyzes the current state of science editing and devotes a good part of its content to open access. Prestigious journals such as Nature have also published several monographs on open access, including the recent “The Future of Publishing” [12].

Open access advocates are convinced that scientific com­ munication would be improved if all academic content were accessible on the Internet, unrestricted and free of charge.

But, when will this vision become reality? How long will it take for all or most scientific publications to be openly accessible? Until recently, the growth of open access had to be assessed qualitatively and indirectly. But today there are estimates on the quantitative impact of open access in the science communication system. These estimates have been made with respect to either the total number of journals or the total number of articles, which serve as two different kinds of indicators.

As for the number of journals, in 2013, Ulrich’s directory, which included scientific journals from all over the world, listed 8,000 open access titles, corresponding to 13.5 % of all peer-reviewed journals (some 60,000 worldwide). If we focus exclusively on the elite journals, those listed by the Web of Science (WoS) or by Scopus, the percentages are a bit lower but in no case are they negligible. In 2013, out of the 10,763 titles in the WoS database, 1,111 (10.3 %) were open access journals (figures taken from the Ulrich directory), while according to Scopus among the 18,500 indexed journals some 1,800 (9.7 %) were open access titles (figures taken directly from Scopus). These similar, substantial percentages provide proof that the quality of open access journals has been acknowledged.

The distribution of open access titles across countries is not homogeneous; rather two extremes are evident. At the lower end are countries with an important tradition in commercial publishing, especially the USA, the UK, the Netherlands, and Germany; on the opposite extreme are emerging economies, for example, Brazil, where over 90 % of the journals published are open access [11].

As for the number of articles in open access, several studies have provided data-based estimates, in both cases derived from samples. Laakso-Bjork [10] focused on articles indexed in Scopus, reporting in 2012 that 17 % were open access (12 %

ABADAL

immediately after publication, and 5 % after an embargo period). A study conducted two years earlier and referring to the total number of articles published [4] estimated that 20 % were open access (8.5 % in portals from publishers and 11.9 % in repositories). Thus it has taken some twelve years to have approximately 20 % of all scientific content unrestrictedly and freely accessible from the Web. This is remarkable progress, even if it is still insufficient to totally transform the science communication system.

For open access to become widely adopted and cover all manner of scientific content, political measures that prioritize this means of publication and dissemination should be instituted. Two mechanisms were advocated by the Budapest Initiative (2002). The aim of what was later referred to as the “gold road” was to ensure that most journals are open access; this is in contrast to what was later called the “green road,” in which the focus is on archiving articles in repositories, as a transitional stage until full implementation of the open access model. These two mechanisms have been equally defended by the open access movement, as, by necessity, they are considered as being complementary.

The UK’s Finch Report [7,8], published in 2012, advocated the exclusive adoption of the gold road in order to reach open access. Its conclusions have generated heated debate as to whether either of the two options should be given priority.

The document has had a remarkable impact not only within the academic world but also among the general public, thanks to its dissemination through the media. In the following, we describe and assess the proposals included in the Finch Report and analyze their possible application to other countries, and particularly to Spain.

The Finch Report The British government charged Janet Finch, Professor of Sociology at the University of Manchester, to conduct a study aimed at determining how all publicly funded research could be made accessible without restrictions and at no cost. The determining factors that had to be respected from the start were: (i) to maintain the high level of quality of the scientific publications (by means of peer review) and (2) to not harm the important British publishing industry.

In the Finch Report, access to scientific information in the UK is analyzed, including a quantification of research and of journal subscriptions costs. Both the communication and dissemination of results as an integral part of research itself and the need for research budgets to include publication

Open Access policies

fees are recognized [8]. After establishing that open access is the horizon for science communication, the Finch Report suggests that the gold road provides a strategy for all science communications in the UK. Specifically, it recommends that the costs of science communication and dissemination be included in research budgets and the launch of a system in which open access journals are funded through author payments. This proposal respects the mandate of the Government while counting on the support of British science publishers.

The Finch Report was released on 18 June 2012. A month later, the British Government announced that it had accepted its recommendations, a move accompanied by changes in the open access policies of the Research Councils, which are the institutions that fund research in the UK. However, the Finch Report generated intense controversy among academicians specialized in open access, because its recommendations did not take into account the function of repositories (thereby distancing itself from that segment of the open access movement that advocates the adoption of both roads) and it laid the burden of article processing charges exclusively on authors.

Underestimation of repositories The Finch Report focused primarily on journal articles, leaving aside monographs and “grey literature,” despite referring to both in several parts of the document. In addition, when it deals with repositories the Finch Report points out several already-known weaknesses, including the small volume of documents they contain, the lack of indexing of their contents in databases, and the often insufficient quality of the access services offered. The role of repositories is, in the end, to facilitate access to research, theses, and grey literature.

Strengthening of the role of repositories to ensure a change in the model of science communication has been encouraged from many quarters. For institutions, the latest recommendations of the Budapest Open Access Initiative [5] maintain the validity of the two roads (gold and green) and insist on the need for repository infrastructures: “3.1 Every institution of higher education should have an OA repository, participate in a consortium with a consortial OA repository, or arrange to outsource OA repository services.”

From the academic sphere, John Houghton and Alma Swan [9] agree that in a fully open access system the net

Int. Microbiol. Vol. 16, 2013

201

benefits of the gold road are higher than those of the green one. However, taking into account that we are in a transitional phase, those authors concluded that repositories are still the most economical and flexible way to make progress towards open access, based on two advantages. Firstly, the green road makes it possible to include any research work, even those that are not strictly journal articles (i.e., doctoral theses, books, working papers, reports, and congresses), which is especially relevant in the humanities and the social sciences, in which research is not disseminated exclusively by means of scientific journals. Secondly, the obligation of depositing scientific production is a political decision that can be adopted unilaterally (which therefore makes it faster than the gold road, in which a more complex global agreement is required) by any funder or institution as well as at the state level, and at relatively low cost. Peter Suber [14] added a further, economic argument in favor of repositories: they entail no costs for the depositor.

Article Processing Costs The Finch model is based on author payment of publication fees. This decision has been welcomed by publishers, as their businesses will be maintained even if the collection of fees is shifter from users to authors. Among academicians, however, the concept of author payment has led to heated discussion as well as to doubts about the viability of the model since it is not entirely clear how authors without funds for their research will manage to pay publication fees.

It is worth noting that publication in open access journals can be funded not only by the authors themselves but also by the publisher or, even, by libraries (as would be the case in the SCOAP3 project). In this regard, the Budapest Initiative is very clear; its recommendation 3.5 proposes a model of reasonable article processing costs and, importantly, favors institutional funding of open access journals.

“3.5. Universities and funding agencies should help authors pay reasonable publication fees at fee-based OA journals, and find comparable ways to support or subsidize no-fee OA journals.” [5] The proposal of the Finch Report can be understood and appreciated in countries with a powerful and consolidated publishing market (as is the case in the UK, the USA, the Netherlands, and Germany), with strong national funding agencies, both public and private, that sustain R+D. In those

202

Int. Microbiol. Vol. 16, 2013

countries, it is not difficult for authors to obtain financial resources for publishing. What happens, however, in countries and in disciplines where financial aid for research is in short supply? In such cases, the proposals of the Finch Report are not feasible and other ways, tailored to the particular conditions and circumstances, must be found. This is the case of Brazil, where open access is near 90 % (as stated above), and of other emerging countries but also of Spain and other countries in southern Europe.

As mentioned above, the same problems confront the humanities and social sciences, since research in either field is only modestly funded. Scientists in these disciplines typically support open access but are quick to point out that the authorpays system is a serious disadvantage. According to the SOAP study [13], this problem was mentioned by 39 % of researchers who would like to publish in open access journals but have difficulties in finding the financial resources to cover the necessary publication fees.

Open access in Spain Spanish support of open access has given rise to state legislation and university regulations that deal with this issue.

Article 37 of the Science, Technology and Innovation Act [6] cites the obligation of depositing the results of research funded by the state’s budget in open access repositories, taking into account limitations based on author’s copyrights.

In addition, the latest Royal Decree on Doctoral Studies (2011) includes the obligation of depositing all theses in open access repositories.

University mandates regarding open access require that the scientific output of academic staff be published in open access journals or placed in open access repositories. These regulations apply broadly and not only to publications resulting from funded projects, as indicated in the Spanish law. One of the first Spanish universities to approve the mandate policies was the Technical University of Catalonia, in 2009. Since then, twelve other centers have joined in [1].

Both legislation and mandates give priority to the green road, i.e., the archiving of scientific production in repositories.

While publication in open access journals is also valued, there are neither incentives nor state funding proposals, in contrast to the Finch Report.

In Spain, the ‘author pays’ model is rarely used, although some journals offer the option of freeing articles. Spanish open access journals account for 35 % of the total—quite a bit higher than the above-mentioned worldwide average of 14 %. Most of

ABADAL

these journals are funded by institutions linked to the public sector, such as universities and public research centers, or learned societies and academies, e.g., the Institute for Catalan Studies. In the sphere of the humanities and social sciences, no part of the scant funds devoted to research is allocated to the payment of publication fees.

Currently, there are 112 repositories, according to the BuscaRepositorios directory. Most universities and research centers have this type of infrastructure, which is well known among the scientific community. According to Webometrics, seven of these Spanish repositories rank among the top 100 in the world. They are those of the Autonomous University of Barcelona, the Technical University of Catalonia, The National Science Research Council (CSIC), The Complutense University of Madrid, the University of Alicante, the University of Salamanca, and the Technical University of Madrid [http:// repositories.webometrics.info/en/Europe/Spain].

Thus, current legislation and regulations in Spain clearly advocate the green road, as it is consistent with the country’s science communication system, in which many journals have little commercial presence (only 28 %), a very low implementation rate of the ‘author pays’ system, but a good repository infrastructure.

Conclusions Open access has grown moderately yet steadily over the last 15 years such that it is currently estimated to comprise 20 % of the total of the science communication system (journals and articles). To date, policies favoring open access have been based on two strategies, fostering publication in OA journals (the gold road) and the archiving of publications in repositories (the green road). The recommendations of the Finch Report, which exclusively supported the gold road, have ignited controversy.

The merit of the Finch Report is its defense of a clear, global, and overwhelming policy supporting open access by the public administration. However, it has been criticized because it exclusively advocates the gold road and the payment of publication fees by authors, thus overlooking the role of repositories and access to materials that are not articles.

In the case of Spain, state legislation and existing uni­ versity mandates generally favor the green road. This model fits well with the characteristics of Spanish science communication, i.e., a significant presence of the humanities and social sciences (for which the article is not the essential item for publication), a low presence of commercial publishers

Open Access policies

of scientific journals, and a good existing infrastructure for repositories.

In considering open access policies, we should carefully analyze the performance of the science communication system in each country to determine the most suitable approach to providing open access. Accordingly, the recommendations of the Finch Report should be confined to the UK and other countries with a powerful publishing industry and wellfunded research. Finding the best road to open access in other countries is not possible without studying their research systems in detail.

Acknowledgements. This study is a part of the activity of Acceso abierto a la ciencia research group [http:// accesoabierto.net]. It was funded by the Spanish Plan Nacional de I+D+i CSO2011-29503-C02-01/SOCI.

References 1. Abadal E, Ollé-Castellà C, Abad-García F, Melero R (2013). Políticas de acceso abierto a la ciencia en las universidades españolas. Rev Españ Document Cient 36:e007 [http://dx.doi.org/10.3989/redc.2013.2.933] 2. Bailey CW Jr (2010) Transforming scholarly publishing through open access: A bibliography. Digital scholarship, Houston [http://digitalscholarship.org/tsp/ transforming.htm] 3. Bailey CW Jr (2005-2010) Open access bibliography: Liberating scholarly literature with e-prints and open access journals. Assoc Res Libraries, Washington DC [http://www.digital-scholarship.org/oab/oab.pdf] 4. Björk BC, et al. (2010) Open access to the scientific journal literature: Situation 2009. PLoS One 5(6) [http://dx.doi.org/10.1371/journal. pone.0011273]

Int. Microbiol. Vol. 16, 2013

203

5. BOAI (2012) Budapest Open Access Initiative: Ten years on from the Budapest Open Access Initiative: setting the default to open [http://www. opensocietyfoundations.org/openaccess/boai-10-recommendations] 6. BOE (2011) Ley 14/2011, de 1 de junio, de la Ciencia, la Tecnología y la Innovación. BOE 131, 2nd June 2011 [http://www.boe.es/boe/ dias/2011/06/02/ pdfs/BOE-A-2011-9617.pdf] 7. Finch, J (2012) Accessibility, sustainability, excellence: How to expand access to research publications. Report of the Working Group on Expanding Access to Published Research Findings [http://www. researchinfonet.org/wp-content/uploads/2012/06/Finch-Group-reportFINAL-VERSION.pdf] 8. Finch J, et al. (2013) Accessibility, sustainability, excellence: how to expand access to research publications. Executive summary. Int Micro­ biol 16:125-132 9. Houghton J, Swan A (2013) Planting the green seeds for a golden harvest: comments and clarifications on ‘Going for Gold’. D-lib magazine 19(12) [http://dx.doi.org/ 10.1045/january2013-houghton] 10. Laakso M, Björk B-C (2012) Anatomy of open access publishing - a study of longitudinal development and internal structure. BMC Medicine 10:124 [http://dx.doi.org/10.1186/1741-7015-10-124] 11. Rodrigues R, Oliveira AB (2012) Periódicos científicos na America Latina: títulos em acesso aberto indexados no ISI e Scopus. Perspectivas em Ciência da Informação 17(4):76-99 [http://portaldeperiodicos.eci. ufmg.br/index.php/pci/article/view/1593] 12. Several authors. The future of publishing (2013) Nature 495 (7442) [http://www.nature.com/news/specials/scipublishing/index.html] 13. SOAP (2011) Report from the SOAP Symposium, 2011: Berlin. SOAP (Study of Open Access Publishing) [http://project-soap.eu/report-fromthe-soap-symposium] 14. Suber P (2012) Open Access. MIT Press, Boston [http://mitpress.mit. edu/ books/open-access] 15. Ware M, Mabe M (2009) The STM report: An overview of scientific and scholarly journals publishing. STM, Oxford [http://www.stm-assoc.org/ 2009_10_13_ MWC_STM_Report.pdf] 16. Ware M, Mabe M (2012) The STM report: An overview of scientific and scholarly journals publishing. 3rd ed. STM, Oxford [http://www.stmassoc.org/2012_12_11_ STM_Report_2012.pdf]

MEETINGS International Microbiology (2013) 16:205-209 doi: 10.2436/20.1501.01.195 ISSN 1139-6709 www.im.microbios.org

The 24th Congress of the Spanish Society for Microbiology (L’Hospitalet de Llobregat, Barcelona, 10–13 July 2013) Ester Fusté, Miguel Viñas Laboratory of Molecular Microbiology and Antimicrobials, Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of Barcelona-IDIBELL, Campus of Bellvitge, L’Hospitalet, Barcelona, Spain E-mails: esterfustedominguez@ub.edu; mvinyas@ub.edu

The 24th Congress of the Spanish Society for Microbiology (SEM) took place on 10–13 July, 2013 at the Bellvitge Cam­ pus of the University of Barcelona (UB), in L’Hospitalet de Llobregat, Barcelona [http://congresosem2013.semi­ cro­ biologia.org] (Fig. 1). This meeting brought together 618 micro­ biologists from several prestigious universities and research centers throughout Spain, as well as experts from 24 countries including the United States, the United Kingdom,

Germany, Australia, Canada, France, Italy, Belgium, Mexico, Austria, Chile, China, Denmark, Slovenia, the Netherlands, Peru, Sweden, Scotland, Turkey, Uruguay, and Venezuela.

In addition to the 17 symposia, there were around 300 free communications, in the form of oral and poster presentations, with contributions from 1008 authors. This scientific summit meeting was the effort of an organizing committee led by one of us (MV), and other members of the Department of

Fig. 1. Barcelona’s skyline, with the Bellvitge Campus of the University of Barcelona, at the left. © M.Berlanga

Int. Microbiol. Vol. 16, 2013

fuste, viñas

Int Microbiol

206

Fig. 2. Ceremonial Main Hall (Paranimph) of the University of Barcelona (left), and upper-floor cloister of the Institute for Catalan Studies (right), where the opening and closing ceremonies, respectively, of the conference were held.

Pathology and Experimental Therapeutics, University of Barcelona-IDIBELL, and by a scientific committee headed by Albert Bosch, President of the Spanish Society for Virology.

One of the main goals of the meeting was to stimulate the participation of young microbiologists. The topics covered by the program included new frontiers in research on the molecular basis of pathogenicity and bacterial resistance, fungal virulence, antimicrobial agents in biodegradation and bioremediation in polluted environments, “-omics” techniques in food microbiology, and bacteriophages in industrial microbiology.

The opening ceremony was presided over by Enric Canela, Vice-Rector of the University of Barcelona; Ricardo Guerrero, current President of the SEM; Miguel Viñas, President of the Congress; and Antonio Ventosa, President-elect of the SEM. This event was held at the Ceremonial Main Hall (Paranimph), at the historical site of the University of Barcelona. Andrés Moya, Professor at the University of Valencia, delivered the opening lecture, “From minimal cells to microbiome.” David Rodríguez Lázaro, from the University of Burgos, gave the closing address, “Molecular approaches to food security,” at the Institute for Catalan Studies (IEC) (Fig. 2).

The different sessions of the conference were held at the modern premises of the Faculty of Medicine, Campus of Bellvitge, of the University of Barcelona (Fig. 3).

Microbial biotechnology A series of talks examined the key role of microbes in the deterioration of buildings and monuments around the

world (S. Betts, M. Urizal, Thor Especialidades, S.A.; J.M. Vaquero, BASF; A. de los Ríos, National Museum of Natural Science, CSIC, Madrid). To combat the effects of fouling microorganisms, mainly fungi and algae, biocides are typically used. These include encapsulated biocides to control cement biodeterioration, antimicrobial paints to avoid the dissemination of pathogenic bacteria, and widespectrum biocides to protect stone monuments and minimize environmental damage.

Microbial diseases and antimicrobial resistance Our knowledge on recent as well as more well-established infectious diseases and antimicrobial resistance was brought up to date in a series of lectures. Among them, the pathogenesis of diseases caused by Candida species, the genetic variability of this fungus, and its strong resistance to antifungal agents were discussed (G. Larriba, University of Extremadura; G. Quindós, University of the Basque Country).

Brucellosis, one of the most common bacterial zoonoses worldwide, was a health problem until a few years ago in Spain but currently of great importance in other countries of the Mediterranean basin, such as those in northern Africa (I. López, University of Navarra, Pamplona). It was pointed out that although there are several vaccines to control this disease in animals, none of them is still completely effective.

Improved efforts to reduce the incidence of human brucellosis will require an understanding of the coordinated role of virulence factors.

24th SEM Congress

207

capture in cells undergoing stress, while preserving the cassette arrangement in stable environments.

Finally, recent results were presented on a new family of mobile genomic islands (MGIs) identified in Vibrio species and other marine Gammaproteobacteria and on the contribution of these MGIs to virulence (V. Burrus, University of Sherbrooke, Canada).

Industrial and food microbiology The conference included talks on the state of the art of industrial microbiology, including the differentiation and development of Streptomyces in bioreactors and the involvement of these bacteria in the production of secondary metabolites (A. Manteca, University of Oviedo), the response to oxidative stress in Actinobacteria (L.M. Mateos, University of Leon), and the relevance of genomic analysis in the search for peroxidases of industrial interest (A. Martínez, Biological Research Centre, CIB, CSIC, Madrid).

The symposium on food microbiology was devoted to the “-omics” techniques (proteomics, genomics, and meta­­genomics) commonly used in this field (E. Jiménez, Complutense

Int Microbiol

The most common antimicrobial resistance mechanisms of pathogens such as Streptococcus pneumonia and Haemophilus influenzae, frequently isolated from patients with chronic obstructive pulmonary disease (COPD), were also reviewed (J.A. Martínez, University of Barcelona; A. Domènech, Bellvitge University Hospital). Other lectures discussed the relevance of new sequencing platforms in determining the phylogeny of Clostridium species (S. Valdezate, National Center of Microbiology, CNM, Madrid), a new method of fast genotyping for Coxiella burnetii, the microorganism that causes Q fever (I. Jado, CNM), and the increasing interest in Europe in hepatitis E, a complex infectious disease currently considered as both zoonotic and imported (J.M. Echevarría, CNM).

The importance of plasmid-encoded proteins associated with chromatin, i.e., H-NS proteins, in the regulation of conjugation and the selection of some genes was highlighted.

In that same talk, integrons, the genetic elements responsible for the capture and spread of antibiotic resistance determinants among gram-negative bacteria, were shown to be linked to the SOS response (D. Mazel, Institut Pasteur, Paris). This coupling enhances the potential for cassette swapping and

Int. Microbiol. Vol. 16, 2013

Fig. 3. Artistic interpretation of the main buildings, hospital and Faculty of Medicine of the Campus of Bellvitge, University of Barcelona, L'Hospitalet, where the different sessions of the conference were held.

208

Int. Microbiol. Vol. 16, 2013

fuste, viñas

University of Madrid; B. Sánchez and S. Delgado, Dairy Institute of Asturias, Villaviciosa, CSIC), including their advantages and disadvantages compared with conventional techniques. Also discussed were the software tools used in data analysis and the relevance of the integration of different “-omics” techniques to study microorganism of interest in food microbiology (E. Smid, Wageningen University, the Netherlands).

Symposium in memoriam Margulis (1938–2011)

of

Prof.

Lynn

The conference included the Symposium “Microbes, symbiosis and evolution”, chaired by R. Guerrero (University of Barcelona), as a tribute to the world-renowned American biologist Lynn Margulis (1938–2011), Distinguished Professor of the University of Massachusetts-Amherst, who in 1967 proposed the endosymbiotic theory to explain the origin of the eukaryotic cell.

All animals and plants carry in their bodies very diverse microbial communities. The development of mole­ cular techniques has allowed the identification of these microorganisms, whose fidelity has continued, generation after generation, throughout the evolutionary history of their hosts. The same tools have been used to elucidate the chemical signals that mediate host-microbe communication.

The symposium took note of the new trends in biology, and in microbiology in particular, focusing on the continuous cooperation and interdependence between microbes and “macrobes.” Margulis saw evolution as a race in which progress is achieved not by those organisms that seek to dominate but by those that cooperate for a common purpose.

She showed the friendly face of evolution, that of a world that has thrived through cooperation and altruism.

Symbiosis, a term coined by Heinrich Anton de Bary (1831–1888), refers to the co-habitation of “differently named organisms.” Symbiotic relationships are long-term physical associations that occur under specific environmental conditions. In endosymbiosis, which can be viewed as a topological state, one of the symbiotic partners lives inside the other. The process by which long-term stable symbiosis leads to evolutionary changes is called “symbiogenesis,” a term coined by the Russian botanist K.S. Merezhkovsky and rescued by Lynn Margulis. It refers to the appearance of new behaviors, morphologies, tissues, metabolic pathways, and taxa (including species), or other recognizable evolutionary novelties.

Examples of symbiogenesis in the animal kingdom, in which animals have adapted to specialized ecological niches, were provided in the talks by I. Uriz (Centre for Advanced

Studies of Blanes, CEAB, CSIC) and M. Berlanga (University of Barcelona) on sponges and xylophagous insects, respectively. The symposium ended with the contributions of N. Skinner (Institute for Catalan Studies, IEC, Barcelona) and A. Omedes (Natural History Museum of Barcelona) talking about communication and diffusion of science, other field in which Lynn Margulis was both outstanding and world-wide known. In her talk N. Skinner (Institute for Catalan Studies) addressed the role of the Internet, and examined the impact of social networks on scientific research and the dissemination of science. Finally, A. Omedes (Natural History Museum of Barcelona) presented an example of public outreach, in the form of the new permanent exhibition of the Museu Blau in Barcelona. Microbes, symbiotic relationships among organisms, and environmental interactions are the driving organizational forces in the newly renovated museum’s fascinating exhibits.

Symposium in memoriam of Prof. Miquel Regué (1953–2012) The conference also included a plenary session in memoriam to Miquel Regué (1953–2012), Professor of Microbiology at the Faculty of Pharmacy of the University of Barcelona, who passed away last year. M. Viñas, A. Juárez, and J. Tomás (University of Barcelona) reflected on Miquel Regué’s personal and professional life. R. Benz, Wisdom Professor of Biophysics and Microbiology at Jacobs University (Bremen, Germany) and Doctor Honoris Causa recipient from the University of Barcelona, Spain, and University of Umeå, Sweden, reviewed the structure and role of porins in gram-positive bacteria. Many gram-positive bacteria have an unusual cell envelope. Besides the thick peptidoglycan layer, they contain large amounts of lipids in the form of mycolic acids and free lipids in their cell walls. The mycolic acids are linked through ester bonds to the arabinogalactan attached to the murein of the cell wall. The chain length of these 2-branched, 3-hydroxylated fatty acids varies considerably within the mycolic-acid-containing taxa. Thus, especially long mycolic acids have been found in Mycobacterium spp. and Tsukamurella spp. they are medium-sized in Gor­do­­nia spp. and Nocardia spp., and small in the genus Corynebacterium.

The mycolic acid layer is considered to be functionally similar to the outer membrane of gram-negative bacteria.

Small hydrophilic solutes can permeate through the outer membrane of gram-negative bacteria, either via channel forming proteins or by receptor mediated uptake mechanisms.

Similarly, the cell wall of gram-positive bacteria from the

24th SEM Congress

order Corynebacteriales was recognized as a permeable barrier containing pores for the permeation of hydrophilic solutes. These channel-forming proteins represent the natural main passage of hydrophilic solutes across the cell wall of these bacteria.

Microbiology and the media How can the information emerging from microbiological research—and from scientific research in general—be disseminated to society? Is communicating science to lay audiences as difficult as often portrayed? Are journalists primarily interested in the more sensationalist aspects of science, as a means to attract the attention of their audiences?

Is it true, as scientists tend to say, that most journalists are poor science communicators because they lack a scientific background? Is it true, as journalists tend to say, that scientists are not usually good science communicators because they have not developed the communication skills needed to make their work understandable by the general public?

Answers to these questions were sought in a symposium on microbiology and journalism, chaired by M. Sánchez (Uni­­ versity Miguel Hernandez, Alicante) and I. López (University of Navarra, Pamplona), with contributions from C. Ribas (President of the Catalan Association for Science Communication, ACCC), M. Piqueras (ACCC past-President), and A. López (Autonomous University of Madrid). The participants agreed that when the message exists, it is crucial to find a messenger capable of appropriately communicating it.

Access to high-quality microorganisms The aim of the Microbial Resource Research Infrastructure (MIRRI) project is to establish a Pan-European research infrastructure that provides access to high-quality micro­ organisms, their derivatives, and the associated data for purposes of research, development, and applications. The MIRRI connects resource holders with researchers and policy makers to more effectively deliver resources and services and to efficiently meet the needs of innovation in biotechnology.

Int. Microbiol. Vol. 16, 2013

209

Microbiology and cosmetics Applied microbiology also had its place in the conference.

A symposium on microbiology and cosmetics, organized

by S. Leranoz (Reckitt Benckiser España, SL, Barcelona) and P. Orús (Colomer BPP, SL, Barcelona), discussed the policies related to the microbiological control of cosmetics.

The interest in this topic was readily apparent from the large number of attendees. *** In summary, the Spanish Society for Microbiology had a very successful and productive meeting, complemented by activities such as concerts and a closing dinner held at the premises of the Institute for Catalan Studies, a Baroque building from mid-17th Century, in the heart of Barcelona.

Looking at the future, in the SEM General Assembly it was approved that the next SEM Congress (with the significant number of being the 25th) will be held in Logroño in 2015, under the presidency and organization of Elena González Fandos, from the University of La Rioja.

Instructions for authors Preparation of manuscripts General information Research articles and research reviews should not exceed 12 pages, including tables and figures. The text should be typed in 12-point, Times New Roman font, with one and a half line spacing, left justification, and no line numbering. All pages must be numbered consecutively, starting with the tile page.

The Title page should comprise: title of the manuscript, first name and surname and affiliation (department, university, city, state/province, and country) for all authors. The address, telephone and fax numbers, and e-mail address of the corresponding author should also be included.

The Summary should be informative and completely comprehensible, briefly present the topic, state the scope of the experiments, indicate significant data, and point out major findings and conclusions. It should not exceed 200 words. Standard nomenclature should be used and abbreviations should be avoided or defined. No references should be cited. Immediately following the Summary, up to five Keywords should be provided; these will be used for indexing purposes.

The Introduction should be concise and define the objectives of the work in relation to other work done in the same field. It should not give an exhaustive review of the literature.

Materials and methods should provide sufficient detail to allow the experiments to be reproduced. However, only truly new procedures should be described in detail; previously published procedures should be cited, and important modifications of published procedures should be mentioned briefly. The suppliers of chemicals and equipment should be indicated if this might affect the results. Subheadings may be used. Statistical techniques used must be specified.

Results should be presented with clarity and precision. The results should be written in the past tense when describing findings in the author’s experiments. Previously published findings should be written in the present tense.

Results should be explained, but largely without referring to the literature.

The Discussion should be confined to interpretation of the results (not to recapitulating them), also in light of the pertinent literature on the subject.

When appropriate, the Results and Discussion sections can be combined.

This will be the case in research notes.

Acknowledgements should be presented after the Discussion section.

Personal acknowledgements should only be made with the permission of the person(s) named.

Competing interests should be declared by authors at submission indicating whether they have any financial, personal, or professional interests that could be construed to have influenced their paper.

References should be listed and numbered in alphabetical order. In the text, citations should be indicated by the reference number in square brackets. The list of references should include only works that are cited in the text and that have been published or accepted for publication. Unpublished work in preparation, Ph.D. and Masters theses, etc., should be mentioned in the text only, in parentheses. The author(s) must obtain written permission for the citation of a personal communication or other’s researchers’ unpublished results. References cited in the text should be numbered and placed within square brackets, referring to an alphabetized list at the end of the paper.

References should be in the following style: Published papers Venugopalan VP, Kuehn A, Hausner M, Springael D, Wilderer PA, Wuertz S (2005) Architecture of a nascent Sphingomonas sp. biofilm under

varied hydrodynamic conditions. Appl Environ Microbiol 71:2677-2686 Books Miller JH (1972) Experiments in molecular genetics. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA Book chapters Lo N, Eggleton P (2011) Termite phylogenetics and co-cladogenesis with symbionts. In: Bignell DE, Yves R, Nathan L (eds) Biology of termites: a modern synthesis, 2nd ed. Springer, Heidelberg, Germany, pp.27-50 Please list the first eight authors and then add “et al.” if there are additional authors. Citation of articles that have appeared in electronic journals is allowed if access to them is unlimited and their URL or DOI number to the full-text article is supplied.

Tables and Figures should be restricted to the minimum needed to clarify the text; a total number (F + T) of five is recommended. Neither tables nor figures should be used to present results that can be described with a short statement in the text. They also must not be integrated into the text. Figure legends must be typed double-spaced on a separate page and appended to the text. Photographs should be well contrasted and not exceed the printing area (17.6 × 23.6 cm). Magnification of micrographs should be shown by a bar marker. For color illustrations, the authors will be expected to pay the extra costs of 600.00 € per article. Color figures may be accepted for use on the cover of the issue in which the paper will appear. Tables must be numbered consecutively with Arabic numerals and submitted separately from the text at the end of the paper. Tables may be edited to permit more compact typesetting. The publisher reserves the right to reduce or enlarge figures and tables.

Electronic Supporting Information (SI) such as supplemental figures, tables, videos, micrographs, etc. may be published as additional materials, when details are too voluminous to appear in the printed version.

SI is referred to in the article’s text and is ported on the journal’s website (www.im.microbios.org) at the time of publication.

Abbreviations and units should follow the recommendations of the IUPAC-IUB Commission. Information can be obtained at: http://www.chem.qmw.ac.uk/iupac/.

Common abbreviations such as cDNA, NADH and PCR need not to be defined. Non-standard abbreviation should be defined at first mention in the Summary and again in the main body of the text and used consistently thereafter.SI units should be used throughout.

For Nomenclature of organisms genus and species names must be in italics. Each genus should be written out in full in the title and at first mention in the text. Thereafter, the genus may be abbreviated, provided there is no danger of confusion with other genera discussed in the paper. Bacterial names should follow the instructions to authors of the International Journal of Systematic and Evolutionary Microbiology. Nomenclature of protists should follow the Handbook of Protoctista (Jones and Bartlett, Boston).

Outline of the Editorial Process Peer-Review Process All submitted manuscripts judged potentially suitable for the journal are formally peer reviewed. Manuscripts are evaluated by a minimum of two and a maximum of five external reviewers working in the paper’s specific area. Reviewers submit their reports on the manuscripts along with their recommendation and the journal’s editors will then make a decision based on the reviewers.

Acceptance, article preparation, and proofs Once an article has been accepted for publication, manuscripts are thoroughly revised, formatted, copy-edited, and typeset. PDF proofs are generated so that the authors can approve the final article. Only typesetting errors should be corrected at this stage. Corrections of errors that were present in the original manuscript will be subject to additional charges. Corrected page proofs must be returned by the date requested.

General Information International Microbiology is a quarterly, open-access, peer-reviewed

journal in the fields of basic and applied microbiology. It publishes two kinds of papers: research articles and complements (editorials, perspectives, books reviews, etc.).

Submission Manuscripts must be submitted by one of the authors of the manuscript by e-mail to int.microbiol@microbios.org. As part of the submission process, authors are required to comply with the following items, and submissions may be returned if they do not adhere to these guidelines: 1. The work described has not been published before, including publication on the World Wide Web (except in the form of an Abstract or as part of a published lecture, review, or thesis), nor is it under consideration for publication elsewhere.

2. All the authors have agreed to its publication. The corresponding author signs for and accepts responsibility for releasing this material and will act on behalf of any and all coauthors regarding the editorial review and publication process.

2. The submission file is in Microsoft Word, RTF, or OpenOffice document file format.

3. The manuscript has been prepared in accordance with the journal’s accepted practice, form, and content, and it adheres to the stylistic and bibliographic requirements outlined in “Preparation of manuscripts.”

4. Illustrations and figures are placed separately in another document.

Large files should be compressed.

Copyright and license If an article is accepted for publication in International Microbiology, the authors (or other copyright holders) must transfer the copyright to the journal, which covers the right—not exclusive—to reproduce and distribute the article including reprints, translations, photographic reproductions, microforms, electronic form (offline, online) or any other reproduction of similar nature.

Creative Commons The journal is published under a Creative Commons Attribution-NonComercial-ShareAlike 3.0 Unported License. http://creativecommons.org/licenses/by-nc-sa/3.0/

Open access publishing provides immediate, permanent, free online access to the full texts of all the journal’s peer-reviewed research articles. It allows all interested readers to view, download, print, and/or redistribute any article without requiring a subscription on the principle that making research freely available to the public supports a greater global exchange of knowledge.

International Microbiology’s open access policy enables a far greater distribution and impact of an author’s work and is in the interest of the scientific community worldwide. The journal’s expenses for providing immediate, permanent, free online access to the full text of research articles are recovered partly from article-processing charges.

Currently many research funding agencies not only allow these expenses to be paid from their grants, but also encourage open access publication. The journal’s standard processing fee is 800.00 €. If a manuscript requires extensive editorial work, an extra charge may be requested. The acceptance of a paper, however, will not depend on the authors’ ability to pay these charges. Individual waiver requests must be done during the submission process and will be considered on a case-to-case basis.

Information for Subscribers International Microbiology is published quarterly. Volume 16 will consist of 4 issues—March, June, September and December—and will be published during 2013.

Recommended annual subscription is 300.00 € plus shipping and handling. Single-issue prices are available upon request. SAL (Surface Air Lifted) delivery is mandatory for Japan, Northeast and Southeast Asia, India, Australia and New Zealand. Airmail delivery to all other countries is available upon request. Orders or claims can be sent directly to: International Microbiology Poblet, 15 08028 Barcelona, Spain Tel. & Fax +34-933341079 E-mail: int.microbiol@microbios.org Cancellations must be received by 30 September to take effect at the end of the same year. Change of address: allow six weeks for all changes to become effective. Please contact int.microbiol@ microbios.org if you have any questions regarding your subscription.

Information for advertisers For advertising inquiries, please contact us at int.microbiol@microbios. org. All advertisements are subject to the publisher’s approval.

All articles in International Microbiology will be available on the Internet to any reader at no cost. The journal allows users to freely download, copy, print, distribute, search, and link to the full text of any article provided the authorship and source of the published article is cited and it is not used for commercial purposes. We recommend authors read about the Creative Commons Attribution-NonComercial-ShareAlike 3.0 Unported License before submitting their paper.

Open access and article processing charges

Disclaimer While the contents of this journal are believe to be true and accurate at the date of its publication, neither the authors and editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no guarantee, expressed or implied, with regard to the material contained therein. p3

INTERNATIONAL MICROBIOLOGY Official journal of the Spanish Society for Microbiology Volume 16 · Number 3 · September 2013 RESEARCH REVIEW

Guerrero R, Margulis L, Berlanga M Symbiogenesis: the holobiont as a unit of evolution

133

RESEARCH ARTICLES

Molina MC, Divakar PK, Zhang N, González N, Struwe L Non-developing ascospores in apothecia of asexually reproducing lichen-forming fungi

145

Zahran HH, Chahboune R, Moreno S, Bedmar EJ, Abdel-Fattah M, Yasser MM, Mahmoud AM Identification of rhizobial strains nodulating Egyptian grain legumes

157

Guzmán K, Campos E, Aguilera L, Toloza L, Giménez R, Aguilar J, Baldoma L, Badia J Characterization of the gene cluster involved in allantoate catabolism and its transcriptional regulation by the RpiR-type repressor HpxU in Klebsiella pneumoniae

165

Plasencia A, Gich F, Fillol M, Borrego CM Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

177

Barros J, Grenho L, Manuel CM, Ferreira C, Melo LF, Nunes OC, Monteiro FJ, Ferraz MP A modular reactor to simulate biofilm development in orthopedic material

191

PERSPECTIVES

Abadal E Gold or green: The debate on Open Access policies

199

MEETINGS

Fusté E, Viñas M The 24th Congress of the Spanish Society for Microbiology (L’Hospitalet de Llobregat, Barcelona, 10–13 July 2013)

INDEXED IN

Agricultural and Environmental Biotechnology Abstracts; ASFA/Aquatic Sciences & Fisheries Abstracts; BIOSIS; CAB Abstracts; Chemical Abstracts; SCOPUS; Current Contents®/Agriculture, Biology & Environmental Sciences®; EBSCO; EMBASE/Elservier Bibliographic Databases; Food Science and Technology Abstracts; ICYT/CINDOC; IBECS/BNCS; ISI Alerting Services®; MEDLINE®/Index Medicus®; Latíndex; MedBioWorldTM; SciELO-Spain; Science Citation Index Expanded®/SciSearch®

205