Campylobacter

Campylobacter Third Edition

Editors

Irving Nachamkin Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Christine M. Szymanski Institute for Biological Sciences National Research Council Ottawa, Ontario Canada and

Martin J. Blaser Departments of Medicine and Microbiology New York University School o f Medicine and New York Harbor Veterans Affairs Medical Center New York, New York

ASM

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PRESS

Washington, DC

CONTENTS

Contributors Preface

I.

ix xv

The Organism

1. Taxonomy of the Family Campylobacteraceae 3 Lies Debruyne, Dirk Gevers, and Peter Vandamme 2. Population Biology of Campylobacter jejuni and Related Organisms 27 Martin C. J. Maiden and Kate E. Dingle 3. Complexity and Versatility in the Physiology and Metabolism of Campylobacter 41 jejuni David J. Kelly 4. Comparative Genomics of Campylobacter 63 jejuni Olivia L. Champion, Suaad Al-Jaberi, Richard A. Stabler, and Brendan W. Wren

5. Comparative Genomics of Campylobacter Species Other Than Campylobacter 73 jejuni William G. Miller

7. Clinical Significance of Carnpylobacter and Related Species Other Than Campylobacter jejuni and Campylobacter coli 123 Albert J. Lastovica and Ban Mishu Allos

8. Burden of Illness of Campylobacteriosis and Sequelae 151 Kdre M d b a k and Arie Havelaar 9. Epidemiology of Carnpylobacter jejuni Infections in Industrialized Nations 163 Christine K. Olson, Steen Ethelberg, Wilfrid van Pelt, and Robert V. Tauxe 10. Molecular Epidemiology of Campylobacter Species 191 Stephen L. W. On, Noel McCarthy, William G. Miller, and Brent J. Gilpin 11. Isolation, Identification, Subspecies Differentiation, and Typing of Campylobacter 213 fetus Marcel A. P. van Bergen, Jos P. M. van Putten, Kate E. Dingle, Martin J. Blaser, and Jaap A. Wagenaar

12. Diagnosis and Antimicrobial Susceptibility of Campylobacter Species 227 Collette Fitzgerald, Jean Whichard, and Irving Nachamkin

Clinical and Epidemiologic Aspects of 11. Campylobacter Infections

13. Guillain-BarrC Syndrome and Campylobacter Infection 245 Bart C. Jacobs, Alex van Belkum, and Hubert P. Endtz

6. Clinical Aspects of Carnpylobacter jejuni and Campylobacter coli Infections 99 Martin J. Blaser and Jargen Engberg

14. Mechanisms of Antibiotic Resistance in Campylobacter 263 Qijing Zhang and Paul J. Plummer

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CONTENTS

15. National Molecular Subtyping Network for Food-Borne Bacterial Disease Surveillance in the United States 277 Peter Gerner-Smidt, Steven G. Stroika, and Collette Fitzgerald

111.

IV.

Glycobiology

25. N-Linked Protein Glycosylation in Campylobacter 447 Harald Nothaft, Saba Amber, Markus Aebi, and Christine M. Szymanski

Pathogenesis and Immunity 26. 0-Linked Flagellar Glycosylation in Campylobacter 471 Susan M. Logan, Ian C. Schoenhofen, and Patricia Guerry 9

16. Interaction of Campylobacter jejuni with Host Cells 289 Robert 0. Watson andlorge E. G a l h

Cell Biology of Human Host Cell Entry by 17. Campylobacter jejuni 297 Lan Hu and Dennis]. Kopecko 8

Campylobacter jejuni Secretes Proteins via 18. the Flagellar Type I11 Secretion System That Contribute to Host Cell Invasion and Gastroenteritis 3 15 Charles L. Larson, Jeffrey E. Christensen, Sophia A. Pacheco, Scott A. Minnich, and Michael E. Konkel

27. Campylobacter jejuni Lipooligosaccharides: Structures and Biosynthesis 483 Michel Gilbert, Craig T. Parker, and Anthony P. Moran 28. Campylobacter jejuni Capsular Polysaccharide 505 Andrey V. Karlyshev, Brendan W. Wren, and Anthony P. Moran 29. Campylobacter Metabolomics 523 Evelyn C. Soo, David J. McNally, lean-Robert Brisson, and Christopher W. Reid

19. Innate Immunity in Campylobacter Infections 333 Nicole M. Iovine

V.

20. Chemosensory Signal Transduction Pathway of Campylobacter jejuni 351 Victoria Korolik and Julian Ketley

Regulation of Flagellar Gene Expression 30. and Assembly 545 David R. Hendrixson

21. Animal Models of Campylobacter jejuni Infections 367 Linda S. Mansfield, David B. Schauer, andJames G. Fox

Natural Competence and Transformation in 3 1. Campylobacter 559 Rebecca S. Wiesner and Victor]. DiRita

Genes and Gene Expression

22. Rabbit Model of Guillain-BarrC 381 Syndrome Nobuhiro Yuki

Survival Strategies of Campylobacter jejuni: 32. Stress Responses, the Viable but Nonculturable State, and Biofilms 571 Sarah L. Svensson, Emilisa Frirdich, and Erin C. Gaynor

23. Pathogenesis of Campylobacter fetus 401 Martin 1.Blaser, Diane G. Newell, Stuart A. Thompson, and Ellen L. Zechner

33. Iron Metabolism, Transport, and Regulation 59 1 Alain Stintzi, Arnoud H. M. van Vliet, andlulian M. Ketley

24. Development of a Human 429 Vaccine David R. Tribble, Shahida Baqar, and Stuart A. Thompson

Regulation of Genes in Campylobacter 34. jejuni 611 Marc M. S. M. Wosten, Andries van Mourik, and 10s P. M. van Putten

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CONTENTS

VI.

Food Safety and Intervention

35. Campylobacter in the Food 627 Supply Wilma Jacobs-Reitsma, Ulrike Lyhs, and Jaap Wagenaar Transmission of Antibiotic Resistance from 3 6. Food Animals to Humans 645 Frank M. Aarestrup, Patrick F. McDermott, and Henrik C. Wegener

37. Poultry Colonization with Campylobacter and Its Control at the Primary Production Level 667 Jaap A. Wagenaar, Wilma Jacobs-Reitsma, Merete Hofshagen, and Diane Newell 38. Bacteriophage Therapy and Campylobacter 679 Ian F. Connerton, Phillippa L. Connerton, Paul Barrow, Bruce S. Seal, and Robert J. Atterbuy Index

695

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CONTRIBUTORS

Frank M . Aarestrup National Food Institute, DK-1790 Copenhagen V, Denmark

Martin J. Blaser Department of Medicine, New York University School of Medicine, New York, NY 10016

Suaad Al-Jaberi Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, United Kingdom

Jean-Robert Brisson NRC-Institute for Biological Sciences, Ottawa, Ontario K1A OR6, Canada Olivia L. Champion School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom

Ban Mishu Allos Division of Infectious Diseases, Vanderbilt University School of Medicine, Nashville, TN 37232

Jeffrey E. Christensen School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234

Markus Aebi Department of Biology, Institute of Microbiology, Eidgenossische Technische Hochschule (ETH) Zurich, CH-8093 Zurich, Switzerland

Ian F. Connerton School of Biosciences, Division of Food Sciences, University of Nottingham, Loughborough, Leics LE12 SRD, United l n g d o m

Saba Amber Department of Biology, Institute of Microbiology, Eidgenossische Technische Hochschule (ETH) Zurich, CH-8093 Zurich, Switzerland

Phillippa L. Connerton School of Biosciences, Division of Food Sciences, University of Nottingham, Loughborough, Leics LE12 5RD, United Kingdom

Robert J. Atterbury Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 ~ D U Y United Kingdom

Lies Debruyne Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Shahida Baqar Enteric Diseases Program, Naval Medical Research Center, Silver Spring, MD 20910-7500

Kate E. Dingle Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom

Paul Barrow School of Veterinary Medicine and Science, Division of Food Sciences, University of Nottingham, Loughborough, Leics LE12 SRD, United Kingdom

Victor J. DiRita Department of Microbiology and Immunology and Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI 48 103 ix

x

CONTRIBUTORS

Hubert P. Endtz Laboratory Sciences Division, International Centre for Diarrheal Disease Research Bangladesh, Dhaka 1212, Bangladesh

Patricia Guerry Naval Medical Research Center, Silver Spring, MD 20910

Jargen Engberg Department of Clinical Microbiology, Hvidovre Hospital, DK-2650 Hvidovre, Denmark

Arie Havelaar Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands

Steen Ethelberg Department of Epidemiology and Department of Bacteriology, Statens Serum Institut, DK-2300 Copenhagen S, Denmark

David R. Hendrixson Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Collette Fitzgerald Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333

Merete Hofshagen National Veterinary Institute, Oslo, Norway

James G. Fox Division of Comparative Medicine and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Lan Hu Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892

Emilisa Frirdicb Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada

Nicole M . Iovine Department of Medicine, New York University School of Medicine, Veteran’s Administration Medical Center, New York, NY 10010

Jorge E. Gal& Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536

Bart C . Jacobs Departments of Neurology and Immunology, Erasmus University Medical Center, Rotterdam 3015 CE, The Netherlands

Erin C. Gaynor Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada Peter Gerner-Smidt Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333 Dirk Gevers Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Michel Gilbert Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada Brent J. Gilpin Water Management Programme, Institute of Environmental Science and Research Ltd., Christchurch, New Zealand

Wilma Jacobs-Reitsma RIKILT Institute of Food Safety, 6708 PD Wageningen, The Netherlands Andrey V. Karlysbev London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom David J. Kelly Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom Julian Ketley Department of Genetics, University of Leicester, Leicester LE1 7RH, United Kingdom Michael E. Konkel School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234

CONTRIBUTORS

Dennis J. Kopeck0 Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892 Victoria Korolik Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia Charles L. Larson School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234 Albert J. Lastouica Department of Biotechnology, University of the Western Cape, Bellville 7535, South Africa

xi

Scott A. Minnich Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, MOSCOW, ID 83844-3 052 a r e Mslbak Department of Epidemiology, Statens Serum Institute, Copenhagen, Denmark Anthony P. Moran Department of Microbiology, National University of Ireland, Galway, Ireland, and Institute for Glycomics, Griffith University, Queensland, Australia Irving Nachamkin Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283

Susan M . Logan Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

Diane G. Newel1 Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom

Ulrike Lyhs Seinajoki Unit, Ruralia Institute, University of Helsinki, FI-60320 Seinajoki, Finland

Harald Nothaft Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

Martin C . J. Maiden Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom

Christine K. Olson Enteric Diseases Epidemiology Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333

Linda S. Mansfield Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Stephen L. W . On Food Safety Programme, Institute of Environmental Science and Research Ltd., Christchurch, New Zealand

Noel McCarthy Department of Zoology, University of Oxford, Oxford OX1 3SY, United Kingdom

Sophia A. Pacheco School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234

Patrick F. McDermott National Antimicrobial Resistance Monitoring System, Center for Veterinary Medicine, U.S. Food & Drug Administration, Laurel, MD 20708

Craig T. Parker Produce Safety and Microbiology Research, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710

David J. McNally NRC-Institute for Biological Sciences, Ottawa, Ontario K1A OR6, Canada

Paul J. Plummer Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, JA 50011

William G. Miller Produce Safety and Microbiology Research Unit, USDA, ARS, Albany, CA 94710

Christopher W. Reid NRC-Institute for Biological Sciences, Ottawa, Ontario K1A OR6, Canada

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CONTRIBUTORS

David B. Schauer Division of Comparative Medicine and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

David R. Tribble Preventive Medicine and Biometrics Department, Uniformed Services University of the Health Sciences, Bethesda, MD 208 14-5119

Ian C . Schoenhofen Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

Alex van Belkum Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam 3015 CE, The Netherlands

Bruce S. Seal Poultry Microbiological Safety Research Unit, Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30605 Evelyn C . So0 NRC-Institute for Marine Biosciences, Halifax, Nova Scotia B3H 321, Canada Richard A. Stabler Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, United Kingdom Alain Stintzi Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada Steven G. Stroika Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333 Sarah L. Svensson Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada Christine M . Szymanski Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada Robert V. Tauxe Division of Foodborne, Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333 Stuart A. Thompson Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912

Marcel A. P. van Bergen Division of Infectious Diseases, Animal Sciences Group, OIE Reference Laboratory for Campylobacteriosis, 8200 AB Lelystad, The Netherlands Peter Vandamme Laboratory of Microbiology, Ghent University, B9000 Gent, Belgium Andries van Mourik Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands Wilfrid van Pelt Department of Epidemiology and Surveillance, Rijksinsituut voor Volksgezondheid en Milieu, 3721 MA Bilthoven, The Netherlands Jos P. M . van Putten Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, 3508 TD Utrecht, The Netherlands

Arnoud H . M . van Vliet Institute of Food Research, Norwich NR4 7UA, United Kingdom Jaap A . Wagenaar Division of Infectious Diseases, Animal Sciences Group , OIE Reference Laboratory for Campylobacteriosis and WHO Collaboration Centre for Campylobacter, 8200 AB Lelystad, and Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, 3.508 TD Utrecht, The Netherlands Robert 0. Watson Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536 Henrik C . Wegener National Food Institute, DK-1790 Copenhagen V, Denmark

CONTRIBUTORS

xiii

Jean Whichard Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333

Nobuhiro Yuki GBS Laboratory, 3-9-5 Kamiikedai, Ota-ku, Tokyo 145-0064, Japan

Rebecca S. Wiesner University of Michigan, Ann Arbor, MI 48103

Ellen L. Zechner Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria

Marc M . S. M . Wosten Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands Brendan W. Wren Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, United Kingdom

Qijing Zhang Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011

PREFACE

late Gerald Aspinall, has provided a structural basis for important polysaccharides and glycolipids from the organism and great insight into glycosylation systems, which also are present in other prokaryotes and eukaryotes. This work, combined with an increased understanding of the molecular biology of gene expression in Campylobacter, is helping to form a more complete picture about the organism and its interaction with the host and environment. Ultimately, research will help improve human health through understanding the immunology of campylobacter infection and the pertinent host defenses. These will lead to the development of strategies to reduce infection, through either effective vaccines or improved food safety. The ecology of food safety also has seen a dramatic increase in research, with emphasis on understanding the extent of campylobacter in the food supply, transmission of antibiotic resistant campylobacters from food animals to humans, and control of campylobacter at the food source. The above advances have helped to form the nucleus for the 3'd edition of Campylobacter. We are grateful to the scientific groups from around the world that have contributed to the preparation of these outstanding chapters. These represent the generous sharing of our growing knowledge of these important zoonotic pathogens of humans. Finally, we especially thank Greg Payne at ASM Press for his help and guidance throughout the process of developing this volume. Irving Nachamkin Christine M. Szymanski Martin J. Blaser February 2008

Our understanding of the clinical aspects, epidemiology, and pathogenesis of Campylobacter infection has increased dramatically since publication of the second edition of Campylobacter in 2000. As the number of species within the family has expanded, so has our knowledge of this group of organisms in terms of their physiology, population biology, and diversity. The ability to understand genomic diversity in Campylobacter is due in great part to improvements in technology that have advanced comparative analyses. The sequencing of the first C. jejuni strain in 2000 was a milestone in Campylobacter genetics and brought to light aspects of biology that previously could not have been identified. Since then, additional complete Campylobacter genome sequences have been published and have provided new insights about the genomic diversity of these organisms, which now is covered extensively in this new edition. Campylobacter infections and their complications, such as Guillain-BarrC syndrome, cause significant morbidity in specific populations. There is growing recognition of Campylobacter among clinicians as well as by the lay public. Antimicrobial resistance also continues to increase and poses new issues regarding therapies. Of particular note are the impact that researchers in our field have had on government regulation of antimicrobial agent use in food animals and how effecting change will ultimately improve human health. Our understanding of the pathogenesis of Campylobacter infections has advanced greatly, especially in the area of signal transduction pathways and cell biology of the organism. The emerging field of Campylobacter glycobiology, which was pioneered by the

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I. THE ORGANISM

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 1

Taxonomy of the Family Campylobacteraceae LIESDEBRUYNE, DIRKGEVERS,AND PETER VANDAMME

fer, and species-specific core genomes and strainspecific accessory genomes have been described for several bacterial species (Dagan and Martin, 2006; Kunin et al., 2007). It would be unrealistic to expect that in the near future bacterial species will be described on the basis of whole-genome sequences only. Several recent studies (Goris et al., 2007; Konstantinidis et al., 2006), however, demonstrated that multiple parameters in whole-genome sequences reflect the traditional delineation of species at the 70% DNA-DNA hybridization level. In particular, multilocus sequence analysis (MLSA), a technique based on sequence analysis of fragments of multiple household genes present in the core genome, has been put forward as a novel approach for the classification of bacteria (Gevers et al., 2005). Konstantinidis et al. (2006) demonstrated that phylogenetic trees based on MLSA data can bear the same phylogenetic signal as trees that are based on sequence analysis of the entire common gene pool, implying that one does not need entire genome sequences for all strains under comparison to design taxonomic frameworks equivalent to frameworks that are based on whole-genome content. MLSA schemes have been developed for a growing number of bacteria, including some members of the family Campylobacteraceae (see below). Additional studies are needed to provide pragmatic criteria for determining how we can use MLSA data to define species and subdivisions within species for poorly studied or newly discovered taxa.

This chapter will present a traditional overview of the biological diversity of Campylobucter and Arcobacter but will also address the taxonomic information that has become available through whole-genome sequence analysis. Starting in the 1990s, genomic sequencing drastically changed our understanding of the relationships between microorganisms. Many new papers have been evaluating diversity within and among species by exploiting whole-genome information content, and the bacterial species concept emerged from obscurity into the spotlight (Doolittle and Papke, 2006; Gevers et al., 2005; Konstantinidis et al., 2006). Since the 1960s, the level of whole-genome DNA-DNA hybridization is the accepted standard for species delineation, with 70% DNA-DNA reassociation being the recommended species cutoff. Genomic DNA-DNA hybridization has been used as the key technology for the phylogenetic delineation of bacterial species because whole-genome sequences were assumed to provide a rational and objective basis for the description and delineation of bacterial species. While waiting for easy access to whole-genome sequences to emerge, the genomic DNA-DNA hybridizations were the best approach to comparing wholegenome sequences. Meanwhile, sequencing technology improved so dramatically that several hundreds of bacterial genomes have been-or are in the process of beingsequenced and annotated. It has become apparent that apart from the traditional nucleotide changes, other selective forces such as gene deletion, gene duplication, horizontal gene transfer, and chromosomal rearrangements shape the bacterial genome and that considerable fractions of the genome can be strain specific. Numerous studies, however, have stressed the dominance of vertical over horizontal gene trans-

TAXONOMIC HISTORY The early history of the genus Campylobacter, starting with the first description by Theodor Escher-

Lies Debruyne, Dirk Gevers, and Peter Vandamme Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Bioinformatics and Evolutionary Genomics, Ghent University/VIB, Technologiepark 927, B-9052 Gent, Belgium. Dirk Gevers Gent, Belgium.

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4

DEBRUYNE ET AL.

ich in 1886, the first isolation of a Vibrio-like organism from aborted ovine fetuses by McFadyean and Stockman in 1913, the emergence of several novel “Vibrio” species in a range of animals with and without clinical symptoms, and the creation of the genus Campylobacter in 1963, has been outlined in detail in several previous reviews (Butzler, 2004; Skirrow, 2006). In 1963, Sebald and VCron transferred two of these Vibrio species, Vibrio fetus and Vibrio bubulus, into the new genus Campylobacter as Campylobacter fetus and Campylobacter bubulus, respectively. The former was the bacterium first isolated by McFadyean and Stockman (1913), which was subsequently also isolated from aborted bovine fetuses (Smith and Taylor, 1919) and from blood cultures of women who aborted (Vinzent et al., 1947); the latter was an organism isolated from the bovine vagina and semen (Florent, 1953). The reasons for transferring these two species into the novel genus Campylobacter included their low DNA base composition, their microaerophilic growth requirements, and their nonfermentative metabolism. Ten years later, VCron and Chatelain (1973) published a more comprehensive study on the taxonomy of the microaerophilic Vibrio-like organisms and considered four distinct species in the genus Campylobacter: C. fetus (the type species), Campylobacter coli (a vibrio isolated from feces of pigs with diarrhea [Doyle, 1948]), Campylobacter jejuni (a vibrio isolated from the feces of cattle with diarrhea Uones et al., 19311, blood cultures of humans with gastroenteritis [King, 1957; Levy, 19461 and aborted sheep fetuses [Bryans et al., 1960]), and Campylobacter sputorum. The latter comprised two subspecies, C. sputorum subsp. sputorum (a vibrio isolated from sputum of a patient with bronchitis [Prihot, 1940; Tunicliff, 19141) and C. sputorum subsp. bubulus (a vibrio isolated from the bovine vagina and semen [Florent, 19531). The application of a filtration technique used in veterinary microbiology allowed Butzler and coworkers in the early 1970s to exploit the small cell dimensions and the vigorous motility of Campylobacter cells to selectively isolate them from stools of humans with diarrhea (Butzler et al., 1973; Dekeyser et al., 1972). The main breakthrough, however, was provided a few years later by Skirrow, who described a selective supplement comprising a mixture of vancomycin, polymyxin B, and trimethoprim that was added to a basal medium (Skirrow, 1977). This simple isolation procedure thus enabled routine diagnostic microbiology laboratories to isolate campylobacters and to evaluate their clinical role. The availability of adequate isolation procedures led to a renewed interest in Campylobacter research

during the early 1980s. As a consequence, manifold Campylobacter-like organisms were isolated from a variety of human, animal, and environmental sources, and gradually, new species were described (Benjamin et al., 1983; Fox et al., 1989; Gebhart et al., 1985; Lawson et al., 1981; Marshall et al., 1984; McClung et al., 1983; Neil1 et al., 1985; Tanner et al., 1981). Classical biochemical tests routinely used for the identification of clinical bacteria often yielded negative or variable results within Campylobacter species. This poor biochemical reactivity and lack of clear-cut differential characters led to the wide application of vernacular names for many groups of Campylobacter-like organisms, and the term CLO (Campylobacter-like organism) became widely used. Some of these CLO groups were later classified as novel species, but several were identified as biochemical variants of well-known species (Vandamme, 2000). Still others remained unnamed: for example, the CLO-3 strain that was isolated in the early 1980s was not formally classified pending the isolation and characterization of additional strains belonging to the same taxon (Totten et al., 1985). An overview of these empirical names with their final classification has been given previously (Vandamme, 2000). Also in the 1980s, the study of bacterial phylogeny as imprinted in the degree of rRNA cistron similarity became more popular and was increasingly used to evaluate and revise bacterial classification schemes. Long-standing genera like Pseudomonas, Flavobacterium, Bacteroides, and many others were found to be extremely heterogeneous, and their classification was gradually revised to conform to the new phylogenetic (or “natural”) insights. The first rRNA-based phylogenetic studies of Campylobacter species were published in the late 1980s and demonstrated that Campylobacter too was heterogeneous, as it comprised three distinct phylogenetic lineages that also included members of the genus Wolinella and two Bacteroides species, Bacteroides gracilis and Bacteroides ureolyticus, which appeared phylogenetically distinct from the true Bacteroides species (Vandamme, 2000). In 1989, Goodwin et al. summarized genotypic and phenotypic arguments to exclude the gastric species Campylobacter pylori (humans) and Campylobacter mustelae (ferrets) from the genera Campylobacter and Wolinella and proposed a novel genus, Helicobacter, to accomodate both. Two years later, a complete revision of the taxonomy and nomenclature of the genus Campylobacter and related bacteria was proposed by Vandamme et al. (1991a). The results of an extensive DNA-rRNA hybridization study of over 60 strains representing all known Campylobacter species, CLO groups, and putative relatives such

CHAPTER 1

as Wolinella, Bacteroides, and “Flexispira” species corroborated and extended the 16s rRNA sequencing data. Carnpylobacter, together with Wolinella and “Flexispira” and B. gracilis and B. ureolyticus, was found to represent a separate, sixth rRNA superfamily sensu De Ley within the group of the gramnegative bacteria. This phylogenetic lineage is now known as the epsilon subdivision of the proteobacteria. Within this lineage, C. fetus, Campylobacter hyointestinalis (Gebhart et al., 1985), Campylobacter concisus (Tanner et al., 1981), Campylobacter mucosalis (Lawson et al., 1981; Roop et al., 1985a), C. sputorum, C. jejuni, C. coli, Campylobacter lari (Benjamin et al., 1983), Campylobacter upsaliensis (Sandstedt and Ursing, 1991), Wolinella curva (Tanner et al., 1984), Wolinella recta (Tanner et al., 1981), B. gracilis, and B. ureolyticus constituted the first rRNA homology cluster. Campylobacter nitrofigilis (McClung et al., 1983), Campylobacter cryaerophila (Neil1 et al., 1985), and several other aerotolerant CLO strains (which were later classified as Arcobacter butzleri and Arcobacter skirrowii vandamme et al., 1992]), constituted the second rRNA homology cluster. Helicobacter pylori (Goodwin et al., 1989; Marshall et al., 1984), H. mustelae (Fox et al., 1989; Goodwin et al., 1989), Campylobacter cinaedi (Totten et al., 1985), Campylobacter fennelliae (Totten et al., 1985), W. succinogenes (Wolin et al., 1961), “Flexispira rappini“ (Bryner et al., 1986), and the CLO-3 strain (Totten et al., 1985) constituted a third rRNA homology cluster. In addition, the free-living saprophytic Campylobacter strains of Laanbroek et al. (1977) and Wolfe and Pfennig (1977) (later reclassified as Sulfurospirillum species) belonged to the same phylogenetic lineage but did not belong to one of the three major rRNA homology clusters. A revised classification was proposed that took into account the phylogenetic results derived from the DNA-rRNA hybridization experiments and that published data on the genotype and phenotype of these organisms. The genus Carnpylobacter was restricted to those species belonging to the rRNA homology cluster containing C. fetus, the type species of the genus Campylobacter. The generically misnamed W. curva and W. recta, and in a subsequent study, B. gracilis (Vandamme et al., 1995), were included in the emended genus Campylobacter as Campylobacter curvus, Campylobacter rectus, and Campylobacter gracilis, respectively. B. ureolyticus was obviously a close relative of the emended genus Campylobacter, but its taxonomic status was not changed pending further study of this taxon and the isolation of similar bacteria (Vandamme et al., 1991a, 1995). The name Arcobacter (with Arcobacter nitrofigilis as the type species) was proposed for the organisms be-

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TAXONOMY OF THE CAMPYLOBACTERACEAE

5

longing to the second rRNA homology cluster. Finally, C. cinaedi and C. fennelliae were transferred into the genus Helicobacter as H. cinaedi and H. fennelliae, respectively, and an emended genus description was proposed. Wolinella succinogenes remained the only species of the genus Wolinella. Several new Campylobacter, Arcobacter, and Helicobacter species have been reported since then. Campylobacter and Arcobacter species will be discussed below. A detailed overview of Helicobacter species is beyond the scope of the present chapter. Finally, the genera Campylobacter and Arcobacter were accomodated in the new bacterial family, the Campylobacteraceae (Vandamme and De Ley, 199l), because they shared several genotypic and phenotypic characteristics. The family Campylobacteraceae currently also comprises the generically misclassified species B. ureolyticus and the genus Sulfurospirillum (Stolz et al., 2005). In addition, a growing number of free-living Epsilonproteobacteria occupy phylogenetic positions among the Campylobacter, Arcobacter, Sulfurospirillum, and Wolinella-Helicobacter clades. The allocation of these bacteria to a bacterial family such as the Campylobacteraceae or others needs further study. Figure 1 represents a traditional phylogenetic tree of the family Campylobacteraceae that is based on percentage of 16s rRNA gene sequence similarity.

WHOLE-GENOME TAXONOMY OF CAMPYLOBACTER AND RELATED BACTERIA Species Tree of Campylobacter and Related Bacteria The progress in the whole-genome studies of Campylobacter and related bacteria presents an opportunity to produce a more robust species tree. Data sets consisting of a single gene or a small number of concatenated genes have a significant probability of supporting conflicting topologies. By contrast, a fully resolved species tree with maximum support can come from the concatenation of a large number of carefully selected genes, which whole-genome sequences provide in abundance. A total of 25 complete and incomplete genome sequences have been obtained for 15 species belonging to the Campylobacteraceae and related bacteria (A. butzleri, C. coli, C. concisus, C. curvus, C. fetus, C. jejuni, C. lari, C. upsaliensis, Helicobacter acinonychis, H. hepaticus, H. mustelae, H. pylori, Sulfurimonas denitrificans, Thiornicrospira crunogena, and W. succinogenes; http: //www.genomesonline.org/). These genomes were subjected to a comparative genome analysis to

6

DEBRUYNE ET AL.

-

-Sulfurospirillum cavolei Phe91T(AB246781) -Sulfurospirillum sp. DSM 806 (AF144694) Sulfurospirillum barnesii SES-3' (AF038843) ulfurospirillum deleyianum Spirillum 5 17.5' (Y13671) Sulfurospirillum multivorans K' (X82931) Sulfurospirillum halorespirans PCE-M2' (AF218076) "CandidatusArcobacter sulfidicus" (AY035822) Arcobacter butzleri ATCC 49616T(AY621116) obacter cibarius LMG 21996T(AJ607391) Arcobacter skirrowii CCUG 10374T(L14625) Arcobacter cryaerophilus CCUG 17801' (L14624) Arcobacter nitrofigilis CCUG 15893T(L14627) Arcobacter halophilus LA3 lBT(AF513455) Campylobacter hyointestinalis subsp. lawsonii CHY 5' (AF097685) -CCampylobacier lanienae NCTC 13004T(AF043425) Campylobacter hyointestinalis subsp. hyointestinalis NCTC 11608' (AF097689) mpylobacterfetus subsp.fetus ATCC 27374' (LO4314) Campylobacterfetus subsp. venerealis ATCC 19438T(L14633) Campylobacter canadensis L266T(EF621894) Campylobacter upsaliensis CCUG 14913' (L14628) -CCampylobacter helveticus NCTC 12470T(U03022) Campylobacter coli CCUG 11283' (LO4312) Campylobacter lari CCUG 23947' (LO4316) Campylobacter insulaenigrae NCTC 12927T(AJ620504) Campylobacterjejuni subsp. doylei CCUG 24567T(L14630)

: L

-

-I

-

c

Figure 1. Phylogenetic tree of the family Campylobacteruceae and related bacteria, based on percentage of 16s rRNA gene sequence similarity.

screen for protein families that together might reveal a major vertical component of species phylogeny (D. Gevers, unpublished data). Orthologous gene families were selected as phylogenetic markers if they met the following criteria: (i) had exactly one copy in each of

the 25 strains, (ii) had a minimum length of 100 amino acids after ambiguous positions were marked out, and (iii) showed no noisy phylogenetic signal from recombination or horizontal gene transfer. This resulted in a carefully selected set of 60 core genes

CHAPTER 1

that could be used to estimate the phylogeny of these bacteria by maximum likelihood (Fig. 2). In general, this species tree correlates nicely with the phylogenetic tree that is based on 16s rRNA gene sequences, but compared with the latter, it has a superior resolution to distinguish closely related bacteria such as C. jejuni subsp. jejuni, C. jejuni subsp. doylei, and C. coli. This tree also confirms and widens previous insights in the evolutionary relationships based on 12 concatenated protein data sets including only seven species (Fouts et al., 2005). By including more C. jejuni genomes, it is not only confirmed that C. jejuni RM1221 is more closely related to the C. jejuni subsp. jejuni strains than to C. coli, but it is also shown that it is closer to any C. jejuni subsp. jejuni than to C. jejuni subsp. doylei 269.97. Further, it confirms that W. succinogenes DSM 1740Tis more closely related to Helicobacter spp. than to any Campylobacter species. In addition, it was found that S. denitrificans ATCC 3 3 889= (reclassification from Thiomicrospira denitrificans) is more closely related to A. butzleri and Campylobacter than to T. crunogena.

0.2

I -

I

I

TAXONOMY OF T H E CAMPYLOBACTERACEAE

7

Whole-Genome Comparisons

As in other groups of bacteria, bioinformatic analyses of the growing number of whole-genome sequences of Campylobacter strains is explaining to which extent strains within species and strains representing different species are different. Goris et al. (2007) demonstrated that the recommended cutoff of 70% DNA-DNA reassociation for species delineation corresponds to 69% conserved DNA and 95% average nucleotide identity (ANI), a measure for evolutionary relatedness that is based on sequence similarity between orthologous genes. As shown in Fig. 3 , the AN1 values among C. jejuni strains do fall above this cutoff and range between 95.24 and 99.68%, with a significant partition between its two subspecies: intrasubspecies values for C. jejuni subsp. jejuni are on average 97.85% ( _ t 0.62%), and intersubspecies values for C. jejuni subsp. doylei versus C. jejuni subsp. jejuni are on average 95.80% (k0.28%). In comparison, Helicobacter pylori comprises a more heterogenous set of strains, with lower AN1 values ranging between 94.05 and 95.57%. The AN1 values

H . pylori 26695 H. p ylori H PAG 1 H . pylori399 H. acinon ychis str. Sheeba H. mustelae ATCC 43772 H . hepaticus ATCC 51449 Wolinella succinogenes DSM 1740 Sulfurimonas denitrificans ATCC 33889 Arcobacter butzleri 40 18 C. fetus subsp. fetus 82-40

HB93-13 81-176 260.94 C. j e j u n i subsp. j e j u n i CG8486 .C. j e j u n i RM1221 C. j e j u n i subsp. j e j u n i NCTC 11168 C. jejuni subsp. jejuni 84-25 CF93-6 Thiomicrospira crunogena X C L- 2

Figure 2. Whole-genome-based phylogeny of Campylobacter and related bacteria. Out of 365 Campylobacter core gene families, a concatenation of 60 carefully selected genes was used to estimate the phylogeny of these bacteria by maximum likelihood. Orthologous protein families were determined by OrthoMCL (Li et al., 2003). Protein sequence alignments were generated by Muscle (Edgar, 2004), and poorly conserved regions were trimmed by Gblocks (Castresana, 2000). Phylogenetic trees with 100 bootstrap replicates were. obtained by the PhyML algorithm (Guindon and Gascuel, 2003) with a JIT amino acid substitution matrix and a discrete gamma model. All nodes had 100% bootstrap support.

8

DEBRUYNE ET AL.

pylori-acinonychis 0

u 1

s!

60

0 5

0

80

90

100 100 I

95

90

85

80

75

70

Average Nucleotide Identity (ANI)

Figure 3. Genetic diversity among genomes of Cumpylobacter and related bacteria. Every data point represents a wholegenome comparison between two genomes (both intra- and interspecies) and shows the percentage AN1 on the x axes plotted against the percentage of gene content similarity on the y axes. Only comparisons with AN1 values above 70% are shown because this is the range in which AN1 is considered to be a robust and sensitive method for measuring evolutionary relatedness (Konstantinidis and Tiedje, 2005). AN1 values were calculated as described previously (Konstantinidis and Tiedje, 2005), and gene content similarity values were calculated on the basis of reciprocal best BLASTp hits.

are useful for quantifying evolutionary relatedness between different species and correspond to wholegenome-based phylogenies. The closest related species of c. jejuni among the included genomes is c. coli (ANIs of 83.23 -t 0.29%), and the second closest is C. upsaliensis (ANIs of 72.89 0.10%). In comparison, the evolutionary distance between H. pylori and H. acinonychis is smaller than any two species of Campylobacter included in our analyses (ANIs of 89.25 ? 0.18). The gene content similarity values, shown in Fig. 3, were determined to reveal the effect of ecology on this group of organisms. It has been reported before that the environment is one of the main factors in shaping the evolution of the gene content of an organism (Konstantinidis et al., 2006). Larger intraspecific genomic differences could be the outcome of a differential evolution of those strains in different environments. The intraspecific values of the C. jejuni

*

strains (excluding the C. jejuni subsp. jejuni CG8486 strain) go as low as 74%, indicative of an ecologically versatile organism. These differences are likely to be determined by the broad host range of C. jejuni, which includes human, cattle, and poultry as well as a large variety of wild bird species (Schouls et al., 2003). Similar large genomic differences within a species have been reported for Escherichia coli (Konstantinidis et al., 2006). The unfinished C. jejuni subsp. jejuni CG8486 genome sequence has significantly fewer annotated genes in comparison with other strains of this species (1,421 versus 1,750 2 100, respectively), which explains why comparisons with this strain result in more extreme gene content similarity values, and thus its gene content similarity values should be considered with caution. The AN1 values do not require the same reservation because they are not affected by large genome size differences in the same way. In contrast to the intraspecific values

CHAPTER 1

of the C. jejuni strains, the H. pylori strains show substantially smaller genomic differences, ranging between 83 and 90%, even though their AN1 values indicate a larger evolutionary distance. This is a strong indication of its narrower ecological nichethat is, it is limited to the human host. It is well established that the phenotypes and genotypes of C. jejuni and C. coli are remarkably similar, resulting in an important taxonomic and diagnostic problem. Figure 3 provides us with a quantitative measure of this similarity. Although the AN1 values reflect a clear distinction between these two species, c. coli seems to have a large set of genes in common with some of the C. jejuni strains, likely explaining the confusing phenotypic test results. Which and How Many Genes Are Needed To Predict the Whole-Genome-Based Phylogeny? Because of the limitations of 16s rRNA gene analysis for accurate species-level identification of campylobacters (see below), several other genes were examined for taxonomic and identification purposes. Karenlampi et al. (2004) demonstrated that partial groEL gene sequences could be used to construct a phylogeny similar to that obtained by 16s rRNA gene sequences, but with lower interspecies and high intraspecies similarities. Korczak et al. (2006) used rpoB sequences and also obtained a good congruence between rpoB and 16s rRNA gene sequence-based trees. Again, resolution for the rpoB gene was much higher than for the 16s rRNA gene, enabling differentiation of even closely related Campylobacter species, as well as of most subspecies. In 2001, Dingle and coworkers (2001) reported a first multilocus sequence typing scheme that is based on seven housekeeping loci to study population biology in C. jejuni. Since then, the analysis of polymorphisms in multiple coding gene sequences has been used in several other Campylobacter species for typing purposes (Dingle et al., 2005; Litrup et al., 2007; van Bergen et al., 2005a). Miller et al. (2005) reported the first multispecies, multilocus sequence-based scheme that allowed analysis of multiple Campylobacter species (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus) but used it for strain typing and not for studying the phylogeny and identification of these bacteria. Yet the analysis of multilocus sequences currently represents the most realistic approach for studying species diversity and evolution at large (Gevers et al., 2005). But how do a few genes correlate with whole-genome-based phylogeny in the case of the Campylobacteraceae, and what genes should preferentially be used to improve phylogenetic accuracy in a cost-effective way? Genes currently used in multi-

TAXONOMY OF THE CAMPYLOBACTERACEAE

9

locus sequencing approaches are often selected primarily on the basis of being used previously in other taxa, being essential, being unlinked in the genome, and having a successful primer design. However, their phylogenetic robustness is overlooked, and a selection based on their predictive power for the whole-genome-based phylogeny would be better. Taking such an approach, it has been shown that a more accurate phylogeny for the Escherichia coli group can be obtained from three best-performing genes compared with the concatenated alignment of eight commonly used markers (Konstantinidis et al., 2006). The availability of 17 Campylobacteraceae genomic sequences allows a similar analysis. We evaluated the correlation between all the 548 Campylobacteraceae core genes separately, and we assessed the whole-genome-based phylogeny by performing comparisons both in terms of tree topology (Robinson-Foulds symmetric difference distance) and branch length (branch score distance of Kuhner and Felsenstein) with the FTREEDIST (http: //emboss. sourceforge.net/) and TOPD (Puigbo et al., 2007) software tools. This methodology guided the selection toward a total of 60 best-performing genes with tree topologies and branch lengths highly similar to the whole-genome-based tree, i.e., the species tree. However, single-gene-based trees correlate poorly with the species tree at the shorter evolutionary scales, i.e., among the very closely related strains of C. jejuni, where only the concatenation of multiple loci provides enough resolving power. This can be explained by the finding that C. jejuni has a weakly clonal population structure as a result of high levels of intraspecific recombination (Suerbaum et al., 2001). Among the genes that are chosen as good phylogenetic markers, there appears to be no functional bias (Table 1). By use of the general functional classification from the COG (Cluster of Orthologous Genes) database (Tatusov et al., 1997), it was found that the genes are evenly distributed over the categories of information storage and processing (25%), cellular processes and signaling (28%), and metabolism (32%), with the remainder classified in the “poorly characterized” category or not classified in any COG at all. For a gene to be practically useful as a phylogenetic marker in an MLSA approach, it should allow the development of a set of conserved primers of limited degeneracy. To identify regions corresponding to the most highly conserved segments of each of the 60 best-performing genes, amino acid alignments were submitted to the BLOCKS Multiple Alignment Processor (Henikoff et al., 1995; http://

Table 1. Functional classification of 60 selected phylogenetic markers for the Campylobacteraceae" Gene name

Product description

P7fA miaA" miaB dusB

Peptide chain release factor 1 tRNA delta(2)-isopentenylpyrophosphatetransferase tRNA-i(6)A37 thiotransferase enzyme tRNA-dihydrouridine synthase B Ribosomal protein L25 GTP-binding protein Glycyl-tRNA synthetase beta subunit Methionyl-tRNA formyltransferase DNA gyrase, B subunit Exodeoxyribonuclease VII, large subunit Excinuclease ABC subunit C DNA polymerase type I Holliday junction DNA helicase RuvB DNA gyrase, A subunit DNA processing protein A PP-loop family protein Chromosome partitioning protein, ParA family Mur ligase family protein Membrane-associated zinc metalloprotease, putative Methyltransferase GidB Outer-membrane lipoprotein carrier protein precursor Permease, putative Peptidase, M23 /M37 family Lipopolysaccharide heptosyltransferase I Penicillin-binding protein DNA repair protein ATPase subunit of heat shock protein H s l W Heat shock protein 90 Mg chelatase-related protein Trigger factor Preprotein translocase, SecY subunit Signal recognition particle-docking protein Homoserine dehydrogenase Aspartate kinase Carbamoyl-phosphate synthase small chain Carbamoyl-phosphate synthase, large subunit CTP synthetase HIT family protein Phosphatase, Ppx/ GppA family Fructose-1,6-bisphosphatase ATP-NAD kinase, putative Dephospho-CoA kinase 4-Hydroxybenzoate octaprenyltransferase Glutamyl-tRNA reductase Dihydropteroate synthase Bifunctional 3,4-dihydroxy-2-butanone 4-phosphate Synthase/ GTP cyclohydrolase I1 protein Oxygen-independent coproporphyrinogen 111 oxidase Riboflavin synthase subunit alpha D-3-phosphoglycerate dehydrogenase Acetyl-CoA carboxylase beta subunit Phosphatidylserine decarboxylase Peptidase, putative GTP-binding protein, GTPl/ Obg family tRNA modification GTPase Conserved hypothetical protein Porphobilinogen deaminase Translation elongation factor Ts Multidrug resistance efflux transporter, putative Delta-aminolevulinic acid dehydratase Peptidase, U32 family

7p"'

ychF dYS fmt" gY7B

xseA" uvrc polA ruvB gY7A

dprA" b b

murF

gidB' lolA* z.

waaC radA hslU htpG

*

tig secY ftsY* hom lysC carA carB PYrG

*

fbP b

coaE* ubiA hemA folP ribB hemN ribE* serA accD psdb b

obg tmzE hemC tsf hemB

COG

J J J J J J J J

L L L L

L L LU D D M M M M M M M M

0 0 0 0 0 U U E E EF EF F FGR FP G G H H

H H H

H H HE I I R R R S

X X X X X

Genes for which automatic primer design was not successful are indicated with an asterisk. hFunctional annotation as the Cluster of Orthologous Genes (COG) database (Tatusov et al., 1997; http://www. ncbi.nlm.nih.gov/ COG).

a

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Next Page CHAPTER 1

bioinformatics.weizmann.ac.il/blocks/).The output from BLOCKS was subsequently subjected to automatic PCR primer design by CODEHOP with parameters maintained at their default settings (Rose et al., 1998). For a total of 41 of 60 genes, a primer combination could be generated whose predicted products ranged from 400 to 1,500 bp (Table 1). The strength of an MLSA approach lies in the concatenation of multiple genes buffering against the distorting effects of recombination at a single locus and resulting in an increased resolving power. Knowing a list of best-performing genes raises the question of how many genes should be used to come up with a cost-effective consensus phylogeny. Figure 4 shows the results of an in silico MLSA analysis that is based on a random combination of 2 to 15 genes selected from the best-performing gene set. When we look at the averages of 100 repetitions per evaluated loci count, we find that each additional gene used improves the correlation with the whole-genome-based phylogeny. However, it does this in a more prominent way, by comparing the lower loci count, whereas the gain in the use of higher loci count is less pronounced. These results also show that combinations of two genes exist that are more congruent to the whole-genome-based phylogeny than some combinations of seven or more genes. However, when only a few genes are used, the buffering capac-

I

I

I

I

I

1

2

3

5

7

I

1

0

I

1

5

Number of genes

Figure 4. In silico MLSA analysis with variable loci count. For a variable number (n = 1-15) of concatenated loci (x axis) randomly selected from the set of best-performing genes (Table l), the differences in tree topology and branch lengths with the wholegenome-based phylogeny was determined and expressed as a tree distance value (y axis). For each number of loci, the procedure was repeated 100 times, and the distributions of the distances between the maximum likelihood trees are plotted as box and whisker plots.

TAXONOMY OF THE CAMPYLOBACTERACEAE

11

ity against unexpected conflicting signals in even a few lineages is too limited. Moreover, in interpreting this analysis, it should be kept in mind that this set of genes was defined by using the same set of strains whose phylogeny we are trying to estimate. Therefore, the analysis does not guarantee similar performance for an independent set of strains. Modern statistical approaches have the potential to make the choice of genes less critical by using sequence information to extract a phylogenetic signal while simultaneously identifying the genes that show discordant signals for particular stains and incorporating this information in the analysis (Didelot and Falush, 2007).

GENERAL DESCRIPTION The Family Campylobacteraceae The genera Campylobacter, Arcobacter, and Sulfurospirillum and the generically misclassified B. ureolyticus form a family of gram-negative, nonsaccharolytic bacteria with microaerobic growth requirements and a low G+C content. This definition is in complete agreement with the original criteria used by Sebald and V&on (1963) to separate several Vibrio species from the genuine vibrios, and to include the former into the new genus Campylobacter. Members of the family Campylobacteraceae occur as commensals or parasites in humans and domestic animals (Campylobacter and Arcobacter) or as free-living environmental bacteria (Arcobacter and Sulfurospirillum). The type genus is the genus Campylobacter Sebald and Vkron 1963. Members of the family Campylobacteraceae have following general characteristics. Cells are curved, S-shaped, or spiral rods that are 0.2 to 0.8 p m wide and 0.5 to 5 p m long. They are gram negative and do not form spores. Cells in old cultures may form spherical or coccoid bodies. They are typically motile, with a characteristic corkscrew-like motion performed by means of a single polar unsheathed flagellum at one or both ends of the cell. Campylobacter and Arcobacter cells grow under microaerobic conditions and have a respiratory and chemoorganotrophic type of metabolism. Some grow under aerobic or anaerobic conditions. Energy is obtained from amino acids or tricarboxylic acid cycle intermediates, not carbohydrates. Carbohydrates are neither fermented nor oxidized. The optimum growth temperature is 30 to 37°C. Typical biochemical characteristics are reduction of fumarate to succinate; negative methyl red reaction and acetoin and indole production; and for most species, reduction of nitrate, absence of hippurate hydrolysis, and presence of oxidase activity.

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DEBRUYNE ET AL.

Menaquinones are the only respiratory quinones detected, with menaquinone-6 (three different structural types) and menaquinone-5 as major components. rRNA genes (16s and 23s) of some strains comprise internal transcribed spacers or intervening sequences. The presence of these internal transcribed spacers is strain specific, not species specific. The DNA base ratio ranges between 27 and 47 mol%. The Genus Campylobacter General characteristics

Campylobacter cells are mostly slender, spirally curved rods, 0.2 to 0.8 p m wide and 0.5 to 5 p m long. Cells of some species are predominantly curved or straight rods. Cells in old cultures may form coccoid bodies; these are considered degenerative forms rather than a dormant stage of the organism. Cells of most species are motile, with a characteristic corkscrew-like motion performed by means of a single polar unsheathed flagellum at one or both ends of the cell. Cells of some species are nonmotile (C. gracilis) or have multiple flagella (Campylobacter showae). In general, biochemical characteristics are as described for the family Campylobacteraceae. Several species grow in anaerobic conditions with fumarate or nitrate as electron acceptor; these species grow only in microaerobic conditions if hydrogen, formate, or succinate is supplemented as electron source. Gelatin, casein, starch, and tyrosine are not hydrolyzed. Oxidase activity is present in all species except C. gracilis. No lipase or lecithinase activity is present. Some species are pathogenic for humans and animals. This organism is found in the reproductive organs, intestinal tract, and oral cavity of humans and animals. The G + C of the DNA ranges from 29 to 47 mol%. At present, there are 17 validly named Campylobacter species, with Campylobacter fetus Sebald and VCron 1963 as the type species. There is no simple gold standard for the routine isolation of all Campylobacter species. Simultaneous application of a microaerobic atmosphere containing hydrogen with a filtration method and a selective base is methodologically the optimal solution. However, the predominant species in human infection can be readily grown in a microaerobic atmosphere on selective media without the necessity to use hydrogen. To evaluate the presence of less common species, appropriate culture conditions need to be applied. Menaquinone-6 (2-methyl-3-farnesyl-farnesyl1.4-naphthoquinone) and a methyl-substituted menaquinone-6 (2,[5 or 81-dimethyl-3-farnesyl-far-

nesyl-1.4-naphthoquinone)have been reported as major respiratory quinones in Campylobacter species. List of the species of the genus Campylobacter C . fetus. The first isolated Campylobacter was almost certainly C. fetus (McFadyean and Stockman, 1913). The species comprises two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis, both of which are considered primary pathogens. C. fetus subsp. fetus has been associated with abortion in sheep and sporadic abortion in cattle, and it has been isolated from a wide range of sources, including ungulates, fowl, reptiles, and humans. It is considered an uncommon human pathogen. It causes systemic infection in immunodeficient patients, or, rarely, neonatal sepsis and septic abortion. C. fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, an infectious venereal disease that can lead to infertility, abortion, and embryo death. It is mainly isolated from the bovine genital tract. Differentiation between the two subspecies is not straightforward. Biochemical differentiation is based on a single phenotypic test, i.e., tolerance to 1% glycine. It was suggested that, additionally, tolerance to 0.1% potassium permanganate, 0.005% basic fuchsin, and 64 mg literp1 cefoperazone may be useful for discrimination between the two subspecies (On and Harrington, 2001). Whole-cell protein electrophoresis, cellular fatty acid analysis, serotyping, and ribotyping do not separate the two taxa. Molecular methods used for discrimination between the two subspecies include numerical analysis of pulsedfield gel electrophoresis (PFGE) DNA profiles (On and Harrington, 2001), amplified fragment-length polymorphism (AFLP) (Duim et al., 2001), randomly amplified polymorphic DNA (RAPD) analysis (Tu et al., 2005), and several PCR tests (e.g., van Bergen et al., 2005b). C. fetus strains of reptile origin are divergent from C. fetus strains from mammalian origin, based on 16s rRNA, recA and sapD gene sequences, RAPD, PFGE, and DNA-DNA hybridization experiments. This suggests that they might represent a distinct taxon (Tu et al., 2005).

C. hyointestinalis. C. hyointestinalis was originally isolated from pigs with intestinal disorders and includes two subspecies: C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii. The former has been isolated from the intestines of pigs and hamsters, fecal samples from cattle, deer, and humans, and occasionally from the blood of patients with bacteremia. C. hyointestinalis subsp. hyointestinalis is associated with porcine proliferative

CHAPTER 1

enteritis and diarrhea in animals and humans, but its pathogenic role is unclear. C. hyointestinalis subsp. lawsonii has been isolated from the stomach of pigs. Its clinical significance is unknown. When grown on common blood agar bases, only some C. hyointestinalis strains grow microaerobically without hydrogen (all strains grow microaerobically in the presence of hydrogen). Both whole-cell protein and macrorestriction profiles, and AFLP analysis can be used for high-resolution identification and for determination of epidemiological relationships in this species (Duim et al., 2001; On and Harrington, 2000).

Campylobacter lanienae. During a routine screen of healthy abattoir workers, Campylobacterlike organisms were isolated from fecal samples of two individuals. Cells were slender, slightly curved rods, with single, bipolar unsheathed flagella (Logan et al., 2000). C. lanienae was also isolated from fecal samples from healthy pigs and has been detected by means of conventional and quantitative PCR in bovine fecal samples. C. sputorum. Bacteria currently classified in the species C. sputorum (On et al., 1998), were originally described as multiple Vibrio species. In 1914, Tunicliff observed a vibrio, later classified as Vibrio sputorum (PrCvot, 1940), in sputum of a patient with bronchitis. Similar bacteria isolated from the bovine vagina and semen were classified as Vibrio bubulus (Florent, 1953). These two species were thought to be related at the subspecies level on the basis of their extensive biochemical similarities and were renamed C. sputorum subsp. sputorum and C. sputorum subsp. bubulus (VCron and Chatelain, 1973). In addition, Firehammer (1965) described a fecal-type vibrio isolated from sheep that differed mainly from V. bubulus by its strong catalase activity, for which the name Vibrio fecalis was proposed. Subsequent DNA-DNA hybridization studies (Roop et al., 1985b) revealed a high level of DNA-DNA relatedness between C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and V. fecalis, and also indicated that C. sputorum subsp. mucosalis (see below) represented a distinct species. Limited biochemical variation between these taxa had been noted (catalase production, and growth on 3.5% NaCl and 1.0% ox-bile media), and therefore Roop et al. (1985b) proposed that these taxa be referred to as source-specific biovars of C. sputorum (C. sputorum bv. sputorum, C. sputorum bv. bubulus, and C. sputorum bv. fecalis). Subsequent studies by On et al. (1998) questioned the legitimacy of C. sputorurn bv. bubulus as a distinct taxon be-

TAXONOMY OF THE CAMPYLOBACTERACEAE

13

cause the tests used to distinguish this taxon from other biovars were found to be poorly reproducible. The identification of urease-positive variants of C. sputorum isolated from cattle feces (Atabay et al., 1997) resulted in the proposal of a new biovar structure, defined by reactions in two simple and reproducible tests: presence of catalase and urease activity. Strains of C. sputorum bv. sputorum produce neither catalase nor urease. They are found in the oral cavity, feces (normal and diarrheic), and abscesses and other skin lesions of humans; the genital tract of bulls; abortions in sheep; and the feces of sheep and pigs. Their pathogenicity is unknown. Strains of C. sputorum bv. faecalis produce catalase, but not urease. They have been isolated from the feces of sheep and cattle, and their pathogenicity is unknown. Finally, strains of C. sputorum bv. paraureolyticus produce urease, but not catalase. Strains are isolated from the feces of cattle and from human diarrhea; their pathogenicity is unknown.

C. mucosalis. The name C. sputorum subsp. mucosalis was originally proposed for a group of strains thought to be a causal agent of proliferative enteritis in pigs (Lawson et al., 1981). However, subsequent studies have identified Lawsonia intracellularis as the principal pathogen in this disease, and DNA-DNA hybridization demonstrated that this organism represented a distinct Campylobacter species (Roop et al., 1985a). C. mucosalis strains do not grow microaerobically on common agar bases in an atmosphere without hydrogen. Three distinct serovars, A, B, and C, have been described (Lawson et al., 1981), which were shown to have strikingly distinct whole-cell protein patterns (Costas et al., 1987; Vandamme et al., 1990). C. mucosalis strains have been isolated from the intestinal mucosa of pigs with porcine intestinal adenamatosis, necrotic enteritis, regional ileitis, and proliferative hemorrhagic enteropathy; and from the porcine oral cavity. Their clinical significance is unknown. Human infections supposedly caused by this organism were shown to be misidentified C. concisus infections (On, 1994). More recently, Tyrrell et al. (2003) reported the isolation of C. rnucosalis, identified by biochemical means, from the tongue dorsum of patients with oral malodor. C . concisus and C:curvus. Tanner et al. (1981, 1984) described two groups of strains isolated from gingival crevices of humans with gingivitis, periodontitis, and periodontosis, which were named C. concisus and C. curvus (the latter was originally described as w. cuwa; see above). Subsequent studies identified C. concisus in stool and blood samples of

14

DEBRUYNE ET AL.

children and adults with and without diarrhea, and more recently also in patients with Barrett’s esophagus, a complication of chronic gastroesophageal reflux disease. The high prevalence of C. concisus in clinical settings has been demonstrated when appropriate isolation conditions are used. Whole-cell protein electrophoresis and DNA-DNA hybridization experiments (Van Etterijck et al., 1996; Vandamme et al., 1989) revealed that C. concisus is a heterogeneous species, which has been confirmed by a range of molecular techniques, including RAPD (Engberg et al., 2005; Matsheka et al., 2006; Van Etterijck et al., 1996), ribotyping (Engberg et al., 2005), and PFGE (Matsheka et al., 2002). AFLP analysis revealed at least four distinct genomospecies within C. concisus (Aabenhus et al., 2005). At present, no clear differences have been observed between isolates from patients with diarrhea and isolates from healthy carriers; therefore, the role of C. concisus as a human enteric pathogen remains unclear. C. cuwus has been rarely isolated from human fecal samples, and it has been implicated in hepatic and bronchial abscesses. Strains of both species require hydrogen for growth in microaerobic conditions.

C. rectus, C . gracilis, and C. showae. C. rectus, C. gracilis, and C. showae are hydrogen-requiring campylobacters for which optimal growth is obtained in anaerobic conditions. Their cell morphology is most unusual. C. rectus (Tanner et al., 1981) is a rather plump, straight rod; its cellular surface is covered with a distinctive array of hexagonal, packed, macromolecular subunits. Strains have been isolated from gingival crevices of humans, in cases of appendicitis, in patients with Barrett’s esophagus, and from extraoral abscesses. It is considered a putative periodontal pathogen. It has been suggested that periodontal disease may contribute to preterm low birth weight, but because many confounding factors may influence preterm delivery, there is no conclusive evidence that periodontal pathogens like C. rectus are actually involved (Buduneli et al., 2005). C. gracilis cells are straight and nonmotile rods (Tanner et al., 1981). C. gracilis is the only oxidasenegative Campylobacter species, although the pattern of cytochromes found in C. gracilis resembled that reported for other campylobacters. Strains have been isolated from gingival crevices; from visceral, head, and neck infections; in soft tissue abscesses; in pneumonia and empyema; and in an ischial wound in humans. The association of C. gracilis with serious deep tissue infection, coupled with a high frequency of antibiotic resistance, suggests that it is an underesti-

mated pathogen, even though conclusive evidence for pathogenicity is lacking. C. showae cells are straight rods and have polar bundles of two to five unsheathed flagella (Etoh et al., 1993). Strains have been isolated from human dental plaque, from infected root canals, and from periodontitis lesions. Pathogenicity is unknown.

Campylobacter hominis. C. hominis was initially described as “Candidatus Campylobacter hominis,” an uncultivated species found in the gastrointestinal tract of healthy human individuals (Lawson et al., 1998). After modifications of the isolation methodology, the organism was subsequently isolated by anaerobic culture. Similar to C. gracilis, C. hominis cells are nonmotile straight rods without flagella (Lawson et al., 2001). C. horninis has been detected by speciesspecific PCR in both healthy and diarrheic human fecal samples. C. jejuni and C. coli. In 1927, Smith and Orcutt described a group of Vibrio-like bacteria from the feces of cattle with diarrhea. Jones and coworkers (193 1) showed a causal relationship between these microaerophilic vibrios and bovine dysentery and subsequently named the organism Vibrio jejuni. Similar organisms were detected in blood cultures of humans with gastroenteritis and aborted sheep fetuses. In 1944, Doyle isolated yet another vibrio from feces of pigs with diarrhea and classified it as Vibrio coli (Doyle, 1948). At present, C. jejuni and C. coli are by far the most important human enteropathogens among the campylobacters. C. jejuni infection is also a known antecedent of Guillain-BarrC syndrome. Within C. jejuni, two subspecies are recognized: C. jejuni subsp. jejuni and C. jejuni subsp. doylei. Strains of the latter species differ biochemically from the former by the absence of nitrate reduction, cephalothin susceptibility, and often catalase activity (a weak reaction may also be observed). Many common molecular-based methods are not suitable for subspeciation, but recently a multiplex PCR based on the nap locus was proposed for C. jejuni subspecies differentiation (Miller et al., 2007). The pathogenic role of C. jejuni subsp. doylei is unknown. Although it is isolated infrequently, it is obtained primarily from human clinical samples, and it is often associated with bacteremia, notably in infants. Biochemically, C. jejuni subsp. jejuni and C. coli differ only in their ability to hydrolyze hippurate, for which C. coli is negative. However, hippuratenegative C. jejuni subsp. jejuni strains are well recognized. Because of the limitations of biochemical differentiation, a wide range of alternative, mainly

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genotypic methods has been developed for differentiation between C. coli and C. jejuni subsp. jejuni (e.g., Jensen et al., 2005; Mandrel1 et al., 2005; Nayak et al., 2005; Sails et al., 2001). The complexity of this taxonomic area has been illustrated by data showing that strains first described as a distinct species, c. hyoilei, are in fact c. coli (Vandamme et al., 1997), despite a higher 16s rRNA sequence similarity to C. jejuni (Alderton et al., 1995). A multilocus sequence typing study of C. coli demonstrated that C. hyoilei strains belonged to the sequence type 1017 complex, which comprised the majority of the C. coli sequence types and included strains from Australia, Belgium, Canada, Denmark, Japan, and the United States and which were isolated from humans and a range of animals (Miller et al., 2005).

C. lari. C. luri was originally referred to as the group of nalidixic acid-resistant thermophilic campylobacters (the NARTC group). These strains were primarily differentiated from C. jejuni and C. coli by their resistance to nalidixic acid, their anaerobic growth in the presence of trimethylamine N-oxide hydrochloride, and later also by the absence of indoxy1 acetate hydrolysis. However, nalidixic acidsusceptible strains (NASC strains), urease-producing strains (UPTC strains), and urease-producing nalidixic acid-susceptible strains (Endtz et al., 1997) were also identified as C. luri variants by onedimensional whole-cell protein electrophoresis and semiquantitative DNA-DNA hybridization (Vandamme et al., 1991b). Endtz et al. (1997) reported a striking heterogeneity among and within the different groups of C. luri variants, which was confirmed by Duim and coworkers (2004), who clearly distinguished four subgroups by means of numerical analysis of AFLP profiles and of partial protein profiles. Preliminary DNA-DNA hybridization experiments revealed that some of these groups may represent distinct species, but at present, the taxonomic status of these groups remains unclear. C. luri strains have been isolated from intestinal contents of seagulls and other animals, river water, and shellfish. In humans, C. luri has been occasionally isolated from diarrheic feces and from cases of bacteremia and other extraintestinal infections in both immunocompetent and immunodeficient patients. Its pathogenicity is unknown. Campylobacter insulaenigae. Four Cumpylobacter-like strains were isolated from rectal swabs taken from common seals and the intestinal contents of a porpoise in northern Scotland and represented a distinct Campylobacter species, named C. insulaenigrae (Foster et al., 2004). C. insuluenigrue

TAXONOMY OF THE CAMPYLOBACTERACEAE

15

has also been isolated from free-living and stranded northern elephant seals in California. Although initially reported as nonthermotolerant, several of the Californian C. insuluenigrue strains were able to grow at 42"C, leading to an initial misidentification of some of these strains as C. lari (Stoddard et al., 2007). The pathogenic potential of these bacteria in marine mammals is unknown.

Campylobacter canadensis. During a study of the bacterial flora of the cloacae of whooping cranes (Grus umericuna), 10 atypical Campylobacter isolates were recovered on two separate occasions and were classified as C. canadensis. The cells were polymorphic in shape (sigmoid to coccoid) and possessed a single polar flagellum. The prevalence of C. cunudensis and its role are at present unknown (Inglis et al., 2007). C. upsaliensis and Campylobacter helveticus. Both C. upsaliensis and C. helveticus are catalasenegative or weakly positive thermotolerant campylobacters. C. upsuliensis strains have been isolated from feces of enteric and asymptomatic humans, dogs, cats, and meercats as well as from human blood samples. Strains associated with a human abortion, a breast abscess, hemolytic-uremic syndrome, and a prosthetic knee infection have been described. C. helveticus has been isolated from feline and canine fecal samples, but at present, no strains have been isolated from human sources. Reports have emerged worldwide implicating C. upsuliensis as a human enteric pathogen, and in some regions, it is preceded only by C. jejuni in being the most frequently isolated Cumpylobucter species in human stool samples. Accumulated clinical and epidemiological data support the importance of C. upsuliensis as a primary human pathogen, even though direct evidence is at present still lacking. Differentiation of Campylobacter species Apart from the recent single-locus, multilocus, or whole-genome-based analyses discussed above, a variety of approaches have been used to identify campylobacters to the species level. Some of these are discussed below. Classical phenotypic characteristics. Most routine laboratories use basic biochemical tests to identify campylobacters. The lack of application of highly standardized procedures and the well-known biochemical inertness of campylobacters often make biochemical identification of these bacteria difficult be-

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cause the discrimination among species or subspecies may rely on one differential character, such as presence of hippuricase or urease activity (On, 1996). A computerized scheme of nearly 70 biochemical tests readily differentiated virtually all Campylobacter species (On et al., 1996; On and Holmes, 1995). Traditional taxonomic analyses. In taxonomic studies, the reference method for the delineation of bacterial species is determination of the level of DNA-DNA hybridization (Vandamme et al., 1996). A variety of DNA-DNA hybridization studies have been performed before the phylogenetic relationships of campylobacters were established by means of rRNA-directed studies and focused on the species known since the early 1980s (detailed overviews are given by Vandamme and Goossens [1992] and On [1996]). Chemotaxonomic techniques such as wholecell protein and fatty acid analysis have been used in a range of taxonomic studies for the identification of Campylobacter species. In particular, electrophoresis of whole-cell proteins by highly standardized sodium dodecyl sulfate-polyacrylamide gel electrophoresis has proven to be extremely reliable to identify Campylobacter and other strains at the species level because numerous studies have revealed a correlation between high similarity in whole-cell protein content and level of DNA-DNA hybridization (On, 1996; Vandamme et al., 1996). rRNA-based approaches. Sequence analysis of the rRNA genes became the ultimate tool to study bacterial phylogeny during the 1980s, and international databases have been constructed to collect sequences and to make them publicly available. Nowadays, it is common to identify unknown organisms that are of particular interest by means of 16s rRNA sequence analysis and comparison with rRNA sequences present in international databases (Gorkiewicz et al., 2003). Several important points should be addressed here. Although unsurpassed in its capacity to reveal the phylogenetic neighborhood of an unknown bacterium, comparison of entire 16s rRNA sequences is generally not adequate for the identification of strains to the species level. It has been reported that strains belonging to different species may have identical 16s rRNA sequences and that strains of one species may have 16s rRNA genes that differ up to 3% (Stackebrandt and Goebel, 1994). Harrington and On (1999) demonstrated differences of up to 4.3% of the total 16s rRNA sequence between C. hyointestinalis strains. There is definitely a lack of knowledge, not only of the strain-to-strain variation within a species, but also of the interoperon variation within a single

strain as reported by Clayton et al. (1995). Therefore, concluding that an unknown organism belongs to a particular species because it shares a high percentage of its 16s rRNA gene sequence with another strain of that species, or concluding that an unknown represents a novel species because it shares only 97% of its 16s rRNA gene sequence with its closest neighbor is premature in the absence of other data. Alternatively, sequence information derived from rRNA cistrons has been used successfully to design and validate many species-, group-, or genusspecific primers and probes (Liibeck et al., 2003; On, 1996). Applied in a PCR or a hybridization assay, these primers and probes offer valuable alternatives for identification of Campylobacter taxa. Finally, broad-spectrum molecular identification schemata that are based on restriction fragment analysis of PCR amplicons derived from 16s (Cardarelli-Leite et al., 1996; Marshall et al., 1999) and 23s (Gonzalez et al., 2006; Hurtado and Owen, 1997) rRNA genes have also been described. AFLP analysis. AFLP analysis is based on selective amplification of restriction fragments of chromosomal DNA and has been widely used in a variety of bacterial genera, including Campylobacter, both for identification and typing purposes. It is a reproducible and discriminatory technique, particularly well suited for computer-assisted data analysis. In Campylobacter, AFLP schemes were initially developed for typing purposes, incorporating only C. jejuni subsp. jejuni and C. coli, and two studies revealed clear differences in AFLP profiles between these two species (Duim et al., 1999; Kokotovic and On, 1999). These observations were extended to other Campylobacter species (Duim et al., 2001; On and Harrington, 2000) and demonstrated that numerical analysis of AFLP profiles is well suited for identification at the species, subspecies, and strain levels. Numerous subsequent studies have applied AFLP to explore intraspecies diversity (e.g., Aabenhus et al., 2005; Duim et al., 2004) and epidemiological relationships (e.g., Keller et al., 2007; Siemer et al., 2005) of Campylobacter strains. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In

MALDI-TOF MS, biological samples are mixed with a matrix that is chosen such that it specifically absorbs the energy of a laser beam. The high-energy impact is followed by the formation of ions that are extracted through an electric field; these are subsequently focused and detected as a m / z (mass/charge) spectrum. In a preliminary study, Winkler et al. (1999) analyzed a set of C. jejuni, C. coli, C. fetus,

CHAPTER 1

H. pylori, and H. mustelae strains by means of both whole cells and partially purified extracts from lysed cells, and they reported the detection of discriminatory biomarker ions in the 10- to 20-kDa range. Further research into the potential of MALDI-TOF MS for identification purposes was performed by Mandrell and coworkers (2005). They analyzed whole bacterial cells from a large collection of well-characterized reference strains from C. coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis, and they observed species-identifying biomarker ions in the 9- to 14-kDa range for each of the six species. Most difficulty was observed for differentiating the closely related C. upsaliensis and C. helveticus. The Genus Arcobacter General characteristics

Arcobacter cells are slender, spirally curved rods 0.2 to 0.9 pm wide and 0.5 to 3 p m long; S-shaped or helical cells are often present. Cells in old cultures may form spherical or coccoid bodies and loose spiral filaments up to 20 p m long. Arcobacter is motile, with a characteristic corkscrew-like motion performed by means of a single polar unsheathed flagellum at one or both ends of the cell. In general, biochemical characteristics are as described for the family Campylobacteraceae. In addition, arcobacters are able to grow in aerobic and anaerobic conditions and at 15°C. Hydrogen is not required. Indoxyl acetate is hydrolyzed. No hydrolysis of gelatin, casein, starch, hippurate, and tyrosine is present. Habitat is extremely diverse. Arcobacter is unique among the Epsilonproteobacteria in that it combines plant and animal associated species and free-living environmental species within one genus. Some species are associated with humans and animals and are found in the reproductive organs and abortions of various animals and in the intestinal tract of humans and animals. Other sources include water reservoirs, river and surface water, sewage, oil field communities, marine and hypersaline environments, and deep-sea hydrothermal vents; one species is a plant-associated nitrogen fixer. The G+C of the DNA ranges from 27 to 35 mol%. Menaquinone-6 (2 -methyl-3 -farnesyl-farnesyl- lY4-naphthoquinone) and a second menaquinone-6, the detailed structure of which has not been determined, were reported as major respiratory quinones. The genus Arcobacter presently comprises six validly named species, with Arcobacter nitrofigilis Vandamme, Falsen, Rossau, Hoste, Segers, Tytgat, and De Ley 1991 as the type species.

TAXONOMY OF THE CAMPYLOBACTERACEAE

17

Isolation procedures for Arcobacter spp. are often based on filtration methods or selective media developed for Campylobacter, Yersinia, or even Leptospira species (Vandamme, 2000). In addition, several isolation methods were developed specifically for Arcobacter spp. (deBoer et al., 1996; Houf et al., 2001a; Johnson and Murano, 1999a, 1999b) by using a variety of selective media, in some cases preceded by an enrichment step. However, several of these methods are suboptimal because arcobacters may be overgrown by campylobacters present in the same specimens, and they may be susceptible to some of the antimicrobial agents used in the selective media (Houf et al., 2001b). In 2001, an Arcobacter enrichment broth and Arcobacter selective agar were developed for the isolation of arcobacters from poultry products, which allowed the recovery of A. butzleri, Arcobacter cryaerophilus, and A. skirrowii and successfully inhibited the contaminating flora (Houf et al., 2001a). In recent years, this procedure was validated for several other matrices, including animal feces (Van Driessche et al., 2003), human feces (Houf and Stephan, 2007), and poultry intestinal content (Van Driessche and Houf, 2007). Species of the genus Arcobacter A. nitrofigilis. A. nitrofigilis strains occur in the roots and rhizosphere of the salt marsh plant Spartina alternifiora, where they fix nitrogen (McClung et al., 1983). They differ from most other members of the family Carnpylobacteraceae by their preference for a high salt concentration (optimum growth is in the presence of 10 to 40 g/liter NaCl), and by the presence of nitrogenase and urease activity. A. cryaerophilus. In 1985, Neil1 and coworkers performed an extensive phenotypic characterization of aerotolerant Campylobacter strains isolated from various animal sources. They concluded that the aerotolerant strains were only distantly related to strains of the other Campylobacter species examined and emphasized that they formed a heterogeneous group. Their findings were confirmed by integrated phenotypic, genotypic, and chemotaxonomic studies of, in part, the same isolates (Kiehlbauch et al., 1991; Vandamme et al., 1991a, 1992), which entailed in the description of a novel genus Arcobacter, with three separate animal-associated species: A. cryaerophilus, A. butzleri, and A. skirrowii. Within A. cryaerophilus, two subgroups, referred to as subgroup 1 or group 1A and subgroup 2 or group lB, have been described (Kiehlbauch et al., 1991; Vandamme et al., 1992). Strains of these subgroups differ in their whole-cell protein and fatty acid

18

DEBRUYNE ET AL.

patterns, and in the restriction fragment length polymorphisms of the rRNA genes. Moderate levels of DNA-DNA hybridization were detected between strains of the different subgroups, suggesting that they could be given a distinct taxonomic rank. However, at present, no formal nomenclatural modifications have been proposed. A. cryaerophilus strains have been isolated from cases of human bacteremia and diarrhea; from chicken, pork, and duck carcasses; from bovine, ovine, and porcine abortions; from fecal samples from cattle, pigs, and horses; and from mastitis in cattle. One study reported its isolation from a naturally infected rainbow trout. Their pathogenicity is unknown. A. butzleri. A. butzleri strains have been isolated from human blood and diarrheic feces; from feces of various animals with diarrhea including nonhuman primates, pigs, horses, cattle, an ostrich and a tortoise; from bovine and porcine abortions; from various food products including ground pork, chicken, and turkey; from surface and drinking water reservoirs and canal waters; from seawater; and from plankton. This species is associated with enteritis, abdominal cramps, bacteremia, and appendicitis in humans and with enteritis and abortion in animals. It was identified as the fourth most isolated Campylobacter-like organism from human diarrheal stool in Belgium and France (Prouzet-Mauleon et al., 2006; Vandenberg et al., 2004). Screening for arcobacters in asymptomatic humans yielded no positive results for A. butzleri (Houf and Stephan, 2007).

A. skirrowii. A. skirrowii strains have been isolated from preputial fluids of bulls; from bovine, ovine, and porcine abortions; from diarrheic feces of various animals including sheep and cattle; and from chicken carcasses. At present, there is one report on the isolation of A. skirrowii from a patient with chronic diarrhea, but it was unclear whether the strain was the etiological agent (Wybo et al., 2004). Their pathogenicity remains unknown. Arcobacter cibarius. A. cibarius strains were first isolated during a long-term study of Arcobacter contamination on poultry carcasses. Twenty isolates, recovered from 13 unrelated broiler carcasses, yielded no Arcobacter species-specific PCR amplicon, but they did yield a genus-specific PCR amplicon (Houf et al., 2005). Additional data demonstrated that the 20 isolates represented a novel Arcobacter species. The organism has also been isolated from porcine and ovine feces (K. Houf, unpublished data) and pig-

gery effluent (Chinivasagam et al., 2007). At present, their pathogenic potential is unknown.

Arcobacter halophilus. A. halophilus is an obligately halophilic Arcobacter species isolated from a hypersaline lagoon on Laysan Atoll in the northwestern Hawaiian Islands, and currently comprises a single strain. Phenotypic and genotypic data placed the type strain LA3 lBT within the genus Arcobacter, but it can be distinguished from all recognized arcobacters by its obligate halophily and fatty acid composition (Donachie et al., 2005). “Candidatus Arcobacter sulfidicus.” “Candidatus A. sulfidicus” is a highly motile sulfide-oxidizing bacterium, unique among prokaryotes in that it excretes elemental sulfur in the form of hydrophilic rigid filaments, as opposed to globular or amorphous sulfur found with other sulfur oxidizers (Wirsen et al., 2002). The organism currently remains a candidate species that is difficult to cultivate, not because of the nutritional aspects of cultivation, but because of the required growth apparatus and conditions (Wirsen et al., 2002). The formation of filamentous sulfur appears to be a specific adaptation permitting these organisms to colonize the specialized niche, characterized by dynamic fluid movement and high sulfide and low oxygen concentrations, required for their survival. The worldwide occurrence of microbial filamentous sulfur formation in marine ecosystems indicates that it is likely to play an important yet undefined role in global sulfur and carbon cycles (Sievert et al., 2007). As opposed to the chemoorganotrophic growth found in other Arcobacter spp., this organism has a chemoautotrophic mode of nutrition. Experiments demonstrated that it has a microaerobic growth preference and that it aggregates within the oxic-anoxic interface by exhibiting a chemotactic response (Sievert et al., 2007). Other Organisms On and coworkers (2002, 2003) reported the isolation of five strains associated with pig abortions and one from duck feces, which were believed to represent a novel Arcobacter species, as assessed by AFLP profiling, phenotype, and 16s rRNA gene sequence analysis. However, formal description was deferred until the identification of at least 10 isolates, in accordance with recommendations for minimal taxonomic standards for this group of bacteria (Ursing et al., 1994). Also, a number of culture-independent studies have documented the existence of additional

CHAPTER 1

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TAXONOMY OF THE CAMPYLOBACTERACEAE

19

Arcobacter taxa, as assessed by 16s rRNA gene phylogeny, in environmental samples including oil field communities (Gevertz et al., 2000; Sette et al., 2007; Voordouw et al., 1996), cyanobacterial mats (Teske et al., 1996), activated sludge of a large municipal wastewater treatment plant (Snaidr et al., 1997), and deep-sea hydrothermal vents (Moussard et al., 2006). Other potentially novel species have been detected in the tunic matrix of the colonial ascidian Diplosoma migrans (Schuett et al., 2005) and as part of the Chilean oyster (Tiostrea chilensis) microbiota (Romero et al., 2002). Because none of these novel taxa was cultivated, they were not formally named.

PCR for identification of A. butzleri and A. cryaerophilus (Brightwell et al., 2007). As for the aforementioned PCR assays, A. cibarius was not included in the evaluation of these DNA-based identification methods. The nucleotide sequences of the gyrA gene for A. butzleri, A. cryaerophilus, A. cibarius, and A. skirrowii have been determined. Phylogenetic analysis of the generated gyrA sequences provided a result similar to 16s rRNA gene phylogeny. Phylogenetic analysis allowed a good separation of these species from each other and from related Campylobacter and Helicobacter species (Abdelbaqi et al., 2007).

Differentiation of Arcobacter Species

The Genus Sulfurospirillum

Basic biochemical tests are still routinely used to identify arcobacters to the species level, and, like for campylobacters, this is hampered by their biochemical inertness and intraspecies variability. In particular, the separation of A. cryaerophilus subgroup 2 and A. butzleri is tedious (Vandamme et al., 1992). As mentioned for Campylobacter, numerical analysis of a large number of phenotypic characteristics (On et al., 1996; On and Holmes, 1995) and whole-cell protein electrophoresis can also be used for Arcobacter identification. Whole-cell fatty acid analysis did not allow discrimination between A. butzleri from A. cryaerophilus subgroup 2, but this technique did permit differentiation of all other Arcobacter taxa (Vandamme and Goossens, 1992). Several PCR assays for the discrimination of animal-associated Arcobacter species have been developed in the last decade, often in the form of a multiplex reaction (Brightwell et al., 2007; Houf et al., 2000; Kabeya et al., 2003). At present, no PCR assay has been developed that included A. cibarius. Restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16s rRNA (Cardarelli-Leite et al., 1996) or 23s rRNA (Hurtado and Owen, 1997) differentiated A. butzleri from other arcobacters but did not differentiate A. cryaerophilus from A. skirrowii. A PCR-restriction fragment length polymorphism analysis that used either a 16s or 23s rRNA amplicon in combination with only one restriction enzyme permitted genuslevel discrimination between Arcobacter, Campylobacter, and Helicobacter (Gonzalez et al., 2006). Numerical analysis of AFLP profiles allowed discrimination of four Arcobacter species (i.e., A. butzleri, A. cryaerophilus, A. skirrowii and A. nitrofigilis) but did not include A. cibarius or A. halophilus (On et al., 2003). Other DNA-based methods include 16s rRNA-based DNA probes (Wesley et al., 1995), ribotyping (Kiehlbauch et al., 1994), and real-time

The genus Sulfurospirillum was established by Schumacher et al. (1992) to accommodate two freeliving Campylobacter-like strains (Spirillum 5 17.ST and Campylobacter sp. “Veldkamp”), which formed a distinct clade within the rRNA superfamily VI (Vandamme et al., 1991a). At present, there are seven named Sulfurospirillum species, with Sulfurospirillum deleyianum (Wolfe and Pfennig 1977) Schumacher, Kroneck, and Pfennig 1993 as the type species. Strain Spirillum 5 175T, isolated from anoxic mud from a forest pond near Heiningen, Germany, was assigned as the type strain of the species Sulfurospirillum deleyianum. Sulfurospirillum arcachonense was isolated from oxidized surface sediment in an intertidal mud flat near Arcachon, France (Finster et al., 1997). Stolz et al. (1999) described Sulfurospirillum barnesii, formerly referred to as “Geospirillum barnesii,” and Sulfurospirillum arsenophilum. The type strain of S. barnesii, SES3T, was isolated from a selenate-contaminated freshwater lake in western Nevada, USA (Oremland et al., 1994), and is able to use selenate and arsenate as terminal electron acceptors. The type strain for S. arsenophilum, MIT-13T, was isolated from arsenic-contaminated watershed sediments in eastern Massachusetts, USA (Ahmann et al., 1994), and is able to reduce arsenate. Sulfurospirillum halorespirans PCE-M2T, isolated from a polluted anaerobic soil, and Sulfurospirillum multivorans KT (formerly Dehalospirillum multivorans), isolated from activated sludge, both have the ability to reduce the pollutant tetrachloroethylene (Luijten et al., 2004; Scholzmuramatsu et al., 1995). Sulfurospirillum cavolei Phe9 lTwas isolated from petroleum-contaminated groundwater in an underground crude oil storage cavity in Japan (Kodama et al., 2007). Sulfurospirilla are extremely fastidious, and very few strains have been isolated and studied. Speciation is primarily based on differences in 16s rRNA gene

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DEBRUYNE ET AL.

sequences, although there are obvious differences in the metabolism of these organisms too. Cells of all of these species are slender, spirally curved rods 0.1 to 0.5 p m wide and 1.0 to 3 p m in length. They may form helical chains of two or more cells. As far as the data available indicate, their general biochemical profiles conform to that of the family Campylobacteraceae. All species show oxidase activity. Growth occurs between 8 and 36°C. The G+C of the DNA ranges from 32 to 42 mol%. Menaquinone-6, a methyl-substituted menaquinone-6 (referred to as thermoplasmaquinon-6), and menaquinone-5 have been reported as respiratory quinones in S. deleyianum; menaquinone-6 and thermoplasmaquinon-6 were found in S. arcachonense. Members of this genus are all sulfur reducers but exhibit metabolic versatility. Different isolation procedure have been described, all of which involve enrichment procedures (Finster et al., 1997; Oremland et al., 1994; Wolfe and Pfennig, 1977). Several other presently unnamed organisms also belong to the genus Sulfurospirillum. Coleman et al. (1993) isolated a marine sulfur-reducing bacterium, designated strain SM-5, implicated in the reduction of Fe(II1). A nitrate-reducing Sulfurospirillum sp., strain KW, was isolated from an oil field (Hubert and Voordouw, 2007). Strain “Veldkamp” (DSM 806), isolated from activated sludge (Laanbroek et al., 1977), was originally classified as a Sulfurospirillum sp. (Schumacher et al., 1992) and was later reclassified as S. deleyianum, but phylogenetic analysis of the 16s rRNA gene sequence of this strain indicates that it may represent a separate species (Fig. 1). Strain NP4, isolated from groundwater with high arsenic concentrations, is classified as a Sulfurospirillum species on the basis of 16s rRNA gene sequence analysis, and it differs from the other Sulfurospirillurn isolates in its carbon source and electron acceptor usage profiles (MacRae et al., 2007). This organism may adversely affect water quality by reducing arsenate to the more toxic arsenite.

Bacteroides ureolyticus B. ureolyticus (Jackson and Goodman, 1978) cells are nonmotile rods that grow microaerobically when hydrogen is provided. Representative strains of this species were included in a polyphasic taxonomic study to elucidate their taxonomic status (Vandamme et al., 1995). B. ureolyticus resembles campylobacters in respiratory quinone content, DNA base ratio, and most of its phenotypic characteristics, but differs from campylobacters in fatty acid composition and proteolytic metabolism. This organism was, however, not formally reclassified pending a thorough taxo-

nomic characterization of additional B. ureolyticuslike bacteria (Vandamme et al., 1995). Strains have been isolated from superficial ulcers and soft tissue infections, urethritis, vaginosis, and periodontal disease. A pathogenic role is suggested by its predominance in mixed infections and its strong proteolytic activity, which may enable tissue destruction. Acknowledgments. We are indebted to the Fund for Scientific Research-Flanders (Belgium) for research grants (P.V.) and for a position as a postdoctoral fellow (D.G.).

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oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26:1628-1635. Sails, A. D., A. J. Fox, F. J. Bolton, D. R. Wareing, D. L. Greenway, and R. Borrow. 2001. Development of a PCR ELISA assay for the identification of Campylobacter jejuni and Campylobacter coli. Mol. Cell. Probes 15:291-300. Sandstedt, K., and J. Ursing. 1991. Description of Campylobacter upsaliensis sp. nov. previously known as the CNW group. Syst. Appl. Microbiol. 14:39-45. Scholzmuramatsu, H., A. Neumann, M. Messmer, E. Moore, and G. Diekert. 1995. Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroetheneutilizing, strictly anaerobic bacterium. Arch. Microbiol. 163:4856. Schouls, L. M., S. Reulen, B. Duim, J. A. Wagenaar, R. J. Willems, K. E. Dingle, F. M. Colles, and J. D. Van Embden. 2003. Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J. Clin. Microbiol. 41:15-26. Schuett, C., H. Doepke, W. Groepler, and A. Wichels. 2005. Diversity of intratunical bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans. Helgoland Mar. Res. 59:136-140. Schumacher, W., P. M. H. Kroneck, and N. Pfennig. 1992. Comparative systematic study on Spirillum 5 175, Campylobacter and Wolinella species: description of Spirillum 5 175 as Sulfurospirillum deleyianum gen. nov., spec. nov. Arch. Microbiol. 158:287293. Sebald, M., and M. V6ron. 1963. Teneur en bases de I’ADN et classification des vibrions. Ann. lnst. Pasteur 105:897-910. Sette, L. D., K. C. Simioni, S. P. Vasconcellos, L. J. Dussan, E. V. Neto, and V. M. Oliveira. 2007. Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin. Antonie Leeuwenhoek 91:253-266. Siemer, B. L., E. M. Nielsen, and S. L. On. 2005. Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping. Appl. Environ. Microbiol. 71:1953-1958. Sievert, S. M., E. B. Wieringa, C. 0. Wirsen, and C. D. Taylor. 2007. Growth and mechanism of filamentous-sulfur formation by “Candidatus Arcobacter sulfidicus” in opposing oxygensulfide gradients. Environ. Microbiol. 9:271-276. Skirrow, M. B. 1977. Campylobacter enteritis: a “new” disease. Br. Med. J. 2:9-11. Skirrow, M. B. 2006. John McFadyean and the centenary of the first isolation of Campylobacter species. Clin. Infect. Dis. 43: 1213-1217. Smith, T., and M. Orcutt. 1927. Vibrios from calves and their serological relation to Vibrio fetus. J. Exp. Med. 45:391-397. Smith, T., and M. S. Taylor. 1919. Some morphological and biochemical characters of the spirilla (Vibrio fetus n. sp.) associated with disease of the fetal membranes in cattle. J. Exp. Med. 310: 299-312. Snaidr, J., R. Amann, I. Huber, W. Ludwig, and K. H. Schleifer. 1997. Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl. Environ. Microbiol. 63:2884-2896. Stackebrandt, E., and B. M. Goebel. 1994. A place for DNA-DNA reassociation and 16s ribosomal RNA sequence analysis in the present species definition in bacteriology. lnt. J. Syst. Bacteriol. 44~846-849. Stoddard, R. A., W. G. Miller, J. E. Foley, J. Lawrence, F. M. Gulland, P. A. Conrad, and B. A. Byrne. 2007. Campylobacter insulaenigrae isolated from Northern Elephant Seals (Mirounga angustirostris) in California. Appl. Environ. Microbiol. 73:17291735.

Stolz, J. F., D. J. Ellis, J. S. Blum, D. Ahmann, D. R. Lovley, and R. S. Oremland. 1999. Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the epsilon Proteobacteria. lnt. J. Syst. Bacteriol. 49:1177-1180. Stolz, J. F., R. S. Oremland, B. J. Paster, F. E. Dewhirst, and P. Vandamme. 2005. Genus 111. Sulfurospirillum Schumacher, Kroneck, and Pfennig 1993, 188VP (Effective publication: Schumacher, Kroneck, and Pfennig 1992, 291), emend. Fienster, Liesack and Tindall 1997d, 1216, p. 1165-1168. In D. J. Brenner, N. R. Krieg, J. T. Staley, and G. M. Garrity (ed.), BergeySManual of Systematic Bacteriology, vol. 2. Springer-Verlag, New York. Suerbaum, S., M. Lohrengel, A. Sonnevend, F. Ruberg, and M. Kist. 2001. Allelic diversity and recombination in Campylobacter jejuni. J. Bacteriol. 183:2553-2559. Tanner, A. C. R., S. Badger, C. H. Lai, M. A. Listgarten, R. A. Visconti, and S. S. Socransky. 1981. Wolinella gen. nov., Wolinella succinogenes (Vibriosuccinogenes Wolin et. al.) comb. nov., and description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from humans with periodontal disease. lnt. J. Syst. Bacteriol. 31:432-445. Tanner, A. C. R., M. A. Listgarten, and J. L. Ebersole. 1984. Wolinella curva sp. nov.: “Vibriosuccinogenes” of human origin. Int. J. Syst. Bacteriol. 34:275-282. Tatusov, R. L., E. V. Koonin, and D. J. Lipman. 1997. A genomic perspective on protein families. Science 278:63 1-637. Teske, A., P. Sigalevich, Y. Cohen, and G. Muyzer. 1996. Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16s ribosomal DNA fragments as a tool for isolation in pure cultures. Appl. Environ. Microbiol. 62:4210-4215. Totten, P. A., C. L. Fennell, F. C. Tenover, J. M. Wezenberg, P. L. Perine, W. E. Stamm, and K. K. Holmes. 1985. Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men. J. Infect. Dis. 151:131-139. Tu, Z. C., W. Eisner, B. N. Kreiswirth, and M. J. Blaser. 2005. Genetic divergence of Campylobacter fetus strains of mammal and reptile origins. J. Clin. Microbiol. 43:3334-3340. Tunicliff, R. 1914. An anaerobic vibrio isolated from a case of acute bronchitis. J. Infect. Dis. 15:350-351. Tyrrell, K. L., D. M. Citron, Y. A. Warren, S. Nachnani, and E. J. Goldstein. 2003. Anaerobic bacteria cultured from the tongue dorsum of subjects with oral malodor. Anaerobe 9:243-246. Ursing, J. B., H. Lior, and R. J. Owen. 1994. Proposal of minimal standards for describing new species of the family Campylobacteraceae. Int. J. Syst. Bacteriol. 445342445. van Bergen, M. A., K. E. Dingle, M. C. Maiden, D. G. Newell, L. van der Graaf-Van Bloois, J. P. van Putten, and J. A. Wagenaar. 2005a. Clonal nature of Campylobacter fetus as defined by multilocus sequence typing. J. Clin. Microbiol. 435888-5898. van Bergen, M. A., G. Simons, L. van der Graaf-van Bloois, J. P. van Putten, J. Rombout, I. Wesley, and J. A. Wagenaar. 2005b. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. J. Med. Microbiol. 54: 1217-1224. Vandamme, P. 2000. Taxonomy of the family Campylobacteraceae, p. 3-26. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Vandamme, P., M. I. Daneshvar, F. E. Dewhirst, B. J. Paster, K. Kersters, H. Goossens, and C. W. Moss. 1995. Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov. lnt. J. Syst. Bacteriol. 45:145-152.

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Vandamme, P., and J. De Ley. 1991. Proposal for a new family, Campylobacteraceae.Int. J. Syst. Bacteriol. 41:45 1-455. Vandamme, P., E. Falsen, B. Pot, B. Hoste, K. Kersters, and J. De Ley. 1989. Identification of EF group 22 campylobacters from gastroenteritis cases as Campylobacter concisus. J. Clin. Microbiol. 27: 1775-1781. Vandamme, P., E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley. 1991a. Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int. J , Syst. Bacteriol. 41~88-103. Vandamme, P., and H. Goossens. 1992. Taxonomy of Campylobacter, Arcobacter, and Helicobacter: a review. Zentralbl. Bakteriol. 276:447-472. Vandamme, P., B. Pot, E. Falsen, K. Kersters, and J. De Ley. 1990. Intra- and interspecific relationships of veterinary campylobacters revealed by numerical analysis of electrophoretic protein profiles and DNA:DNA hybridizations. Syst. Appl. Microbiol. 13~295-303. Vandamme, P., B. Pot, M. Gillis, P. de Vos, K. Kersters, and J. Swings. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60:407-438. Vandamme, P., B. Pot, and K. Kersters. 1991b. Differentiation of campylobacters and Campylobacter-like organisms by numerical analysis of one-dimensional electrophoretic protein patterns. Syst. Appl. Microbiol. 1457-66. Vandamme, P., L. J. Van Doorn, S. T. a1 Rashid, W. G. Quint, J. van der Plas, V. L. Chan, and S. L. On. 1997. Campylobacter hyoilei Alderton et al. 1995 and Campylobacter coli Veron and Chatelain 1973 are subjective synonyms. Int. J. Syst. Bacteriol. 47~1055-1060. Vandamme, P., M. Vancanneyt, B. Pot, L. Mels, B. Hoste, D. Dewettinck, L. Vlaes, C. van den Borre, R. Higgins, and J. Hommez. 1992. Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. Int. J. Syst. Bacteriol. 42:344-356. Van Driessche, E., and K. Houf. 2007. Discrepancy between the occurrence of Arcobacter in chickens and broiler carcass contamination. Poult. Sci. 86:744-751. Van Driessche, E., K. Houf, J. Van Hoof, L. De Zutter, and P. Vandamme. 2003. Isolation of Arcobacter species from animal feces. FEMS Microbiol. Lett. 229:243-248.

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Van Etterijck, R., J. Breynaert, H. Revets, T. Devreker, Y. Vandenplas, P. Vandamme, and s. Lauwers. 1996. Isolation of Campylobacter concisus from feces of children with and without diarrhea. J. Clin. Microbiol. 34:2304-2306. Vandenberg, O., A. Dediste, K. Houf, S. Ibekwem, H. Souayah, S. Cadranel, N. Douat, G. Zissis, J. P. Butzler, and P. Vandamme. 2004. Arcobacter species in humans. Emerg. Infect. Dis. 10:1863-1867. Viron, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and V6ron and designation of the neotype strain for the type species Campylobacter fetus (Smith and Taylor) Sebald and VCron. lnt. J. Syst. Bacteriol. 23:122-134. Vinzent, R., J. Dumas, and N. Picard. 1947. Septicemie grave eu cours de la grossesse, due iun vibrion. Avortement consecutif. C. R Acad. Med. 131:90. Voordouw, G., S. M. Armstrong, M. F. Reimer, B. Fouts, A. J. Telang, Y. Shen, and D. Gevertz. 1996. Characterization of 16s rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide-oxidizing bacteria. Appl. Environ. Microbiol. 62: 16231629. Wesley, I. V., L. Schroeder-Tucker, A. L. Baetz, F. E. Dewhirst, and B. J. Paster. 1995. Arcobacter-specific and Arcobacter butzleri-specific 16s rRNA-based DNA probes. J. Clin. Microbiol. 33:1691-1698. Winkler, M. A., J. Uher, and S. Cepa. 1999. Direct analysis and identification of Helicobacter and Campylobacter species by MALDI-TOF mass spectrometry. Anal. Chem. 71:3416-3419. Wirsen, C. O., S. M. Sievert, C . M. Cavanaugh, S. J. Molyneaux, A. Ahmad, L. T. Taylor, E. F. DeLong, and C. D. Taylor. 2002. Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur. Appl. Environ. Microbiol. 68:3 16-325. Wolfe, R. S., and N. Pfennig. 1977. Reduction of sulfur by Spirillum 5175 and syntrophism with Chlorobium. Appl. Environ. Microbiol. 33 :427-43 3. Wolin, M. J., E. A. Wolin, and N. J. Jacobs. 1961. Cytochromeproducing anaerobic vibrio, Vibrio succinogenes, sp. n. J. Bacteriol. 81:911-917. Wybo, I., J. Breynaert, S. Lauwers, F. Lindenburg, and K. Houf. 2004. Isolation of Arcobacter skirrowii from a patient with chronic diarrhea. J. Clin. Microbiol. 42:1851-1852.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Biaser 6 2008 ASM Press, Washington, DC

Chapter 2

Population Biology of Campylobacter jejuni and Related Organisms MARTIN

c. J. MAIDEN AND KATE E. DINGLE

must take account of population biology and evolution. Population studies of bacteria comprise three elements: (i) sampling the population, (ii) measuring variation in the population, and (iii) interpreting the variation. Sampling the natural world is a nontrivial problem, and it is impossible to achieve the ideal of a truly random or even wholly representative population sample. In microbiology there are additional technical questions about the reliability of culture methods-or indeed other methods-for accurately capturing all of the variation present in a given sample. For widely distributed multihost bacteria, such as the members of the genus Campylobacter, these issues are particularly relevant (Bolton and Robertson, 1982) and have to be mitigated by the adoption of appropriately designed structured sampling schemes that are driven by the particular question being investigated. Molecular methods can go some way to mitigating problems with culture (Abulreesh et al., 2006), but even these can be subject to sensitivity issues, and the lack of bacterial isolates limits the extent to which biological variation can be accurately characterized. Once bacterial isolates are available, exhaustively measuring the variation present among genomes is possible by the appropriate application of highthroughput nucleotide sequence determination technology (Maiden, 2000). Two approaches can be used. The existence of a complete genome from one example of the population being analyzed enables the nucleotide sequence of genes in many isolates to be determined with PCR-based direct nucleotide sequence determination; this has the advantage that it can also be done on specimens containing noncultur-

Studies of the ecology and evolution of bacterial populations, which require measurement of variation, theory development, and testing, are often carried out independently from the investigation of microbial function that are usually conducted within a reductionist framework by means of controlled experiments. There is a potentially creative tension between these two approaches: population studies require the collection and analysis of large numbers of bacterial isolates, while on the other hand, functional studies commonly involve the detailed examination of one, or at most a few, example isolates, with little or no reference to population variation. The integration of these approaches is necessary if recent technical advances in data collection are to be fully exploited. The availability of whole-genome sequences for many bacteria, including the principal human and animal pathogens (Bentley and Parkhill, 2004), has highlighted the need for better interaction of these approaches. A whole-genome sequence is in many ways the apotheosis of a single organism study, and in principle, the determination of the complete genetic information for any organism enables all aspects of its biology to be deduced. In practice, however, current and anticipated knowledge of biochemistry and genetics is too limited to permit such deductions from genome sequences alone. Biochemistry is still not a high-throughput science in the sense that genome sequence determination is, and detailed investigations of all cistrons from all genomes are impractical. Consequently, most of the information in annotated genomes comes from comparative studies, the quality of which is dependent on the quality of the comparisons (Brenner, 1999). Once a comparative element is introduced into a study, it perforce

Martin C. J. Maiden Department of Zoology, University of Oxford, South Parks Rd., Oxford OX1 3PS, United Kingdom. Kate E. Dingle Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.

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able organisms. Conversely, the complete genome of many samples can be compared with microarray, or better, rapid whole-genome resequencing approaches, although this generally requires the availability of isolates to prepare genomic DNA. The former approach has the advantage of producing defined data rapidly, whereas the latter provides the intriguing prospect of comprehensive comparative analysis. At the present time, the whole-genome sequence determination approach remains impractical as a result of cost constraints. Even if such data were to become available, new analysis algorithms would be required to cope with the quantity of data generated. The use of an evolutionary framework is essential to the interpretation of biological variation, and a number of analytical approaches have been developed. These allow the variation present in a given population to be explored with a view to establishing the biological forces that have generated the variation present. In the case of the bacteria, which are fast growing, have small genomes, and live in intimate contact with their environment, genetic variation can be especially informative in understanding their ecology. The most effective analysis methods are parametric and model based, and in this respect, recently developed analytical tools that are based on coalescent theory are especially valuable (Balding, 2003; Didelot and Falush, 2007; Falush et al., 2003). Informal techniques can also be valuable in the primary analysis of data (Feil et al., 2004). Here we explore the contribution that population studies have made to our understanding of the biology of the Campylobacter, and we argue that such studies have a central role to play in understanding the epidemiology and pathogenesis of this important group of gramnegative bacteria.

ANALYSIS OF POPULATIONS OF CAMPYLOBACTER The factors that make members of the genus Campylobacter difficult to study with conventional single-isolate approaches-in particular their ubiquity and diversity-also make them tractable to, and indeed attractive subjects of, population studies. The biology underlying the diversity of members of the genus Campylobacter, including factors such as host range and specialization, as well as being interesting biological problems in their own right, are also relevant to the main human interaction with these bacteria, namely their propensity to cause gastroenteritis. Carnpylobacter jejuni and Campylobacter coli cause the majority of human cases of Campylobacter-associated gastroenteritis; these two organisms are asso-

ciated with approximately 90 and 10% of cases, respectively (Gillespie et al., 2002). Campylobacter upsaliensis is frequently identified in domestic cats and dogs, and it causes occasional cases of gastroenteritis in humans. Campylobacter helveticus is also found in domestic animals, but it appears not to cause human disease. Campylobacter lari can cause gastroenteritis in humans. It has been isolated from shellfish (Endtz et al., 1997), but it is most commonly found among wild birds. Campylobacter fetus is primarily associated with infections in livestock, which result in abortion and infertility, particularly in sheep and cattle. C. fetus can also be isolated from goats, pigs, horses, fowl, and reptiles and causes occasional human infections. Transmission to humans generally occurs orally through contaminated food or water (Garcia et al., 1983). A range of techniques have been used to study variation within the genus Campylobacter. Indeed, the plethora of isolate characterization techniques available could be said to have obscured rather than illuminated research into populations of these organisms (Wassenaar and Newell, 2000). In this respect, it is necessary to distinguish those bacterial characterization schemes that can be sensibly used for population studies from those that cannot. It is relatively trivial to develop a typing scheme that can classify isolates as distinct or indistinguishable (Achtman, 1996), but it is much more problematic to develop schemes that generate reproducible data that can be analyzed to investigate the biology of the organism in question. Because virtually all variation in bacterial populations is genetic or ultimately genetically determined, the availability of high-throughput nucleotide sequence determination has provided influential advances in isolate characterization. Thanks to the combination of the availability of complete genome sequences of C. jejuni, for example that of isolate NCTC 11168 (Parkhill et al., 2000), and PCR amplification technology, it is now possible to establish definitively the genetic variation present at any part of the genome by PCR-direct nucleotide sequence determination. Other methods, such as multilocus enzyme electrophoresis (Aeschbacher and Piffaretti, 1989), pulsed-field gel electrophoresis analysis (Yan et al., 1991), and amplified fragment-length polymorphism (Duim et al., 1999), are at best proxies for nucleotide sequence diversity and therefore generally provide less information (Sails et al., 2003a, 2003b). In addition, the data produced by these techniques are not as tractable to phylogenetic and statistical analysis as nucleotide sequences themselves. The application of high-throughput nucleotide sequence-based approaches (Maiden, 2000) to large

CHAPTER 2

isolate collections and the analysis of these data have enabled the diversity of C. jejuni populations to be defined (Dingle et al., 2002) and the relationship of this population to other members of the genus compared (Dingle et al., 2005; Miller et al., 2005; van Bergen et al., 2005). At present, the major technique used is multilocus sequence typing (MLST) (Maiden et al., 1998). MLST is a generic approach for the characterization of bacterial isolates that aims to identify lineages in bacterial populations by indexing the variation present in several (commonly seven) housekeeping genes located at various parts of the chromosome (Maiden, 2006). The advantages of using housekeeping genes is their presence in all, or nearly all, isolates, combined with the fact that although they contain measurable variation, they are subject to stabilizing selection for conservation of metabolic function. Given that horizontal genetic exchange is common in the microbial world (Narra and Ochman, 2006), the use of multiple loci is important because single loci can be misleading indicators of relationship by descent. MLST is in effect a nucleotide sequence-based version of multilocus enzyme electrophoresis (Selander et al., 1986) in that it indexes of genetic variation at housekeeping loci with nucleotide sequencing. This approach has a number of advantages in addition to generating definitive, reproducible, and transportable data. For MLST, a viable isolate is not required, and the process is scalable from one isolate to many hundreds or thousands by the exploitation of liquid handling technology and automated DNA analyzers. For a group of organisms as diverse as those making up the genus Campylobacter, this is a particular advantage because it facilitates the analysis of very large numbers of isolates. MLST also introduced the innovation of sharing data via the Internet (Chan et al., 2001; Jolley et al., 2004) and promoted the development of novel analysis tools (Didelot and Falush, 2007; Feil et al., 2004, 1999; Jolley et al., 2001). MLST approaches have the advantage that they can simultaneously address large-scale or more local population questions, especially in combination with other sequence-based typing approaches (Maiden, 2006). In the Campylobacter MLST scheme, the sequences of seven allele fragments are determined after amplification from genomic DNA or a killed cell suspension. In common with other schemes, short gene fragments, generally 400 to 550 bp, are used because these are easily sequenced on both DNA strands by using only one extension primer pair, ensuring rapidity, cost effectiveness, and accuracy. Each unique sequence of an allele fragment is assigned an arbitrary number in order of discovery. These unique

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sequences are stored electronically in the “profiles database,” which is essentially a dictionary that catalogs variation and that is used to assign typing data as they are collected (Jolley et al., 2004). The allele numbers are combined into allelic profiles, or sequence types (STs), which are also arbitrarily assigned. Allele numbers and STs are in effect shorthand codes for bacterial variation, with each ST representing a unique combination of nucleotide sequence of, in the case of C. jejuni, 3,309 bp in length and including the variation present at seven separate loci. STs can be grouped into “clonal complexes,” surrogates of genetic lineages that are described in detail for C. jejuni below. The definitions of these clonal complexes are also stored in the profiles database. Analysis can be conducted either at the level of clonal complex, ST, allele, or nucleotide sequences, including all or a subset of the MLST data, depending on the questions being addressed (Maiden, 2006).

VARIATION WITHIN THE GENUS CAMPYLOBACTER MLST schemes are now available for many members of the genus Campylobacter. The first to be developed was for C. jejuni (Dingle et al., 2001a); schemes are also available for C. coli (Dingle et al., 2005), C. lari, C. upsaliensis, C. helveticus (Miller et al., 2005), and C. fetus (van Bergen et al., 2005). These schemes have established the basic population structure of the members of the genus and are beginning to demonstrate associations of genotypes with particular animal hosts (Dingle et al., 2002; French et al., 2005). The relationships of these different Campylobacter microbiological species with human and veterinary disease are being more precisely defined on the basis of the insights available from MLST data. The level of genetic divergence among Campylobacter housekeeping genes is as high as 80.9% (Miller et al., 2005). Consequently, only C. jejuni can be MLST typed reliably by using the oligonucleotide primers originally described (Dingle et al., 2001b), which were designed by using the first draft of the C. jejuni genome sequence (Parkhill et al., 2000). Two approaches have been used to adapt this MLST scheme to other Campylobacter species. First, the scheme was extended to C. coli and C. fetus by using a large collection of different C. jejuni oligonucleotide primers in PCR amplifications of DNA from C. coli and C. fetus (Dingle et al., 2005; van Bergen et al., 2005). The amplification reactions were performed under conditions of reduced stringency to

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permit the amplification of corresponding housekeeping gene sequences from heterologous species. The sequences of these amplicons were determined, and new primers were designed that were specific to C. coli or C. fetus. The same seven housekeeping genes were used as the C. jejuni scheme to allow comparisons of diversity, population structure, and epidemiology among the different species. The C. coli MLST scheme was validated by using 53 isolates from humans, chickens, and pigs, together with 15 Penner serotype reference isolates (Dingle et al., 2005). The C. fetus MLST scheme was validated by using a diverse collection of 140 geographically diverse C. fetus isolates from a variety of animal hosts and humans (van Bergen et al., 2005). A second approach to extending the C. jejuni MLST scheme to other species was made possible by the availability of the draft genome sequences of C. coli, C. upsaliensis, and C. lari (Fouts et al., ZOOS), enabling the design of degenerate oligonucleotide primers, so that individual primer pairs could be used to amplify MLST loci from up to five different Campylobacter species (Miller et al., 2005). This had the advantage of easily detecting interspecies genetic recombinants, genetic hybrids, within the genus Campylobacter and detecting mixed samples. In addition to C. jejuni, the species targeted were C. coli and C. helveticus, for which the same loci are used as the C. jejuni scheme. Schemes have also been developed for C. lari, for which adk and pgi were substituted for aspA and gltA, and C. upsaliensis, for which adk and pgi were substituted for gltA and pgm. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed, demonstrating the utility of the approach for distinguishing among these bacteria. More than 80% of the isolates had unique STs. Genetic recombinants have since been described between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus (D’lima et al., 2007; Kinana et al., 2007; Miller et al., 2005). The MLST data for all of the above schemes are available at the pubMLST website (http: //pubmlst. org/campylobacter/), stored in web-accessible customized databases (Fig. 1)that permit online analysis and comparison (Jolley et al., 2004; Jolley and Maiden, 2006). The MLST scheme used for C. jejuni and C. coli is represented by a single PubMLST isolate database, which is integrated with typing data from the flaA, flaB, and porA genes used for antigen gene sequencing by AgdbNet software (Jolley and Maiden, 2006) (Fig. lb). Data from the other MLST schemes are also available at pubMLST. Although MLST was originally developed with a view to identifying clones or lineages within bacterial

populations, especially those of pathogens (Maiden et al., 1998), it has many other applications, including addressing the vexed question of bacterial “speciation” (Stackebrandt et al., 2002). Where the same genes have been chosen for different members of the genus, their interrelationships can be investigated in a number of ways, the simplest being a phylogeny based on the nucleotide sequences. A simple phylogeny drawn for those members of the genus Campylobacter for which MLST data are available shows that C. fetus is the most divergent member of the group, with C. jejuni and C. coli sharing a common ancestor, as do C. lari and C. insulaenigrae, and C. upsaliensis and C. helveticus (Fig. 2). Phylogenic trees represent genealogies, with the branches illustrating pathways of descent. Conventional algorithms use genetic distances, calculated in various ways, to reconstruct these phylogenies, by comparing nucleotide sequences on the assumption that the sequence changes occur according to a more or less constant clock rate. Such a representation is unproblematic if there is no, or at least very little, genetic exchange among the taxa being analyzed, as in the case of the named Campylobacter species. However, as the frequency of gene transfer increases among the organisms placed on divergent branches of the tree, the reliability of a conventionally generated treelike phylogeny decreases. This is because a single genetic transfer event, which is common in some bacterial populations, may introduce a large number of sequence changes, resulting in a long branch, while a single mutation, a comparatively rare event, would generate a short branch. The generation of mosaic genes by intragenic recombination further complicates the interpretation of conventional phylogenies (Maiden, 1993). It is now known that genetic exchange (sometimes referred to as lateral gene transfer or “localized sex” [Maynard Smith et al., 19911) is quite common in the bacteria and needs to be taken into account when reconstructing bacterial genealogies (Maynard Smith et al., 1993). In the case of the relationships among the different members of the genus, a conventional tree, such as that shown in Fig. 2, is more or less reliable, as there is relatively little genetic exchange among the bacteria classified as microbiological species. Indeed, bacterial “species” are probably best defined as populations of bacteria that recombine infrequently, rather than the more traditional definitions that are based on DNA homology (Stackebrandt et al., 2002). The problem of such a definition is that it is difficult to define the levels of gene flow that permit the emergence of bacterial species, and in any case, estimating these parameters is complex and can be sensitive to the sampling frame used. Further, the different levels

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31

a

N

N

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% 0

0 0 0

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tn

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Figure 1. Carnpylobacter MLST databases. (a) The growth of the Carnpylobacterjejuni and Campylobacter coli database since its inception in 2001. Open squares represent isolates submitted; closed squares, sequence types. (b) The structure of the pubMLST database network, which is used for all of the Campylobacter databases. It allows the integration of data from various sources in a variety of public or private databases, ensuring that a common typing nomenclature is used.

of horizontal genetic exchange may have different effects on diverse bacterial populations. Wholly asexual populations, i.e., those in which genetic exchange is negligible, will conform to the clonal model of population structure. Clonality is an inevitable consequence of reproduction by binary fission because any mutations that occur in the population are limited to the descendants of the mother cell in which they arose. Diversity reduction events such as bottlenecking (Achtman, 1997) or periodic selection (Levin, 1981) purge the population of diversity, resulting in a population that is dominated by lineages with distinct genotypes and therefore phenotypes. Clonal populations will conform to a num-

ber of predictions: (i) alleles at different loci will exhibit linkage disequilibrium; that is, they will occur in nonrandom combinations; (ii) they will exhibit relationships that conform to a treelike phylogeny, with branches generated by mutation events; and (iii) they will exhibit congruence; that is, the same phylogeny will be recorded at different loci. The population structure of wholly sexual populations is characterized by a lack of lineage structure, with genetic variation that exhibits (i) linkage equilibrium, (ii) a netlike phylogeny, and (iii) incongruence, with each locus recording a unique evolutionary history (Maynard Smith, 1989). In practice, few bacteria occupy the extreme of an entirely clonal or

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C.insulaenigrae \

C. lari \

,

C. jejuni C. upsaliensis

I

w

C. helveticus

C. fetus

Figure 2. Relationships among the different Campylobacter microbiological species. The phylogeny was reconstructed with concatenated sequences from the alleles common to all of the schemes (gln, gly, and tkt).

wholly nonclonal population, and the process of binary fission imposes some element of clonality on all bacterial populations (Feil et al., 1999; Spratt and Maiden, 1999). Understanding bacterial population structure therefore becomes an issue of determining the rate of gene flow and applying an appropriate model, i.e. clonal, partially clonal, or nonclonal. Within the genus Campylobacter, different types of population structure have been observed within bacteria currently assigned to different microbiological species. By far the most studied to date are C. jejuni and C. coli, reflecting their importance in human disease.

Campylobacter jejuni The MLST data collected to date show that C. jejuni is highly diverse with a total of 2,243 distinct STs from some 3,704 isolates deposited in the pubMLST database (http://pubmlst.org/ campylobacter/); many more isolates, perhaps in excess of 10,000, have been characterized by MLST in a variety of studies. In addition to the most widely used scheme, two other C. jejuni MLST schemes have been developed. As expected, given the nature of MLST systems, all three schemes provide similar perspectives on C. jejuni biology (Dingle et al., 2001a; Manning et al., 2003; Suerbaum et al., 2001). Analyses of subsets of the MLST data by maximum likelihood methods show that the phylogenies generated from the different loci are incongruent,

violating the predictions of clonality (Dingle and Maiden, 2005). In addition, there is little evidence for treelike structure in nucleotide sequences of the individual alleles (Dingle et al., 2001a; Suerbaum et al., 2001). There is, however, some evidence of linkage disequilibrium in these data sets (Dingle and Maiden, 2005; Schouls et al., 2003). These features, which are seen in a number of other bacteria analyzed by MLST (Maiden, 2006), are consistent with a partially clonal population, that is, one that contains distinct clusters of related isolates that do not share clonal relationships with each other. In other words, although each member of each cluster shares a common ancestor with other members of the same cluster, cluster recombination has reassorted genetic variation wholesale among clusters. Clusters of this type are recognized in MLST data as clonal complexes, which are defined informally within the MLST databases on the basis of a number of biological properties (Maiden, 2006). Examination of data sets from partially clonal organisms reveals differences in the frequency distribution of STs in that some STs are much more frequent in population samples than others. Typically these STs are also recovered in isolates obtained over prolonged periods of time and from diverse geographic locations. In addition, such STs are often characterized by having many low-frequency relatives that vary from them at one, two, or three loci. These STs correspond to “central genotypes” (sometimes referred to as “founders”) that define and give their name to the clonal complex. The clonal complex then includes all isolates with the central ST plus those with STs sharing up to four identical loci. Thus, the ST45 clonal complex includes ST-45 itself and all of its single, double, and triple locus variants (Fig. 3). In most cases, the variants are generated by recombination rather than point mutation. The influence of recombination on the population structure of C. jejuni is further indicated by the relationship of alleles to STs in the database. Although there were a total of 2,243 distinct STs, organized into 42 clonal complexes at the time of writing, the number of alleles at each locus was much smaller, ranging from 134 (for aspA) to 256 (for pgm). This relationship shows that most genotypes are assembled by reassortment of preexisting alleles, not by the generation of novel alleles by mutation or intra-allele recombination. This in turn implies that the recombination fragment sizes are rather larger than the average size of the MLST alleles (402 to 507 bp in C. jejuni) and, given that these are substantial fractions of the average size of C. jejuni cistrons (948 bp; Parkhill et al., 2000), the genes themselves. This observation is somewhat at odds with some estimates

I

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of average recombination fragment sizes of 225 to 750 bp obtained by approximate likelihood methods (Fearnhead et al., 2005) but consistent with the estimate of 3.3 kbp obtained with apparently less precise methods (Schouls et al., 2003). However, these observations further underline the importance of genetic exchange in the population biology and evolution of Campylobacter. Clonal complexes are surrogates for lineages, and the utility of the clonal complex as a unit of analysis presumably reflects the strength of the lineage structure within populations of C. jejuni. The recent development of the ClonalFrame algorithm (Didelot and Falush, 2007) permits the formal analysis of these lineages for the first time. This program identifies clonal lineages while estimating the likelihood that particular changes have occurred by either recombination or single base-pair mutation, avoiding the problems associated with distance-based methods. The principal disadvantage of this approach is that it is computationally intensive to the extent that it is impractical to apply to all samples in the MLST databases and has to be rerun each time a new ST is added. It is, however, invaluable for testing the robustness of the informal clonal complex designations. Analysis of sample data sets with ClonalFrame shows that C. jejuni clonal complexes correspond to genetic lineages, as predicted. It also confirms the observation that particular clonal complexes are associated with different isolation sources. This probably reflects the fact that clonal complexes represent lineages that are adapted to particular niches that, at least in some cases, correspond to different host species. For example, ST-45 complex C. jejuni are isolated primarily from chickens, environmental sources, or human disease, ST-61 complex mostly from bovine or ovine sources, the environment, or human disease, and ST-42 complex mostly from bovine or ovine sources and human disease (Dingle et al., 2002) (Fig. 4). Some relationships among clonal complexes are apparent from the ClonalFrame analysis; for example, ST-283 complex and ST-45 complex appear to be related, as do ST-48 complex, ST-353 complex, and ST-682 complex (Fig. 4). Most branches, however, converge in a “star phylogeny,” showing that at least for data from seven loci, ClonalFrame analysis cannot resolve deeper phylogenies. This may be a consequence of extensive horizontal genetic exchange disrupting all clonal signal in the population, or it may be a limitation of the numbers of loci used in the analysis. The ST-21 complex is also poorly resolved in this analysis, also appearing as a star phylogeny; this also might be resolved by the inclusion of more loci in future studies. Because the

ST-21 complex is found in a variety of host sources, there may be sublineages within this clonal complex adapted to particular niches. Further support for these ideas comes from the assignment of host types on the basis of MLST data. By use of the structure algorithm (Falush et al., 2003) and a reference set of isolates, the origins of variant alleles in a set of ST-21 complex isolates were assigned and compared with the host origin of the isolate itself. In 18 of 22 cases, the assignment of the variant alleles corresponded with the isolation source, in terms of host animal of the variant strain, indicating that the strain (i) had been resident in the host, and (ii) had preferentially acquired alleles associated with that particular host (McCarthy et al., 2007). Clonal complex, a surrogate for lineage, is therefore a fundamental unit of C. jejuni biology, and much of the confusion present in the field was a consequence of the fact that earlier typing schemes, especially serotyping, did not accurately reflect the clonal complexes (Dingle and Maiden, 2005). The lineages are sufficiently stable to be recovered over periods of time and to be associated with particular hosts. Given that most sampling effort is focused on human disease cases and food animals, it is likely that clonal complexes associated by nonfarm animal sources are poorly represented in current isolate collections, and it is likely that many additional lineages associated with wild animal sources exist (French et al., 2005). In one case a lineage had been identified and named as a discrete subspecies. Isolates characterized as C. jejuni subspecies doylei have been classified as distinct from other C. jejuni isolates on the basis that they are primarily isolated from humans and have a propensity to cause bacteremia. Analysis of MLST data with conventional phylogenetic techniques and microarray-based comparative genome indexing suggested that C. jejuni doylei was distinct from “C. jejuni jejuni” (Parker et al., 2007). This division into two clades is not supported by ClonalFrame analysis, which suggests that the isolates belonging to C. jejuni doylei are just another lineage. This analysis also provides the following explanation for the discrepancy. The C. jejuni doylei isolates are located in a sparsely sampled part of the C. jejuni tree generated by ClonalFrame, which contains isolates mostly of environmental origin. Moreover, the well-sampled part of the tree, which contains mostly isolates derived from humans or food animals, contains a number of major lineages that are related to each other as well as to the ST-21 complex. Thus, a collection of reference isolates that reflected current isolate collections would have very few of the samples of nonhuman and nonhuman food origin, and very many of those from humans and their food animals. There-

CHAPTER 2

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\ C.doylei

Figure 4. Correspondence of clonal complexes with genealogies. Clonal complex designations were used to annotate a phylogeny generated by ClonalFrame. The size of the pie charts represents the number of isolates in each clonal complex, with the color indicating the relative contribution of each source: black, human disease; white, chickens and chicken meat; dark gray, ovines and ovine meat; light gray, environmental sources. Data from Dingle et al. (2002) and Parker et al. (2007).

fore, analyses comprising C. jejuni doylei and other human disease isolates, however performed, will show C. jejuni doylei to be distinct because other isolates with a similar relationship to the humancentered isolates will be absent. An interpretation that is consistent with both sets of data is that C. jejuni doylei isolates represent a lineage associated with an environmental source that very rarely comes into contact with humans, but when it does, it tends to cause more severe disease. The observations on the potentially misleading characterization of the C. jejuni doylei isolates as a subspecies distinct from all other C. jejuni lineages introduces another implication of the population

structure of C. jejuni. Comparisons of the genomes of multiple isolates, especially by microarray methods, are popular and considered to be important (Champion et al., 2005). However, the inferences that can be gained from such studies are dependent on the genes included in the microarrays used and the isolates included in the analysis. It is known that not all C. jejuni isolates contain all of the genes present in the population, and it is likely that different lineages typically have distinct gene complements. A consequence of this is that any comparison of gene complement that is based on a microarray assembled with only one genome sequence can only provide resolution of the C. jejuni population into two groups:

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“like the reference isolate” and “unlike the reference isolate.” Thus, microarray-based “genomotyping” with microarrays that are based on C. jejuni 11168 can only divide C. jejuni collections into two groups: like NTCC1168 (which happens to be an ST-21 complex isolate) and unlike NTCC1168 (Champion et al., 2005). Adding data from more genomes will help in this process, but the MLST data assembled so far indicate that data from a minimum of 42 isolates (one from each clonal complex identified to date) should be used to generate the microarrays if the association of particular clonal complexes with host sources are to be informatively defined. In general, clonal complex structure should be taken into account when choosing isolates for inclusion in functional analyses or multiple genome sequencing. Although human infection is one of the most important practical applications of studies of Campylobacter populations, in terms of Campylobacter population dynamics and evolution, infection is probably irrelevant. Human infection is almost certainly incidental, and little if any importance should be attached in terms of onward evolutionary spread of C. jejuni. Understanding the population biology of Carnpylobacter is, however, crucial in understanding the transmission to humans and developing means for its control. MLST has permitted direct comparisons to be made among studies of C. jejuni epidemiology (Gillespie et al., 2002). The identification of outbreaks and potential sources of infection relies on the precise characterization of isolates and knowledge of their interrelationships. The clonal complex is a valuable unit of analysis for epidemiological investigation, allowing the comparison of data from diverse sources worldwide. This has demonstrated that different clonal complexes are important in different geographical regions of the world, probably reflecting different food animal sources of human infection (Dingle et al., 2002; Duim et al., 2003; Mickan et al., 2007; Sopwith et al., 2006) (Fig. 4). MLST has also been of value in investigating the link of C. jejuni genotypes with specific disease syndromes (Dingle et al., 2001b). Antigen sequence typing generates high levels of discrimination among bacterial isolates by measuring the variation present in antigen genes that are subject to diversifying selection, presumably by virtue of their exposure to the host immune system. The inclusion of these data are therefore useful in identifying very closely related bacterial isolates, for example in an outbreak situation, and in studies of the immune response to bacteria, but their high rates of change make them less useful in unambiguously identifying lineages. Combining MLST and antigen gene sequence data can be a highly effective means of

achieving high-resolution characterization data that also allow long-range comparisons. For the identification of disease outbreaks, clonal complex designation alone is insufficiently precise to reliably detect outbreaks (Clark et al., 2003; Fitzgerald et al., 2001; Sails et al., 2003b). Sequence typing data also provide the resolution and reproducibility necessary to investigate the diversity of Campylobacter isolates obtained from farms and the environment (Colles et al., 2003; French et al., 2005; Manning et al., 2003). These studies have shown that genotypes most like those from human disease are recovered from farm animals (Colles et al., 2003; Manning et al., 2003), while the genotypes from environmental sources seem to be distinct (Fig. 4) (French et al., 2005). Certain clonal complexes are associated with particular food animal sources, with the ST-61 complex especially associated with cattle (Dingle et al., 2002; French et al., 2005) and the ST-403 with pigs (Manning et al., 2003). Large-scale studies of isolates from a wide variety of sources provide the prospect that in the near future, it will be possible to trace the movement of Campylobacter genotypes through the food chain (Allen et al., 2007) and attribute cases of human infection to particular host sources with reasonable precision (McCarthy et al., 2007). MLST has also been used to examine antibiotic-resistant isolates obtained from poultry (Kinana et al., 2006, 2007).

Campylobacter coli C. coli has a similar host range to C. jejuni and is associated with up to 10% of human Campylobacter infections. At the time of writing, the MLST allelic profile database contained 708 C. coli STs. Preliminary analyses of MLST data from C. coli (Miller et al., 2005) identified two putative clonal complexes among a small collection of 37 STs. The first was designated ST-1017 complex (31STs) and the second ST-1052 complex (4 STs). Now that more data are available, ST-1017 has been shown to be part of the ST-828 complex, and ST-1052 is currently unassigned. Two large C. coli clonal complexes have now been confirmed, the largest being ST-828 complex, with 425 STs, and the other ST-1150 complex, with 25 STs (http: //pubmlst.org/campylobacter/). This indicates the difficulty of identifying the central genotype of a clonal complex when only few data are available. Of the 708 STs currently in the PubMLST database, 258 are unassigned to a clonal complex. These STs were analyzed for the presence of clonal complexes by the BURST algorithm (Feil et al., 2004), and a new large lineage was identified, ST-

CHAPTER 2

1172 complex, containing 130 STs. Two smaller possible clonal complexes, ST-2013 (eight STs) and ST2461 (seven STs), were also identified. Five groups of 4 isolates, two groups of 3 isolates, and 10 paired isolates were identified, with 67 singletons remaining. This suggests that the C. coli population structure is similar to but not identical to C. jejuni, C. coli apparently comprising a smaller number of genetic lineages. The two large clonal complexes identified so far appear to resemble ST-21 complex in C. jejuni in that they are multihost and could potentially be better understood by the sequencing of additional loci. Five studies performed in four different countries have used MLST to study C. coli isolated from food animals and human disease (D’lima et al., 2007; Kinana et al., 2007; Litrup et al., 2007; Thakur and Gebreyes, 2005; Thakur et al., 2006). Pigs are considered a common reservoir for C. coli infections in humans; therefore, several studies have used MLST to examine the genotypes associated with pigs. A MLST-based comparison of 151 C. coli isolates from pigs on the farm and at slaughter demonstrated a high level of genetic diversity, with a total of 74 STs identified among the isolates. Most STs found on the farm were not found at slaughter, but different STs were, suggesting an important role for crosscontamination at slaughter (Thakur and Gebreyes, 2005; Thakur et al., 2006). They also compared isolates from animals reared with and without the use of antibiotics. ST-828 complex was described as containing isolates from both the production systems examined. A study of 36 C. coli isolates from chickens in Senegal described a low level of genetic diversity, with 13 STs, and no clear genetic lineages, probably due to the small size of the data set (Kinana et al., 2007); however, 10 of the STs identified were members of ST-828 complex. There was no apparent link between quinolone resistance and ST. In a study of 160 Danish C. coli isolates from humans, broilers, pigs, and cattle, high genetic diversity was observed, with 84 STs being identified. Ten percent of the isolates possessed STs that were found in human, poultry, and pig isolates. Only 10% of the isolates from pigs shared STs with isolates from humans, and these shared STs were found in poultry isolates as well. Therefore, C. coli isolates from pigs were presumed not to be a significant source of human infections in Denmark (Litrup et al., 2007). A study of 59 multidrug-resistant C. coli strains from turkeys demonstrated that only 3 of the 14 STs accounted for 69% of the isolates, and these STs were turkey specific, implying that multidrug-resistant C. coli strains from turkeys in North Carolina, USA, are characterized by a relatively small number of clonal groupings (D’Lima et al., 2007).

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37

The studies by Litrup et al. and D’Lima et al. illustrate the potential of C. coli MLST for tracking the origins of human infection. They also illustrate the requirement for large data sets before host associations start to become apparent. Miller et al. (2006) have made major advances in this respect by typing 488 C. coli strains from four different food animal sources (cattle, chickens, swine, and turkeys) collected at different times over a 6-year period from different geographical locations in the United States. A total of 149 STs were identified. The 185 swine strains were the most diverse, possessing 82 STs. The cattle strains were the most clonal; 52 (83%) of 63 strains possessed a single ST (ST-1068). Most STs and alleles were host associated, i.e., found primarily in strains from a single food-animal source. Only 12 (8%) of 149 STs were found in multiple sources, indicating the potential for MLST to help trace human infections (Miller et al., 2006). C . fetus Population Structure and Molecular Epidemiology

A large and diverse collection of 140 C. fetus isolates, including a wide range of hosts and geographic locations, was used to validate the C. fetus MLST scheme (van Bergen et al., 2005). Fourteen different STs were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. The C. fetus MLST data indicated that this species is genetically homogeneous compared with the heterogeneity of other Campylobacter species for which MLST data are available (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus). A total of 91% of C. fetus isolates was assigned to only four STs. C. fetus is divided into two subspecies: C. fetus subspecies fetus (associated with abortions most often in sheep, and a wide range of other hosts) and C. fetus subspecies venerealis (isolated mainly from cattle). The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis, which appears to represent a bovine clone. Congruence was observed among C. fetus subspecies, sap types, and ST. MLST therefore confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche. No associations among the STs and host or geographic region were identified. Other Species The extended MLST method (Miller et al., 2005) was used to type 20 C. lari isolates, 78 C. upsaliensis, and nine C. helveticus. Each species was ge-

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netically diverse, the majority of the STs being unique (http://pubmlst.org/campylobacter/).A total of 15 C. lari STs, 66 C. upsaliensis STs, and 8 C. helveticus STs were identified.

CONCLUSIONS Since the development of MLST 10 years ago (Maiden et al., 1998), major progress has been made in understanding the population biology of C. jejuni and its relatives. The application of high-throughput nucleotide sequence-based methods, and particularly MLST, has not only improved isolate characterization but also illuminated areas of the biology of these highly diverse organisms that are relevant to understanding and controlling disease. It is instructive to reflect that the first study of C. jejuni and C. coli population structure by multilocus enzyme electrophoresis (Aeschbacher and Piffaretti, 1989) provided many insights that have proved to be correct and that have been extended and deepened by MLST studies. The clonal complex has proved to be a powerful and adaptable unit for analysis of population structure that also enables long-term epidemiological monitoring. Including data from antigen-encoding genes provides a highly discriminatory system for tracking genotypes through the food chain and in epidemiological monitoring. The association of particular genotypes with particular host species provides the prospect of accurately determining the relative importance of different sources of human infection. Ongoing nucleotide sequence-based studies involving large numbers of isolates and improved genealogical analysis tools provide the highly attractive prospect that well within the next 10 years, the population biology of these organisms, at least insofar as it relates to human infection, will be effectively resolved. Acknowledgments. M.C.J.M. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. The United Kingdom Department of Environment, Food and Rural Affairs supported our work (contract numbers 020604 and 020611). We thank Samuel Sheppard for assistance in the preparation of the figures.

REFERENCES Abulreesh, H. H., T. A. Paget, and R. Goulder. 2006. Campylobacter in waterfowl and aquatic environments: incidence and methods of detection. Enuiron. Sci. Technol. 40:7122-7131. Achtman, M. 1996. A surfeit of YATMs? J. Clin. Microbiol. 34: 1870. Achtman, M. 1997. Microevolution and epidemic spread of serogroup A Neisseria meningitidis-a review. Gene 192:135-140. Aeschbacher, M., and J.-C. Piffaretti. 1989. Population genetics of human and animal enteric Campylobacter strains. Infect. lmmun. 57~1432-1437.

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juni clones in Curacao, Netherlands Antilles. 1.Clin. Microbiol. 415593-5597. Duim, B., T. M. Wassenaar, A. Rigter, and J. Wagenaar. 1999. High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting. Appl. Environ. Microbiol. 65:236923 75. Endtz, H. P., J. S. Vliegenthart, P. Vandamme, H. W. Weverink, N. P. van den Braak, H. A. Verbrugh, and A. van Belkum. 1997. Genotypic diversity of Campylobacter lari isolated from mussels and oysters in The Netherlands. Int. J. Food Microbiol. 34:7988. Falush, D., M. Stephens, and J. K. Pritchard. 2003. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164: 1567-1587. Fearnhead, P., N. G. Smith, M. Barrigas, A. Fox, and N. French. 2005. Analysis of recombination in Campylobacter jejuni from MLST population data. J. Mol. Evol. 61:333-340. Feil, E. J., B. C. Li, D. M. Aanensen, W. P. Hanage, and B. G. Spratt. 2004. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol. 186:1518-1530. Feil, E. J., M. C. J. Maiden, M. Achtman, and B. G. Spratt. 1999. The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis. Mol. Biol. Evol. 16:1496-1502. Fitzgerald, C., L. 0. Helsel, M. A. Nicholson, S . J. Olsen, D. L. Swerdlow, R. Flahart, J. Sexton, and P. I. Fields. 2001. Evaluation of methods for subtyping Campylobacter jejuni during an outbreak involving a food handler. J. Clin. Microbiol. 39:23862390. Fouts, D. E., E. F. Mongodin, R. E. Mandrell, W. G. Miller, D. A. Rasko, J. Ravel, L. M. Brinkac, R. T. DeBoy, C. T. Parker, S . C. Daugherty, R. J. Dodson, A. S . Durkin, R. Madupu, S. A. Sullivan, J. U. Shetty, M. A. Ayodeji, A. Shvartsbeyn, M. C. Schatz, J. H. Badger, C. M. Fraser, and K. E. Nelson. 2005. Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species. PLoS Biol. 3:e15. French, N., M. Barrigas, P. Brown, P. Ribiero, N. Williams, H. Leatherbarrow, R. Birtles, E. Bolton, P. Fearnhead, and A. Fox. 2005. Spatial epidemiology and natural population structure of Campylobacter jejuni colonizing a farmland ecosystem. Environ. Microbiol. 7:1116-1126. Garcia, M. M., G. M. Ruckerbauer, M. D. Eaglesome, and W. E. Boisclair. 1983. Detection of Campylobacter fetus in artificial insemination bulls with a transport enrichment medium. Can. J. Comp. Med. 47:336-340. Gillespie, I. A., S . J. O’Brien, J. A. Frost, G. K. Adak, P. Horby, A. V. Swan, M. J. Painter, K. R. Neal, and C. S. S. S. Collaborators. 2002. A case-case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses. Emerg. Infect. Dis. 8:937-942. Joky, K. A., M. S . Chan, and M. C. Maiden. 2004. mlstdbNetdistributed multi-locus sequence typing (MLST) databases. BMC Bioinfomzatics 5:86. Joky, K. A., E. J. Feil, M. S . Chan, and M. C. Maiden. 2001. Sequence type analysis and recombinational tests (START). Bioinfomzatics 17: 1230-123 1. Joky, K. A., and M. C. Maiden. 2006. AgdbNet-antigen sequence database software for bacterial typing. BMC Bioinformatics 7:314. Kinana, A. D., E. Cardinale, I. Bahsoun, F. Tall, J. M. Sire, S. Breurec, B. Garin, C. Saad-Bouh Boye, and J. D. Perrier-GrosClaude. 2007. Campylobacter coli isolates derived from chickens

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in Senegal: diversity, genetic exchange with Campylobacter jejuni and quinolone resistance. Res. Microbiol. 158:138-142. Kinana, A. D., E. Cardinale, F. Tall, I. Bahsoun, J. M. Sire, B. Garin, S. Breurec, C. S . Boye, and J. D. Perrier-Gros-Claude. 2006. Genetic diversity and quinolone resistance in Campylobacter jejuni isolates from poultry in Senegal. Appl. Environ. Microbiol. 72:3309-3313. Levin, B. R. 1981. Periodic selection, infectious gene exchange and the genetic structure of E. coli populations. Genetics 99:l-23. Litrup, E., M. Torpdahl, and E. M. Nielsen. 2007. Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark.1. Appl. Microbiol. 103:210-218. Maiden, M. C. 2006. Multilocus sequence typing of bacteria. Annu. Rev. Microbiol. 60561-588. Maiden, M. C. J. 1993. Population genetics of a transformable bacterium: the influence of horizontal genetical exchange on the biology of Neisseria meningitidis. FEMS Microbiol. Lett. 112: 243-250. Maiden, M. C. J. 2000. High-throughput sequencing in the population analysis of bacterial pathogens. Int. J. Med. Microbiol. 290~183-190. Maiden, M. C. J., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95:3140-3145. Manning, G., C. G . Dowson, M. C. Bagnall, I. H. Ahmed, M. West, and D. G. Newell. 2003. Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni. Appl. Environ. Microbiol. 69:6370-6379. Maynard Smith, J. 1989. Trees, bundles or nets. Trends Ecol. Evol. 4 :3 02-3 04. Maynard Smith, J., C. G. Dowson, and B. G. Spratt. 1991. Localized sex in bacteria. Nature 349:29-31. Maynard Smith, J., N. H. Smith, M. O’Rourke, and B. G. Spratt. 1993. How clonal are bacteria? Proc. Natl. Acad. Sci. USA 90: 4384-4388. McCarthy, N. D., F. M. Colles, K. E. Dingle, M. C. Bagnall, G. Manning, M. C. Maiden, and D. Falush. 2007. Host-associated genetic import in Campylobacter jejuni. Emerg. Infect. Dis. 13: 267-272. Mickan, L., R. Doyle, M. Valcanis, K. E. Dingle, L. Unicomb, and J. Lanser. 2007. Multilocus sequence typing of Campylobacter jejuni isolates from New South Wales, Australia. J. Appl. Microbiol. 102:144-152. Miller, W. G., M. D. Englen, S . Kathariou, I. V. Wesley, G . Wang, L. Pittenger-Alley, R. M. Siletz, W. Muraoka, P. J. FedorkaCray, and R. E. Mandrell. 2006. Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals. Microbiology 152:(Pt. 1):245-255. Miller, W. G., S . L. On, G. Wang, S . Fontanoz, A. J. Lastovica, and R. E. Mandrell. 2005. Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus. J. Clin. Microbiol. 43:2315-2329. Narra, H. P., and H. Ochman. 2006. Of what use is sex to bacteria? Cum Biol. 16:R705-R710. Parker, C. T., W. G. Miller, S . T. Horn, and A. J. Lastovica. 2007. Common genomic features of Campylobacter jejuni subsp. doylei strains distinguish them from C. jejuni subsp. jejuni. BMC Microbiol. 7 5 0 . Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S . Holroyd, K. Jagels, A. V. Karlyshev, S . Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H.

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van Vliet, S . Whitehead, and B. G. Barrel]. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668. Sails, A. D., B. Swaminathan, and P. I. Fields. 2003a. Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strain associations identified by multilocus enzyme electrophoresis. J. Clin. Microbiol. 41:4058-4067. Sails, A. D., B. Swaminathan, and P. I. Fields. 2003b. Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni. J. Clin. Microbiol. 41:4733-4739. Schouls, L. M., S . Reulen, B. Duim, J. A. Wagenaar, R. J. Willems, K. E. Dingle, F. M. Colles, and J. D. Van Embden. 2003. Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J. Clin. Microbiol. 41:15-26. Selander, R. K., D. A. Caugant, H. Ochman, J. M. Musser, M. N. Gilmour, and T. S . Whittam. 1986. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 515337-884. Sopwith, W., A. Birtles, M. Matthews, A. Fox, S . Gee, M. Painter, M. Regan, Q. Syed, and E. Bolton. 2006. Campylobacter jejuni multilocus sequence types in humans, northwest England, 20032004. Emerg. Infect. Dis. 12:1500-1507. Spratt, B. G., and M. C. J. Maiden. 1999. Bacterial population genetics, evolution and epidemiology. Proc. R. SOC. Lond. B Biol. Sci. 354:701-710.

Stackebrandt, E., W. Frederiksen, G. M. Garrity, P. A. Grimont, P. Kampfer, M. C. Maiden, X. Nesme, R. Rossello-Mora, J. Swings, H. G. Truper, L. Vauterin, A. C. Ward, and W. B. Whitman. 2002. Report of the ad hoc committee for the reevaluation of the species definition in bacteriology. Int. /. Syst. Bacteriol. 52(Pt. 3):1043-1047. Suerbaum, S., M. Lohrengel, A. Sonneveld, F. Ruberg, and M. Kist. 2001. Allelic diversity and recombination in Campylobacter jejuni. J. Bacteriol. 183:2553-2559. Thakur, S., and W. A. Gebreyes. 2005. Campylobacter coli in swine production: antimicrobial resistance mechanisms and molecular epidemiology. J. Clin. Microbiol. 435705-5714. Thakur, S., W. E. Morrow, J. A. Funk, P. B. Bahnson, and W. A. Gebreyes. 2006. Molecular epidemiologic investigation of Campylobacter coli in swine production systems, using multilocus sequence typing. Appl. Environ. Microbiol. 725666-5669. van Bergen, M. A., K. E. Dingle, M. C. Maiden, D. G. Newell, L. van der Graaf-Van Bloois, J. P. van Putten, and J. A. Wagenaar. 2005. Clonal nature of Campylobacter fetus as defined by multilocus sequence typing. J. Clin. Microbiol. 435888-5898. Wassenaar, T. M., and D. G. Newell. 2000. Genotyping of Campylobacter species. Appl. Environ. Microbiol. 66: 1-9. Yan, W., N. Chang, and D. E. Taylor. 1991. Pulsed-field gel electrophoresis of Campylobacter jejuni and Campylobacter coli genomic DNA and its epidemiologic application. J. Infect. Dis. 163:1068-1072.

Cumpylobucter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 3

Complexity and Versatility in the Physiology and Metabolism of

Campylobacter jejuni DAVIDJ. KELLY

UNDERSTANDING PHYSIOLOGY AND METABOLISM HELPS TO UNDERSTAND THE COLONIZATION AND DISEASE PROCESS

comparative genomics. The challenge now is to use this information to discover which metabolic pathways or enzyme activities are essential in the different environments that C. jejuni is capable of surviving or growing in. This will give us insights into the strategies it uses for successful colonization, and it will suggest how C. jejuni can be a commensal in the avian gastrointestinal tract, a pathogen in the human gastrointestinal tract, and a persistent survivor in the environment and the food chain. In these different situations, the metabolism of the bacteria may be significantly altered, yet these crucial changes are poorly understood at present. In this chapter, the current knowledge on some of the metabolic aspects of C. jejuni physiology, with emphasis on those features of carbon, nitrogen, and electron flow that are likely to be of importance in understanding growth in the environment and in vivo, will be reviewed. The chapter will focus on catabolic pathways, i.e., those involved in the breakdown of extracellular solutes, yielding energy and key intracellular intermediates that are the building blocks for new cell growth. Since I last reviewed this area (Kelly, 2005), a number of important papers have been published. I have endeavored to include most of these, but because of space limitations, the material is necessarily selective.

The recent availability of the genome sequences of several campylobacters, particularly Campylobacter jejuni, has refocused attention on the physiological and metabolic properties of this medically and economically important group of the Epsilonproteobacteria. For many years, such studies have taken second place to work on virulence and colonization, but it is now becoming clear that the particular metabolic properties of any pathogen are intimately related to its ability to cause disease, and a greater understanding of these relationships is crucial to gain a full picture of pathogenicity. The campylobacters can be difficult organisms to culture, often have complex nutritional requirements, and are microaerophilic, and this combination of features has undoubtedly hindered progress in elucidating many aspects of their physiology and metabolism. Nevertheless, such studies are central to understanding growth in animal and human hosts, survival in the environment and the food chain, and mechanisms of pathogenicity, as well as what carbon sources are being used in vivo. Physiological studies may also provide leads in identifying potential targets for controlling or preventing the growth of campylobacters. The determination of the complete genome sequence of C. jejzkni NCTC 11168 (Parkhill et al., 2000) provided many new insights into its biology, not least in terms of metabolism. Complete genome sequences are now available for additional diverse strains of C. jejuni, for example 81-176 (Hofreuter et al., 2006), RM1221 (Fouts et al., 2005), and CG8486, a recent clinical isolate from Thailand (Poly et al., 2007), which provide a wealth of material for

TRANSPORTING SOLUTES IN AND OUT OF CAMPYLOBACTER JEJUNI Overview

Solute transport is a fundamental prerequisite for metabolism in the cytoplasm of hydrophilic and polar growth substrates. Active solute transport sys-

David J. Kelly Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.

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tems can be broadly classified into two major groups on the basis of their energy-coupling mechanisms. Primary systems are often multicomponent and depend on direct hydrolysis of chemical bonds to release free energy for solute transport, often ATP hydrolysis. In contrast, secondary systems are usually simpler (sometimes a single transport protein) and use a preexisting electrochemical ion-gradient across the cytoplasmic membrane to power solute uptake, often the proton motive force or a sodium ion gradient. C. jejuni contains many types of both classes of transport systems. As an example, the NCTC 11168 genome contains about 92 distinct transport proteins, 37% of which form primary systems and 57% of which form secondary systems. Most primary systems are accounted for by ABC-type transporters (about 30 complete systems, both importers and exporters), with the remaining primary systems comprising three P-type ATPases (the cation transporters Cjll5.S and Cj1161, and the Kdp potassium transporter) and one F-type ATPase (the proton-translocating ATP synthase). Of the secondary transporters, most fall into the major facilitator superfamily group, consisting of a single transport protein, but there are one or more representatives of about 20 other distinct families of secondary transporters, each of which will have specialized roles in solute movements. It is often difficult to accurately predict the substrates used by transporters from sequence homologies. As a general observation, we can conclude that C. jejuni NCTC 11168, for example, has few carbohydrate transporters, but many systems for amino acid and organic acid transport. This reflects the asaccharolytic nature of its metabolism and its reliance on amino acids as major carbon sources (discussed below). Here, there is only space to describe a selection of the transport systems present that relate to the major metabolic pathways in C. jejuni for which some functional data are available. Amino Acid Transport As described below, amino acids are thought to be the most important carbon and energy sources for C. jejuni, and data are now accumulating on the transport of these substrates, which suggests a number of interesting connections between pathogenicity and metabolism. This is particularly evident in the dual roles of several amino acid binding proteins, which are also immunogenic surface protein antigens and adhesins. PEBla and the PEBl transport system: aspartate and glutamate transport In a study designed to identify proteins suitable for use in serology and as vaccine candidates, Pei et

al. (1991) identified four proteins of 28, 29, 30, and 31 kDa in acidic glycine extracts of whole cells of strain 81-176. These were termed PEBl to PEB4, respectively. The structural gene for PEBl (pebla)was cloned by Pei and Blaser (1993) by using polyclonal antibody raised against purified PEBla protein in a screen of a hgtl 1 library of genomic DNA from strain 81-176. The gene was mutated, and the null mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture (Pei et al., 1998). Moreover, mouse challenge studies indicated that the rate and duration of intestinal colonization was significantly lower and shorter, respectively, compared with the wild-type strain. These data have established the PEBla protein as an important colonization and virulence factor in C. jejuni. Western blot analysis showed that PEBla is present in all C. jejuni and C. coli strains examined, but it appears to be absent from C. fetus, C. lari, and Helicobacter pylori (Pei et al., 1991). From database searching that used the deduced amino acid sequence of PEBla, Pei and Blaser (1993) showed that the protein is homologous to the periplasmic-binding protein component of amino acid ABC transporters, for example GlnH (27.8% identity) and HisJ (28.9% identity). A linked open reading frame encodes a protein with homology with the membrane proteins GlnQ and HisP, and the genome sequence of strain NCTC 11168 clearly reveals that pebla in this strain (CjO921c) is part of a four-gene operon encoding a typical ABC transporter. A potential dual role for PEBla as both a surface-exposed adhesin and a periplasmic solute binding protein can be rationalized because of the unusual existence of two predicted processing sites in the signal sequence for signal peptidase I and 11, which may be responsible for the localization of the protein both in the periplasm and on the cell surface (Pei and Blaser, 1993). PEBla is encoded by CjO921c in the C. jejuni NCTC 11168 genome (Parkhill et al., 2000). Overexpression and purification of the recombinant form of Cj092 1 followed by steady-state fluorescence titrations with all 20 amino acids (Leon-Kempis et al., 2006) showed that the protein specifically bound Laspartate and L-glutamate with low (micromolar) Kd values but had a low affinity for L-asparagine and Lglutamine. The other amino acids did not bind. Significantly, a CjO921c mutant was completely deficient in glutamate transport, showed a -20-fold reduction in aspartate transport compared with the parent strain, and was unable to grow on either aspartate or glutamate as a carbon source in minimal media (Leon-Kempis et al., 2006). It is thus clear that PEBla is the periplasmic component of an aspartate/gluta-

CHAPTER 3

mate ABC transporter, essential for optimal microaerobic growth on these dicarboxylic amino acids. The vast majority of PEBla in C. jejuni was found to be periplasmic (Leon-Kempis et al., 2006), consistent with its solute binding function, but some must be surface exposed in order for the protein to act as an adhesin. The presence of a signal peptidase I1 recognition site might suggest that the protein could be anchored in the outer membrane, but no evidence for this was found from fractionation and localization studies (Leon-Kempis et al., 2006). Rather, the presence of PEBla in acidic glycine extracts and in concentrated culture supernatant samples supports the suggestion of Pei and Blaser (1993) that the protein can be exported across the outer membrane. Although the mechanism for this is unclear, the two-component signal peptide of PEBla is extremely similar to that of endoglucanase (Egl) from Pseudomonas solanacearum (Huang and Schell, 1992; Pei and Blaser, 1993), the two-step export of which has been studied in detail. Egl is first directed across the inner membrane to the periplasm by the lipoprotein signal peptide, then is further cleaved at the signal peptidase I site during export across the outer membrane (Huang and Schell, 1992). A similar process may apply to PEBla, although in this case, most of the fully mature protein appears to remain in the periplasm. Recently, the crystal structure of PEBla at 1.5 8, resolution has been obtained (Miiller et al., 2007), allowing insights into the mechanism of solute binding. The protein has a two-domain a / p structure, characteristic of periplasmic extracytoplasmic solute receptors and a chain topology related to the type I1 subfamily. An aspartate ligand, clearly defined by electron density in the interdomain cleft, forms extensive polar interactions with the protein, the majority of which are made with the larger domain. Arg89 and Asp174 form ion-pairing interactions with the main chain a-carboxyl and a-amino groups, respectively, of the ligand, while Arg67, Thr82, Lysl9, and Tyrl56 coordinate the ligand side chain carboxyl. Lysl9 and Arg67 line a positively charged groove, which favors binding of Asp over the neutral Asn. The ligand-binding cleft is also of sufficient depth to accommodate a glutamate. This is the first structure to be determined of an ABC-type aspartate binding protein and explains the high affinity of the protein for aspartate and glutamate and its much weaker binding of asparagine and glutamine. Stopped-flow fluorescence spectroscopy indicates a simple bimolecular mechanism of ligand binding, with high association-rate constants (Miiller et al., 2007). Interestingly, sequence alignments and phylogenetic analyses revealed PEBla homologues to be

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43

present in some gram-positive bacteria. The alignments also suggested a more distant homology with GltI from Escherichia coli, a known glutamate and aspartate binding protein, but Lysl9 and Tyrl56 are not conserved in GltI. These results provide a structural basis for understanding both the solute transport and adhesin/virulence functions of PEBla.

CjaA: cysteine transport Interestingly, PEBla is not the only differentially localized solute binding protein in C. jejuni with an unusual signal peptide. Another C. jejuni immunodominant surface antigen and vaccine candidate, CjaA (Cj0982), has been shown to be a cysteine binding protein component of an ABC system, and the three-dimensional structure of this protein has been determined (Miiller et al., 2005). The signal peptide of CjaA contains a strongly predicted recognition sequence for signal peptidase I1 but a much less convincing atypical signal peptidase I site, so the sorting mechanism may not be the same as for PEBla. Indeed, preliminary data suggest that CjaA is mainly located on the periplasmic side of the inner membrane (Godlewska et al., 2005). CjaA is localized in an operon with CjaB, which encodes a predicted solute:proton symporter (Wyszynska et al., 2006), but there is no evidence that CjaB has a functional role in cysteine transport. Instead, the predicted membrane permease and ABC protein subunits that could make up a functional ABC-type cysteine transporter along with CjaA appear to be localized in an unlinked region of the chromosome, and they have been detected by bioinformatic comparisons with other bacteria (Miiller et al., 2005). Serine transport As described below, serine is a good carbon source for the growth of C. jejuni. The genome sequence of NCTC 11168 reveals a two-gene operon (Cj1624c and Cj1625c) encoding homologues of the E. coli serine dqhydratase (SdaA) and serine transporter (SdaC) for L-serine transport and catabolism. The hydropathy profile of SdaC shows that it contains 10 or 11 potential membrane-spanning segments, and its overall topology is similar to the E. coli SdaC protein and other members of the hydroxyl aromatic amino acid permease family. Biochemical evidence suggests that serine transport in C. jejuni is actually carried out by at least two systems, a lowaffinity L-serine-specific transporter encoded by sdaC, and a high-affinity transporter, which was revealed by analysis of the residual serine transport kinetics in an sdaC mutant (Velayudhan et al., 2004). The iden-

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KELLY

tity of the high-affinity transporter is unknown, but competition studies with other amino acids suggest it could be a dual serine-threonine transporter. This type of system is known in other bacteria-for example the Na+-coupled serine-threonine uptake system in E. coli (Hama et al., 1987). Organic acid transport systems There appears to be a multiplicity of transporters for organic acids encoded in the C. jejuni genome; at least nine distinct secondary systems can be deduced in strain NCTC 11168. The physiological roles for many of these are not clear, but various strains of C. jejuni have been shown to grow on pyruvate (Velayudhan and Kelly, 2002), citrate, malate, fumarate, succinate, and lactate (Hinton, 2006), all of which will require active transport across the cytoplasmic membrane. C4-dicarboxylate transport (malate, succinate, fumarate, and possibly also aspartate) seems to be particularly important in C. jejuni. These substrates can feed directly into the citric acid cycle (CAC), malate and succinate can act as direct electron donors for aerobic respiration, and fumarate is an alternative electron acceptor under oxygen-limiting conditions. There are three genes (CjOO25c, Cj1097, and Cj 1192) that encode members of the dicarboxylate/ amino acid: cation symporter family. Cj1192 is a likely DctA homologue, well known in other bacteria to transport malate/succinate/fumarate for catabolism under aerobic conditions. DcuA (Cj088) and DcuB (Cj0671) are paralogous and partially redundant C4-dicarboxylate secondary uptake systems that are known to function in fumarate:succinate antiport during anaerobic fumarate respiration in enteric bacteria like E. coli (Unden and Kleefeld, 2004). Because C. jejuni is known to carry out fumarate respiration (see below) and the active site of fumarate reduction by the fumarate reductase catalytic subunit FrdA is on the cytoplasmic side of the membrane, the Dcu proteins are essential for this process. Moreover, by acting as electroneutral antiporters, by using the outwardly directed succinate concentration gradient to power the uptake of fumarate, they provide a bioenergetically efficient solution to the need to deliver fumarate to the inside of the cell. Significantly, microarray and reverse transcriptase PCR studies have shown that both dcuA and dcuB are upregulated in expression in C. jejuni grown in the chick gut compared with laboratory media (Woodall et al., ZOOS), suggesting that fumarate respiration may be of increased importance in this oxygen-limited in vivo environment (see below). E. coli also contains another Dcu protein, DcuC. The role of this is thought to be

as a dedicated succinate efflux system during fermentative growth (Unden and Kleefeld, 2004). Interestingly, strains NCTC 11168 and RM1221 each contain a dcuC pseudogene, while in strain 81-176, there are apparently two functional full-length dcuC genes in addition to dcuA and dcuB homologues (Hofreuter et al., 2006). This suggests an increased role for dicarboxylate metabolism in 8 1-176. Whether this might relate to the increased pathogenicity of this strain is not yet clear.

CENTRAL CARBON METABOLISM IN C. JEJUNI Gluconeogenesis and Anaplerotic Reactions Genes encoding enzymes of the Embden-Meyerhof (EM) and pentose phosphate pathways are present in the genome of the currently sequenced strains of C. jejuni, but these strains lack the key enzymes of the Entner-Doudoroff pathway. Most of the reactions of the EM pathway are reversible in vivo, with the notable exceptions of the 6-phosphofructokinase and pyruvate kinase (PYK) reactions, both of which constitute key control points in many organisms. The C. jejuni strains sequenced thus far lack a gene that could encode a 6-phosphofructokinase, which would account for the well-known inability of this species to catabolize glucose (Velayudhan and Kelly, 2002; Kelly, 2005). However, a gene encoding fructose-1,6bisphosphatase is present, which suggests that the EM pathway functions solely in gluconeogenesis. Surprisingly, a homologue of PYK (Cj0392 in strain NCTC 11168) is present in the C. jejuni genome. This is unexpected because PYK catalyzes the physiologically irreversible conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP at the final stage of the glycolytic pathway (Fig. 1).PYK is thus a catabolic enzyme that should not be required if the only function of the EM pathway in C. jejuni is in gluconeogenesis. A p y k mutant displayed a slightly decreased growth rate in complex media but was able to grow with pyruvate, lactate, and malate as carbon sources in defined media (Velayudhan and Kelly, 2002). PYK is present in cell extracts at a high specific activity (>800 nmol min-' mg-' protein), is activated by fructose-l,6-bisphosphate,and displays other regulatory properties strongly indicative of a catabolic role. Therefore, PYK may function in the catabolism of unidentified substrates, which are metabolized through PEP. Thus, the lower part of the EM pathway in C. jejuni may have a catabolic role in some situations (e.g., during in vivo growth) even though the absence of 6-phosphofructokinase does not allow the full catabolism of hexose sugars. In this

CHAPTER 3

PHYSIOLOGY AND METABOLISM OF C. 7E7UNI

45

Serine I

1

+Acetate A

proline

PutA

\ glutama&e

cytoplasm

i GSSG --GG-T Glu

Proline

i

Glutamate Aspartate

Glutathione (GSSG) Figure 1. Major pathways of central carbon metabolism and amino acid utilization in C. jejuni. The cell is represented with an outer membrane (OM) and inner membrane (IM), enclosing the periplasm, and a cytoplasm within which the major pathways of carbon metabolism are shown. Key amino acid transport systems are shown as black rectangles in the inner membrane. The black circle emphasizes the transamination reaction that converts glutamate to aspartate. The major enzymes are shown next to the reactions catalyzed. SdaC, serine transporter; SdaA, serine dehydratase; Pyk, pyruvate kinase; Pyc, pyruvate carboxylase; Pck, PEP carboxykinase; Por, pyruvate:acceptor oxidoreductase; Acs, acetyl-CoA synthetase; Pta, phosphotransacetylase; AckA, acetate kinase; GltA, citrate synthase; Acn, aconitase; Icd, isocitrate dehydrogenase; Oor, 2oxog1utarate:acceptor oxidoreductase; SUC,succinyl-Cod synthetase; Sdh, succinate dehydrogenase; Fum, fumarase; Mqo, ma1ate:quinone oxidoreductase; Mdh, malate dehydrogenase (NAD linked); AspA, aspartase; Aat, aspartate:glutamate aminotransferase; GlnA, gluramine synthase; GltBD, glutamate synthase; PutA, proline dehydrogenase; PutP, proline transporter; GGT, y-glutarnyl transpeptidase. The conversion of glutathione to glutamate ocurrs in the periplasm of some strains only (dotted arrow). The Pebl system is an ABC transporter containing the periplasmic aspartateightamate binding protein Pebla. Fld, flavodoxin; Fd, ferredoxin. OAA, oxaloacetate; PEP, phosphoenol pyruvate; AcP, acetyl-phosphate.

context, it is interesting to note that although a pseudogene in NCTC 11168, strain 81-176 contains a glpT gene encoding a glycerol-3-phosphate transporter homologue (Hofreuter et al., 2006), which may allow the latter strain to grow on this substrate, which can be catabolized by the lower part of the glycolytic pathway. Net synthesis of oxaloacetate (OM) from pyruvate or PEP is required to permit the CAC to fulfill

both biosynthetic and energy-conserving roles. C. jejuni possesses homologues of anaplerotic enzymes that may function in this capacity (Fig. l),the activities of some of which have been detected by nuclear magnetic resonance (Mendz et al., 1997) and direct assay (Velayudhan and Kelly, 2002). Pyruvate carboxylase (PYC) is a key anaplerotic enzyme encoded by pycA (Cj1037c) and pycB (CjO933c) homologues, which correspond to the biotin carboxylase and bi-

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KELLY

otin carboxyl carrier subunits, respectively, of the two-subunit type of carboxylase (Goss et al., 1981). PYC fulfills an important function in many organisms by catalyzing the ATP-dependent carboxylation of pyruvate to yield OAA. Phosphoenolpyruvate carboxykinase (PCK) provides the PEP required for gluconeogenesis and catalyzes the ATP-dependent decarboxylation of OAA to PEP. C. jejuni has a pckA gene (CjO932c), and interestingly, it is contiguous with the pycB homologue. In addition to PYC and PCK, another predicted decarboxylating/carboxylating enzyme located at the metabolic node around pyruvate is malate oxidoreductase (malic enzyme [MEZ]). MEZ catalyzes the oxidative decarboxylation of malate to pyruvate coupled with NAD(P) reduction. Cj 1287c encodes a probable malate oxidoreductase homologous to that of Bacillus stearothermophilus (Parkhill et al., 2000). Insertion mutants in pycA, pycB, and mez have been generated (Velayudhan and Kelly, 2002). However, this could not be achieved for pckA, indicating that PCK could be an essential enzyme in C. jejuni. The lack of PEP synthase and pyruvate orthophosphate dikinase activities in cell-free extracts suggest a unique role for PCK in PEP synthesis. A pycA mutant was able to grow normally in complex media but was unable to grow in defined media with pyruvate or lactate as major carbon sources, thus confirming an important role for PYC in anaplerosis. In view of the high K, of MEZ for malate (-9 mM) and the lack of a growth phenotype of the rnez mutant, MEZ seems to have only a minor anaplerotic role in C. jejuni (Velayudhan and Kelly, 2002). A Complete CAC with Some Characteristics of Anaerobes The genome sequences of the strains of C. jejuni so far sequenced indicate the presence of a complete oxidative CAC (Fig. l),with all of the key enzymes present including homologues of succinate dehydrogenase or a type B fumarate reductase (which could also act in reverse as a succinate dehydrogenase), the a and /3 subunits of succinyl-CoA synthetase (SCS; sucAB genes) and an NAD-linked malate dehydrogenase and a ma1ate:quinone oxidoreductase. Importantly however, genes for the pyruvate and 2-oxoglutarate dehydrogenase complexes usually found in respiratory aerobes are absent. In fact, C. jejuni uses unrelated enzymes more commonly found in obligately anaerobic bacteria to perform the same reactions. This has consequences for understanding the bioenergetics of campylobacters and so will be discussed in some detail.

The entry of carbon from pyruvate into the cycle requires its oxidative decarboxylation to acetyl-CoA, which, in most aerobic bacteria, is carried out by the pyruvate dehydrogenase multienzyme complex, with NAD used as an electron acceptor. However, in many obligate anaerobes, a flavodoxin- or ferredoxin-dependent pyruvate:acceptor oxidoreductase (POR) catalyzes this reaction. Daucher and Kreig (1995) showed that many species and strains of Campylobacter possess this latter type of enzyme, which can be detected by using benzyl or methyl viologen as an artificial electron acceptor in cell-free extracts. The significance of this is that such acceptor: oxidoreductases are iron-sulfur-cluster-containing enzymes, which are usually sensitive to inactivation by molecular oxygen or reactive oxygen species. This has been clearly shown in the related H. pylori by the oxygen lability of the purified pyruvate:flavodoxin oxidoreductase (Hughes et al., 1995). In addition, both H. pylori and C. jejuni contain a related oxygen labile CAC enzyme, 2-oxog1utarate:acceptor oxidoreductase (OOR; Hughes et al., 1998; Kelly, 2001), which replaces the function of the 2-oxoglutarate dehydrogenase multienzyme complex found in conventional aerobes. Campylobacter jejuni is a true microaerophile that exhibits oxygen-dependent growth but is unable to grow at normal atmospheric oxygen tensions, and it has been proposed that the presence of these proteins might contribute to the microaerophilic phenotype of both Helicobacter and Campylobacter (Kelly, 1998, 2001, 2005). However, there is an interesting difference between the POR of C. jejuni and H. pylori in that the former enzyme is a large single-subunit protein containing four separate functional domains that are present as separate gene products in H. pylori. In contrast, the OORs of both bacteria are similar four-subunit enzymes. The situation in C. jejuni and the fact that there is only a rather low overall sequence similarity between the POR and OOR subunit sequences in H. pylori indicate that these two enzymes have evolved independently. An important question is how the reduced flavodoxin or ferredoxin produced in the POR/OOR reactions is reoxidized and thus how the substrate derived electrons are used by the bacteria for energy conservation. In H. pylori, it was shown that in cellfree extracts in the presence of CoA and flavodoxin, pyruvate could support the reduction of NADP, even though NADP is not a substrate of the purified POR enzyme (Hughes et al., 1998). This suggested that an additional redox protein might mediate the transfer of electrons from POR-reduced flavodoxin to NADP, which might then be used as a respiratory substrate. A likely candidate for this enzyme has recently been identified in H. pylori as HP1164, a flavodoxin:qui-

CHAPTER 3

none reductase that can catalyze flavodoxin-dependent NADP reduction in vitro and that appears to be unique to and highly conserved in many Epsilonproteobacteria, including C. jejuni (St Maurice et al., 2007). In NCTC 11168, the flavodoxin:quinone reductase homologue is Cj0559 (St Maurice et al., 2007), but it has not yet been shown whether this might mediate the same reaction. An alternative route for the electrons derived from pyruvate or 2-0x0glutarate via ferredoxin/ flavodoxin is a more direct interaction with the respiratory chain. As discussed below, an interesting possibility is via the unusual NDH-1 complex, which does not contain NAD(P)Hbinding subunits in C. jejuni and related bacteria.

AMINO ACID CATABOLISM AND NITROGEN ASSIMILATION The main carbon sources used by C. jejuni in vivo are likely to be amino acids because the bacterium is unable to metabolize exogenous sugars but does have the transport and enzymatic capacity for amino acid catabolism. There are essentially four enzymatic amino acid catabolic mechanisms that might be operative, depending on the particular chemistry of the amino acid side chain: (i) deamination (oxidative or nonoxidative), (ii) transamination via an oxo-acid acceptor like 2-oxoglutarate or OAA, (iii) p elimination of ammonia via dehydratases or amino acid:ammonia lyases, and (iv) decarboxylation. These four mechanisms must provide intermediates that can readily feed into central metabolic pathways, and some amino acids might require a combination of enzymes to satisfy this requirement. In C. jejuni NCTC 11168, on the basis of the available genome sequence and a consideration of the most likely catabolic pathways in which these enzymes participate, there are enzymes that would be predicted to allow the complete catabolism of only aspartate, asparagine, glutamate, glutamine, serine, and proline (Fig. 1). This is a narrow range of amino acids, but it accords exactly with the amino acid utilization data of Leach et al. (1997), and interestingly, aspartate, glutamate, proline, and serine are the most abundant amino acids in chicken excreta (Parsons et al., 1982). CjOO21c encodes a potential fumarylacetoacetate hydrolase that is the final enzyme in tyrosine catabolism in some bacteria, but the other enzymes of this complex pathway appear to be absent. No amino acid catabolic decarboxylases are present, but the three other principal mechanistic routes are represented (Fig. 1).Asparagine might be deaminated to aspartate by Cj0029 (a predicted asparaginase), and there are two possible aspartate/glutamate transaminases (CjOl50 and

PHYSIOLOGY AND METABOLISM OF C. TETUNI

47

Cj0762). A key aspartate catabolic enzyme is likely be encoded by Cj0087, a predicted aspartate:ammonia lyase (aspartase) that would directly form fumarate by p-elimination of ammonia. Proline could be oxidized to glutamate via a predicted bifunctional proline dehydrogenase PutA (Cj1503) with A'pyrroline-5-carboxylate as an intermediate. The electrons from this reaction may feed into the respiratory chain at the level of menaquinone (Fig. 2). Glutamate catabolism is likely to proceed via transamination to aspartate. Glutamate and glutamine metabolism is also dominated by the glutamine synthase and glutamine:2-oxoglutarate amino-transferase system, which is the major route by which nitrogen (in the form of ammonium ions) is incorporated into cellular amino acids. 2-Oxoglutarate is the primary amino group acceptor in the interconversion of glutamine and glutamate, and it is a crucial intermediate in C. jejuni amino acid metabolism. Some biochemical studies have been carried out on amino acid utilization in both batch and continuous cultures (Karmali et al., 1986; Westfall et al., 1986; Leach et al., 1997; Mendz et al., 1997). In complex media, the most heavily depleted amino acids were serine, aspartate, glutamate, and proline (Leach et al., 1997). In continuous culture, there was a strong growth rate effect on the utilization of different amino acids, with aspartate and serine being metabolized preferentially at high dilution (growth) rates, with a switch to glutamine and proline at lower dilution rates (Leach et al., 1997). L-Serine seems to be a particularly favored amino acid for catabolism. C. jejuni possesses an active serine dehydratase (deaminase) that converts serine to pyruvate and ammonia. The pyruvate can be rapidly and efficiently metabolized via POR and the CAC, which may explain why this amino acid is so well utilized (Fig. 1).Most serine dehydratases from aerobic bacteria are pyridoxal 5'-phosphate (PLP)dependent enzymes. Results (Velayudhan et al., 2004) have shown that the sdaA gene in C. jejuni encodes an L-serine dehydratase that is devoid of PLP; instead, it is an oxygen-labile iron-sulfur enzyme. The enzyme lacks the consensus sequence centered around a lysyl residue to which PLP binds via a Schiff base (Ogawa et al., 1989), in addition to lacking the glycine-rich region reported for all PLP-dependent enzymes (Marceau et al., 1988). Moreover, the PLP-reactive carbonyl reagents phenylhydrazine and hydroxylamine did not significantly affect enzyme activity. The absorption spectrum of the purified protein was, however, typical of that for an iron-sulfur protein, with broad peaks at 300 to 350 nm and at 420 nm (Velayudhan et al., 2004). Consistent with this, the C. jejuni SdaA se-

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KELLY

FldA/Fd

NO3-

NapAB

(Cj078 11782)

Cj0020 and Cj0358

NrfH --* (Cj1358)

NAD(P)H

SorAB Malate Proline

? e Gluconate

Tor/Dor Cj026

(CjO408-410)

f

Succinate 4

TMAO/DMSO

TMADMS

Figure 2. Major electron transport pathways in C. jejuni. Integral membrane oxidoreductases on the electron donor side of the menaquinone (MK) pool include an NDH-1-like complex (Cj1566-1579), the electron donor to which is unknown, hydrogenase, formate dehydrogenase, and succinate dehydrogenase. Peripherally associated oxidoreductases include (among several others) ma1ate:quinone oxidoreductase, proline dehydrogenase, and a lactate dehydrogenase. Reducing equivalents are transferred to menaquinone in the lipid bilayer of the inner membrane. Menaquinol reduces the cytochrome bc, complex, which in turn reduces periplasmic cytochrome c. Cytochrome c is reoxidized by one of the terminal oxidases, a cb-type cytochrome c oxidase. A separate quinol oxidase (CioAB) is also present. Cytochrome c may also be reoxidized by hydrogen peroxide in the periplasm through the activity of two separate CCPs. Several alternative reductases are present in C. jejuni. Fumarate reductase (FrdCAB) catalyzes electron transfer from menaquinol to fumarate as terminal acceptor. Periplasmic nitrate (Nap), nitrite (Nrf), and TMAOiDMSO reductases (ToriDor) are also present. An additional type of DMSO reductase (DmsABC) is present in strain 81-176 (not shown; see text and Fig. 3 for details). Cj0378/379 are homologues of the E. coli YedZ/YedY proteins, which are a b-type cytochrome and a molybdoprotein reductase, respectively, but the substrate reduced is unknown. Solid lines indicate experimentally established or highly likely routes of electron transport; dotted lines indicate uncertainty as to the exact route, possibly with the participation of unidentified additional redox proteins. Figure modified and updated from Kelly (2005).

quence contains four conserved cysteine residues, which could coordinate a [4Fe-4S] cluster. Moreover, both the spectral features and enzyme activity were lost on exposure to air, but activity could be restored by treatment with ferrous iron and a reducing agent (Velayudhan et al., 2004). These properties are similar to those exhibited by the L-serine dehydratases of many anaerobes, which are PLP-independent, iron-sulfur-containing enzymes (Grabowski et al., 1993). They form a family of enzymes that share mechanistic similarities with the dehydration of citrate by aconitase (Grabowski et al., 1993), but they are generally highly specific for L-serine deamination. The use of an oxygen-labile dehydratase for serine catabolism is another example of the use by C. jejuni of a type of enzyme more commonly found in anaerobic bacteria than aerobes.

Mutation of sduA resulted in a 10-fold reduction in the activity of serine dehydratase in cell-free extracts. The residual activity was found to be due to the PLP-dependent biosynthetic L-threonine dehydratase (IlvA; CjO828c), but this activity was unable to support growth of the mutant on L-serine in a minimal medium, and there was no evidence from 'H-nuclear magnetic resonance spectroscopy for significant utilization of L-serine by intact cells (Velayudhan et al., 2004). However, growth of the sduA mutant in a complex medium was indistinguishable from that of the parent strain. This indicates that an inability to deaminate serine does not result in a growth disadvantage when a larger array of carbon sources in addition to amino acids are available. Most significantly, however, it was found that there was a defect in colonization of 2-week-old chickens by the

CHAPTER 3

isogenic sdaA mutant compared with its wild-type colonization-proficient parent strain (Velayudhan et al., 2004). The data suggest that a source of serine is readily available to the bacterium and that its catabolism via SdaA is a determinant of its ability to grow in the chicken cecum. These results may also indicate a high degree of selectivity in the types of amino acid utilized by C. jejuni in this niche. Role for y-Glutamyl Transpeptidase in Amino Acid Catabolism and Colonization in C. j e j m i Although all of the key enzymes discussed above are present in each of the sequenced C. jejuni strains, there is an interesting situation regarding one enzyme, y-glutamyl transpeptidase (GGT), which is present in some strains, e.g., 81-176 (Hofreuter et al., 2006) and 81116 (Barnes et al., 2007), but not in NCTC 11168 or RM1221. Colonization studies with isogenic ggt mutants have clearly shown that this gene is required for persistent colonization of l-day-old chicks (Barnes et al., 2007) and also a mouse model of infection (Hofreuter et al., 2006). GGT catalyzes the transpeptidation of the y-glutamyl group of glutathione, and it is an essential enzyme in glutathione metabolism in most animal tissues. Its role in bacteria, in which it is also widespread, has been less clear because other enzymes of glutathione metabolism and the y-glutamyl cycle are often absent, and also because in gram-negative bacteria, GGT is a periplasmic enzyme. However, GGT also catalyzes the hydrolysis of glutamine to form glutamate and ammonia, and glutathione to form glutamate and cysteinylglycine. The hydrolysis reaction of GGT has been largely overlooked in assessing the function of the enzyme in prokaryotes, but a recent elegant study in H. pylori by Shibayama et al. (2007) has revealed that the kinetics of the hydrolysis reaction are far more favorable than the y-glutamyl transferase reaction, and that the likely role of GGT in H. pylori is to supply the bacteria with glutamate for catabolism by the hydrolysis of extracellular glutathione or glutamine. These substrates would be hydrolyzed in the periplasm and then the glutamate transported inside the cell (Shibayama et al., 2007). Given the importance of amino acid catabolism for the growth of C. jejuni, as emphasized above, and the known abundance of glutathione (and almost certainly glutamine) in avian and mammalian tissues, it is tempting to speculate that in those strains that possess a ggt gene, a similar physiological role in supplying glutamate applies (Fig. 1). Other Sources of Nitrogen Low concentrations of environmental ammonium ions and those produced as a result of amino

PHYSIOLOGY AND METABOLISM OF C. TETUNI

49

acid catabolism by deamination or @elimination reactions as described above can be assimilated via the glutamine synthase and glutamine:2-oxoglutarate amino-transferase system. Other sources of nitrogen are also likely to be available to C. jejuni in the host-for example, urea and uric acid in mammalian and avian hosts, respectively. Unlike H. pylori and some thermophilic campylobacters, C. jejuni does not possess a urease gene to allow the direct hydrolysis of urea to ammonia and carbon dioxide. An alternative route of urea catabolism is via urea carboxylase to allophanate (urea-l-carboxylate), which can then be hydrolyzed to yield ammonia (Kanamori et al., 2004). These reactions may be carried out by Cj1541 and Cj1542 in NCTC 11168, with the cognate genes also being conserved in RM1221 and 81176, although biochemical data are lacking. Degradation of the purine ring system of uric acid is more complex and requires a set of enzymes to oxidize and hydrolyze uric acid to allantoin, allantoate, and then on to urea. Clear homologues of uricase (urate oxidase) and allantoinase appear to be lacking in C. jejuni, but a possible enzyme to hydrolyze the product of the uricase reaction (5-hydroxyisourate) is present in all the genome-sequenced strains (Cj0715 in NCTC 11168). This is hydroxyisourate hydrolase, a protein containing a eukaryotic-like transthyretin domain (Lee et al., 2005).

PHYSIOLOGY OF MICROAEROBIC GROWTH Campylobacters and many related Epsilonproteobacteria are well known as microaerophiles. The term microaerophile refers to microorganisms that, although requiring oxygen for growth, are unable to grow at normal atmospheric oxygen tensions; these organisms are thought to be adapted to particular niches that contain low oxygen concentrations (Kreig and Hoffman, 1986; Kelly et al., 2001). Most strains of C. jejuni are routinely cultured in gas atmospheres containing 3 to 10% (vol/vol) oxygen and 5 to 10% (vol/vol) carbon dioxide. However, during its life cycle, C. jejuni must be exposed to highly variable oxygen concentrations, and it thus presents an interesting paradox; although it is oxygen sensitive, it nevertheless must be able to survive high environmental oxygen tensions, resist the oxidative stresses encountered in vivo, and adapt to the severe oxygen limitation of the gut. The relationship between C. jejuni and oxygen is therefore one of the major defining features of the biology of this food-borne pathogen, yet it is also one of the least studied and understood. Indeed, although many genera of freeliving and pathogenic microaerophilic bacteria are

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known, it is still the case that the molecular and physiological bases of microaerophily are not fully understood for any bacterium. A facile explanation would be a deficiency of oxidative stress defenses. All aerobic bacteria are at risk from damage by molecular oxygen, as the stepwise one-electron reduction of 0, results in the formation of the superoxide radical (02-) and hydrogen peroxide (H,O,), which can be removed by the well-characterized superoxide dismutase and catalaselperoxidase enzymes. Significantly, although some microaerophiles do lack these activities (Kreig and Hoffman, 1986) there are many species-including C. jejuni-that contain them, so additional, more complex explanations need to be invoked. As is apparent from the discussion above, C. jejuni, and indeed related microaerophiles, contain several key enzymes that are oxygen sensitive and that may be major targets for inactivation by both molecular oxygen and reactive oxygen species. This may be one reason for the oxygen sensitivity of growth of these bacteria. Sensing of oxygen is clearly important in microaerophiles both for oxygen avoidance at supraoptimal levels and for maximizing the use of alternative pathways of electron transport (and thus energy conservation) if oxygen concentrations become too limiting for aerobic growth. Mechanisms of oxygen sensing are unclear in C. jejuni; from the genome sequence, it is obvious that many of the global regulatory systems present in “model” aerobic bacteria are absent. There are few transcriptional regulators, only seven two-component systems and no RpoStype stationary-phase sigma factor. Significantly, the SoxRS and OxyR oxidative stress regulators are absent, and there are no Arc or Fnr-type systems (Parkhill et al., 2000). Nevertheless, previous microarray studies have provided evidence that C. jejuni upregulates several alternative electron transport pathways in response to oxygen limitation, both in vitro (Gaynor et al., 2004) and in the chick gut (Woodall et al., 2005). The data from the latter study suggest that there may be a coordinated response to limiting oxygen in the sense that metabolically related sets of genes show upregulation in vivo, i.e., the chick gut, compared with in vitro.

PATHWAYS OF ELECTRON TRANSPORT IN C. JEJUNI Organization and Assembly of Electron Transport Chains The complexity of electron transport systems are a key element in the ability of bacteria to grow under a variety of environmental conditions. This is because

the composition and degree of branching of the electron transport chain determines the metabolic flexibility a bacterium possesses in terms of the variety of electron donors and acceptors that can be used to support growth. The simplest types of electron transport chain consist of a primary dehydrogenase to extract electrons from a substrate electron donor, a membrane soluble quinone as a mobile carrier of the electrons (in the form of hydrogen atoms), and a terminal reductase to reduce an appropriate electron acceptor. Key properties are (i) the degree of branching at both “dehydrogenase” and “reductase” ends of the chain; (ii) the ability to use alternative electron acceptors to molecular oxygen, with suitably positive midpoint redox potentials (EM7);(iii) the presence of a variety of types of cytochromes as additional electron carriers and often more than one type of quinone; (iv) interactions between electron transport pathways, optimizing the possibility of each reductant being paired with a wide choice of oxidants; and (v) the degree to which each electron transport chain contributes to proton translocation and energy transduction. The early detailed studies of Hoffman and Goodman (1982) and Carlone and Lascelles (1982) indicated that the respiratory chain of C. jejuni was complex. This work can now be interpreted in terms of the available genome sequences of C. jejuni. Figure 2 shows a partial reconstruction of the major predicted electron transport pathways in strain NCTC 11168 as deduced from the genome sequence and biochemical evidence. A variety of primary dehydrogenases can be identified that feed electrons to a menaquinone pool. C. jejuni contains a proton-translocating cytochrome bc, complex feeding electrons to a periplasmic c-type cytochrome (probably Cjll53 in NCTC 11168) and then to a high-affinity cb-type oxidase, which allows efficient energy conservation when oxygen is used as electron acceptor. Some periplasmic dehydrogenases ( e g , for sulfite and gluconate; see below) probably feed electrons to the same periplasmic cytochrome c (Fig. 1).C. jejuni also contains a number of terminal reductases that allow respiration with a variety of alternative electron acceptors to oxygen. These are discussed in more detail below. The respiratory chain in C. jejuni is clearly highly branched and is significantly more complex than might be expected for such a small-genome pathogen. It should be noted, however, that this complexity is currently underestimated because although a large majority of the components of the respiratory chains can be fitted into diagrams such the one in Fig. 2, there are still a number of uncharacterized cytochromes and other redox proteins. For example, the

CHAPTER 3

molybdoenzyme encoded by Cj0379 is a homologue of the E. coli YedY protein, the three-dimensional structure of which is known (Loschi et al., 2004), but the physiological substrate has not been identified (Fig. 1).Strain 81-176 contains an additional c-type cytochrome and an associated biogenesis system not present in the other strains (Hofreuter et al., 2006). All the key electron transport enzymes contain redox components like heme, iron-sulfur clusters, or molybdenum, nickel, copper, or other cofactors, which have their own pathways of biosynthesis and incorporation into the relevant apoproteins. The biogenesis of the electron transport chains of C. jejuni is thus critically dependent on metal-ion transport and complex cofactor biosynthesis enzymes. In addition, many of the proteins involved in the various pathways of respiration described below must be exported to the periplasm for their function. In the case of the c-type cytochromes, this is achieved via the Sec translocation system, with covalent heme attachment to the apoprotein occurring posttranslationally in the periplasm. In the case of complex cofactor containing proteins like molybdoenzymes (see below), cofactor attachment occurs in the cytoplasm and the mature proteins are translocated across the cytoplasmic membrane via the “twin arginine” translocation (TAT) system. TAT signal peptides can be easily recognized in the N-terminal regions of such proteins in C. jejuni, which also contains homologues of the tatAIE (Cj1176c), tatB (CjOS79c), and tatC (CjOS78c) translocase genes. Electron Donors Hydrogen and formate may be key energy sources in vivo C. jejuni contains a set of structural genes for a NiFe-type “uptake” hydrogenase, as well as accessory genes for nickel incorporation, which could mediate the transfer of electrons from gaseous hydrogen to the quinone pool (Fig. 2). C. jejuni has been shown to possess hydrogenase activity in membrane fractions (Carlone and Lascelles, 1982; Hoffman and Goodman, 1982). Hydrogen could be an important source of electrons for the growth of campylobacters in the gut because many obligate anaerobes in the intestine produce hydrogen from redox reactions associated with fermentation. It would be of great interest to know whether hydrogenase activity is essential for or contributes to the colonization of the avian gut by C. jejuni, as studies with H. pylori have shown that hydrogenase mutants do not colonize a mouse model of infection, and microelectrode measurements have shown that hydrogen is present in the

*

PHYSIOLOGY AND METABOLISM OF C. TETUNI

51

mouse stomach at concentrations much greater than the K , value of the hydrogenase enzyme (Olson and Maier, 2002). In addition to hydrogen, formate is also a good electron donor because the formate/bicarbonate couple has a highly negative redox potential (EM, -420 mV). Formate is also produced by anaerobes in the gut from mixed-acid-type fermentation reactions, so it is available to an organism like C. jejuni, which has the enzymatic capacity for formate oxidation. Respiratory activities in membrane vesicles were SO to100 times greater with formate or hydrogen as substrates when compared with the rates achieved with other electron donors such as succinate, lactate, malate, or NADH, suggesting that the former are indeed excellent electron donors (Hoffman and Goodman, 1982) and indicating the presence of an active formate dehydrogenase. The genome sequence of C. jejuni 11168 reveals an operon encoding putative formate dehydrogenase subunits IfdhA-D/CjlSllc-CjlS08c).C. jejuni fdhA (Cjl51lc) encodes a large 104-kDa selenocysteine containing molybdoprotein equivalent to the E. coli 110 kDa FdnG (a)subunit. fdhB (Cj1510c) encodes a 24 kDa iron-sulfur subunit equivalent to the E. coli 32-kDa FdnH (p) subunit. fdhC (CjlS09c) encodes a 3.5-kDa cytochrome b subunit equivalent to the E. coli 20-kDa FdnI ( y ) subunit. In addition to these three subunits, both E. coli and C. jejuni encode an FdhD protein (29 kDa) required for activity of the formate dehydrogenase enzyme complex (Berg et al., 1991). Molecular and functional studies are lacking on the C. jejuni enzyme. In particular, it would be of great interest to know whether fdh mutants are deficient in colonization of chickens or other animal models because this would provide evidence for the utilization and importance of formate as an in vivo electron donor. The puzzle of NAD(P)H oxidation and the function of complex I In most organisms, NADH is the major electron donor for oxidative phosphorylation, via interaction with a proton-translocating quinone oxidoreductase (often referred to as complex I, Nuo, NQO, or NDH-l), whereas the usual role of NADPH is as a source of electrons for cytoplasmic biosynthetic reactions. NDH-1 is a complex enzyme with a multitude of redox centers and is most commonly made up of 14 different subunits in bacteria. Electrons are passed from NADH via one FMN and nine Fe centers in NDH-1, to ubiquinone/menaquinone in the respiratory chain. Four protons are translocated

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across the membrane for every two electrons transferred. The genome sequences of C. jejuni show that the bacterium contains a cluster of genes (Cj1566 to Cj1579 in NCTC 11168) encoding a potential NADH:quinone oxidoreductase of the NDH-1 type (Fig. 2). However, examination of the deduced proteins encoded by this gene cluster suggests that the complex may not actually oxidize NADH because of the lack of the N Q O l (NuoE) and N Q 0 2 (NuoF) subunits (Smith et al., 2000; Kelly, 2005). These subunits are thought to be essential components for the function of the NDH-1 complex. NuoF binds NADH and also possesses a bound FMN and an Fe-S center. NuoE, G, By and I have cysteine residues that coordinate Fe-S clusters. In place of the expected nuoE and nuoF genes, C. jejuni (and also H. pylori and related Epsilonproteobacteria) has open reading frames of unknown function. Significantly, these open reading frames encode unique small proteins that do not contain any obvious NAD(P)H-binding motif. There are two possibilities regarding the function of this complex in C. jejuni. The first is that electrons from NAD(P)H are transferred to it via an intermediate protein that interacts with the N Q O l and N Q 0 2 replacements (Finely 1998; Smith et al., 2000). Alternatively, coupling of NAD(P)H with the respiratory chain may not occur via the NDH-1 homologue at all, but through an alternative quinone reductase, which may or may not be proton translocating. This implies that an as yet unidentified electron donor interacts with the Cj1566-1579 complex, and that its function is different from most bacterial NDH-1 systems. The nature of the electron donor and whether this type of enzyme is proton translocating are important unresolved questions for understanding the bioenergetics of C. jejuni. One possibility, discussed above, is that flavodoxin/ ferredoxin reduced by the POR/OOR complexes in the CAC donates electrons directly to the NDH-1-like enzyme. Organic acids as electron donors Several types of organic acids (in addition to formate) appear to be able to act as direct electron donors to the respiratory chain in c. jejuni (Fig. 2). Hoffman and Goodman (1982) demonstrated the activity of a lactate dehydrogenase in oxygen-linked respiration, although analysis of the NCTC 11168 genome sequence suggests no obvious locus for a membrane associated lactate dehydrogenase enzyme in C. jejuni. An annotated L-lactate dehydrogenase (Cj1167) is probably a fermentative enzyme. Malate can act as a direct electron donor to the quinone pool

due to the presence of a flavoprotein-type malate oxidoreductase (Mqo). Analysis of the C. jejuni genome reveals a putative oxidoreductase (Cj0393) with sequence similarity to the E. coli ma1ate:quinone oxidoreductase and to the Mqo of H. pylori, indicating that these bacteria use a similar enzyme for malate oxidation (Kather et al., 2000). This is also consistent with the data of Hoffman and Goodman (1982), which showed that malate stimulated respiration in C. jejuni, producing a H'/O ratio of 2.0. Although Mqo and thus malate oxidation is predicted to be localized on the cytoplasmic side of the inner membrane, an interesting additional potential periplasmic electron donor in each of the sequenced C. jejuni strains is gluconate (Fig. 2), which is based on the prediction of a flavin-containing gluconate dehydrogenase encoded by the Cj0414 and Cj0415 genes in strain NCTC 11168, the former with a TAT signal sequence. It is likely that gluconate-derived electrons will enter the chain at the level of the periplasmic cytochrome c (Fig. 2). In enteric bacteria, gluconate has been shown to be a significant in vivo growth substrate (Chang et al., 2004) and is an abundant component of intestinal mucus. It is highly likely that this substrate will be encountered by C. jejuni in vivo, but it is unlikely to be used as a growth substrate and carbon source because C. jejuni lacks the Entner-Doudoroff pathway for its intracellular catabolism. The interconversion of fumarate and succinate can be carried out by two related enzymes: by succinate dehydrogenase, which is expressed under aerobic conditions, or by fumarate reductase, which is induced under anaerobiosis. Succinate dehydrogenase (succinate:quinone oxidoreductase) catalyzes electron transfer from succinate to quinone, where succinate is oxidized to fumarate as part of the tricarboxylic acid cycle. As noted above, C. jejuni appears to have a complete CAC with genes encoding clearly identifiable homologues of all of the conventional enzymes (Parkhill et al., 2000), including separate sdh and frd operons. Sulfite: an unexpected electron donor in C . jejuni The ability to use sulfite as a respiratory electron donor is usually associated with free-living chemolithotrophic sulfur-oxidizing bacteria. However, it has been shown that C. jejuni has the ability to respire sulfite, with oxygen as the electron acceptor (Myers and Kelly, 2005). The C. jejuni NCTC 11168 CjOOO4c and CjOOOSc genes encode a monoheme cytochrome c and molybdopterin oxidoreductase, respectively, homologous to the su1fite:cytochrome c

CHAPTER 3

oxidoreductase of Starkeya novella (Fig. 2). Western blot tests of C. jejuni periplasm probed with a SorA antibody demonstrated cross-reaction of a 45-kDa band, consistent with the size of Cj0005, and a CjOOO4c mutant was unable to respire with sulfite or metabisulfite as electron donors but showed wildtype rates of formate dependent respiration (Myers and Kelly, 2005). Evidence from inhibitor studies suggested that electrons from sulfite enter the respiratory chain after the bc, complex at the level of cytochrome c (Fig. 2), and a fractionation study confirmed that the majority of the su1fite:cytochrome c oxidoreductase activity is located in the periplasm (Myers and Kelly, 2005). Sulfite is an extremely widespread inorganic anion in many environments, particularly low-oxygen niches in soil or water, where it is more stable than in aerobic conditions. The possession of a sulfite respiration system may thus contribute to the survival of C. jejuni in such environments, particularly because this bacterium has a high-affinity cb-type cytochrome c oxidase, which could allow sulfite respiration at extremely low oxygen concentrations. Sulfite respiration may also provide a detoxification mechanism for this normally growth-inhibiting compound. The presence of a sulfite respiration system in C. jejuni is another example of the surprising diversity of the electron transport chain in this small genome pathogen. Oxygen-Dependent Respiration in C. jejuni Most bacteria possess at least two terminal oxidases, often a quinol oxidase and a cytochrome c oxidase (Poole and Cook, 2000), with distinct catalytic properties and regulation of expression. Whereas only one terminal oxidase, a cb-type cytochrome c oxidase, has been found to terminate the respiratory chain of Helicobacter pylori (Nagata et al., 1996), all sequenced strains of Campylobacter jejuni have two terminal oxidases (Parkhill et al., 2000; Fouts et al., 2005; Hofreuter et al., 2006). This confirms the earlier spectroscopic work of Carlone and Lascelles (1982) and Hoffman and Goodman (1982). One oxidase is a cb-type cytochrome c oxidase, similar to that described in H. pylori, and the other resembles a cytochrome bd-type quinol oxidase. Analysis of the NCTC 11168 genome sequence, for example, reveals the presence of a cydAB-like operon encoding subunits I and I1 of such a quinol oxidase. However, in a recent detailed study of the terminal oxidases of NCTC 11168 (Jackson et al., 2007), it is clear from the lack of optical spectral signals associated with the high-spin hemes b and d that this

. PHYSIOLOGY AND METABOLISMOF c. mmr

53

oxidase is not of the conventional cytochrome bd type. Mutation of the cydAB operon was without significant effect on growth, but it did reduce formatesupported respiratory activity. However, the cydABencoded oxidase enhanced survival of cells cultured in a gaseous environment containing 5% (vol/vol) oxygen and was relatively cyanide resistant and has now been renamed CioAB (cyanide-insensitive oxidase). The oxygen affinity of each oxidaie was determined by means of a highly sensitive assay that exploits globin deoxygenation during respirationcatalyzed oxygen uptake (Jackson et al., 2007). The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K, = 0.8 pM) and a V,, of >20 nmol/ mg/s. The alternative ccoNOQP-encoded cyanidesensitive oxidase, which encodes a cytochrome cbtype enzyme, plays a major role in the aerobic respiratory metabolism of C. jejuni because it appeared to be essential for viability, as judged by the failure of repeated attempts to construct a knockout mutation. The cytochrome cb-oxidase exhibited a high oxygen affinity, with a K,,, value of 40 nM and a V, of 6 to 9 nmol/mg/s. Therefore, C. jejuni has two oxidases with different structural and kinetic properties. The marked differences in affinity for oxygen probably reflect distinct physiological roles in response to, and survival under, various degrees of oxygen provision. In particular, the possession of a cb-type oxidase is likely to endow the bacterium with the ability to continue respiration at extremely low oxygen concentrations, as might be found in the niches that it occupies in the gut of animals or humans. Hydrogen Peroxide as an Electron Acceptor Reduction of oxygen to water is a four-electron reaction, but toxic oxygen intermediates formed by incomplete reduction include the superoxide anion (O,-), hydrogen peroxide (H,O,), and the hydroxyl radical (HO'). Hydrogen peroxide can be degraded to H,O and 0, by the cytoplasmic enzyme catalase, but in the periplasm, H,O, can be broken down to water alone by a periplasmic cytochrome c peroxidase (CCP), with the requirement of reduced cytochrome c as an electron donor (Atack and Kelly, 2007). Proton translocation and energy conservation may thus accompany electron transport from a primary electron donor to hydrogen peroxide via the electron transport chain. Although the periplasmic location of the peroxidase means that the enzyme itself is not energy conserving, the reduction of hydrogen peroxide could allow proton translocation to occur at the level of the primary dehydrogenases and also,

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as in C. jejuni, through the operation of the cytochrome bc, complex. The importance of the energyconserving (as opposed to the peroxide detoxification) function of CCPs is difficult to define, but Goodhew et al. (1988), working with the catalasenegative microaerophile Campylobacter mucosalis, were able to show that addition of hydrogen peroxide resulted in uncoupler-sensitive proton extrusion in classical proton-pulse type experiments, albeit with a low stoichiometry (about 0.6 H+/H202).It was suggested that this may be important in the bioenergetics of the bacterium during growth on formate where periplasmic hydrogen peroxide may be generated as a side reaction of the formate dehydrogenase. The true physiological role of bacterial CCPs is actually not well understood (Atack and Kelly, 2007). It is well known that exposure of bacterial cells to excess oxygen increases the rate of H,O, production (Seaver and Imlay, 2004). Paradoxically, however, bacterial ccp genes appear to be upregulated preferentially under low oxygen (microaerobic) or even anaerobic conditions. This may be relevant in pathogenic bacterialike C. jejuni because a drop in oxygen levels may be a signal of entry into the host and would allow the deployment of a periplasmic first line of defense against the attack of hydrogen peroxide-producing phagocytes, for example. In prokaryotes, CCPs are usually diheme proteins, containing two hemes C, one high potential and one low potential, although triheme CCPs have recently been recognized (Atack and Kelly, 2007). The high-potential heme is the source of the second electron for H,O, reduction, and the low-potential heme acts as a peroxidatic center. C. jejuni strains NCTC 11168, 81-176, and RM1221 each have two separate genes encoding putative CCP homologues. In NCTC strain 11168, Cj0020 and Cj03.58 are 34 kDa and 37 kDa, respectively, with both proteins containing two cytochrome c heme-binding site signatures. Cj0358 appears to be a typical CCP, which clusters phylogenetically with other similar enzymes from the bacteria in the epsilon group of the proteobacteria (e.g., H. pylori). These enzymes are all related to the “classical” Pseudomonas aeruginosa type of CCP, which has been studied in detail mechanistically (Atack and Kelly, 2007). However, Cj0020 appears not to be a closely related paralogue of Cj0358, and it is not contained within any of the four major groups that can be identified by phylogenetic analysis (Atack and Kelly, 2007). It does appear to be distantly related to the triheme enzymes from Zymononas mobilis and Gluconobacter oxydans, even though Cj0020 contains only two heme-binding mo-

tifs. Mutants in the CjOO2Oc gene show increased M. sensitivity to killing by hydrogen peroxide Atack and D. J. Kelly, unpublished data), and mutants in the 81-176 homologue are unable to effectively colonize chicks (Hendrixson and DiRita, 2004). It is not yet known whether this putative CCP also plays a role in virulence. The second CCP in C. jejuni, Cj0358, is also needed for resistance to hydrogen peroxide (J. M. Atack and D. J. Kelly, unpublished data), and its expression is increased significantly in the chick cecum (Woodall et al., 200.5), which is thought to be a low-oxygen niche. Cj0358 is also negatively regulated under iron-limited conditions by the iron response regulator Fur, by its homologue PerR (the peroxide response regulator [Holmes et al., 200.5]), and by the temperature-responsive regulator, RacR (Bras et al., 1999). Taken together, these studies suggest a complex regulatory pattern, which may well be related to colonization and possibly pathogenicity.

u.

Growth of C. jejuni under Oxygen Limitation: Respiration by Alternative Electron Acceptors In addition to oxygen-dependent electron transport, it is also clear that the genome of C. jejuni encodes a number of reductases that would be predicted to allow the bacterium to carry out respiration with several alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, trimethy1amine-Noxide (TMAO), and dimethyl sulfoxide (DMSO) (Kelly, 2001; Sellars et al., 2002; Myers and Kelly, 2005; Pittman et al., 2007). Nevertheless, Sellars et al. (2002) found that although growth with these alternative electron acceptors (see below) was insignificant under strictly anaerobic conditions, such growth was possible under severely oxygen-limited conditions. Their results indicated that some oxygen requiring metabolic reaction or reactions prevented anaerobic growth. Because C. jejuni only contains genes for an oxygen-requiring class I type of ribonucleotide reductase (RNR), it was suggested that the inability to synthesize DNA anaerobically is the most likely explanation. Consistent with this, cells incubated anaerobically with electron acceptors did not divide properly but formed filaments analogous to those seen after treatment of aerobic cells with the RNR inhibitor hydroxyurea (Sellars et al., 2002). Thus, C. jejuni can use alternative electron acceptors in energy-conserving reactions, but only if some oxygen is present to satisfy the requirement for deoxyribonucleotide production. This conclusion is directly relevant for understanding growth of C. jejuni in the gut because it implies that in the niches occupied by

CHAPTER 3

the bacterium, small amounts of oxygen must be present for continued viability. Whether in these severely oxygen-limited niches alternative electron acceptors are being used in addition to or even instead of molecular oxygen will require studies with the relevant mutants in animal models. Fumarate as electron acceptor In the absence of oxygen, fumarate (EM7+30 mV) can be used as an alternative terminal electron acceptor for an electron transport chain by using menaquinol (EM7-74 mV), and fumarate respiration is important in energy conservation in many anaerobic bacteria. C. jejuni strains have an frdCAB operon, encoding subunits highly similar to the thoroughly investigated fumarate reductase of Wolinella succinogenes (Lancaster, 2002). This enzyme is a type B quino1:fumarate reductase, which is reversible and has two b-type hemes bound to the quinol-oxidizing FrdC subunit, which spans the membrane. FrdB is an iron-sulfur cluster containing electron transfer protein, which passes electrons on to the peripheral FrdA subunit, which contains a flavin adenine dinucleotide (FAD) cofactor, the site of fumarate reduction. Because the site of fumarate reduction (which consumes 2H+ per mole of fumarate reduced to succinate) is on the cytoplasmic side of the membrane, and quinol oxidation releases protons into the periplasm, this type of fumarate reductase should be an electrogenic (and energy conserving) enzyme. However, experimental evidence from W. succinogenes has shown that the overall reaction is electroneutral (Kroger et al., 2002). To explain this, the E-pathway hypothesis has been advanced (Lancaster, 2002), which proposes a counterbalancing proton-translocating pathway in the enzyme from periplasm to cytoplasm via a conserved glutamate residue that is coupled to electron transfer between the two b hemes. The operation of this energy-dissipating pathway is essential for the function of the enzyme, even though the overall redox reaction of fumarate reduction by menaquinol is thermodynamically favorable. It is highly likely that similar considerations apply to the homologous C. jejuni Frd complex and that, like the remaining periplasmic reductases described below, net energy conservation occurs only at the level of the primary dehydrogenases. Nitrate and nitrite as electron acceptors

C. jejuni NCTC 11168 has both nitrate (Nap) and nitrite (Nrf) reductases located in the periplasm (Sellars et al., 2002; Pittman et al., 2007). The Nap type of nitrate reductase is a two-subunit enzyme consisting of NapA, a -90-kDa catalytic subunit that

PHYSIOLOGY AND METABOLISM OF C. 7E7UNI

55

binds a bis-molybdenum guanosine dinucleoside cofactor and a [4Fe-4S] cluster, and NapB, a -16-kDa diheme c-type cytochrome. NapA is a substrate for the TAT system and requires an additional protein, NapD, as a “proofreading” chaperone in the export process (Palmer et al., 2005). In C. jejuni, the electron transport pathway to nitrate via NapAB is not immediately obvious because nitrate reduction in other Nap systems is usually coupled to quinol oxidation by a membrane-anchored tetraheme cytochrome, NapC. However, a napAGHBLD operon is present in C. jejuni, which does not contain napC. Analysis of the NCTC strain 11168 genome shows that it does encode a napC homologue (Cj1358c), which is directly upstream of the nrfA nitrite reductase gene (Cj13.57~)~ implying that it is part of the nitrite reductase system. This is the case in Wolinella succinogenes, which encodes a NapC-like subunit (NrfH) similar to the product of Cj1358c (Simon et al., 2000). How, then, is the NapAB complex coupled to menaquinol oxidation in C. jejuni? Pittman et al. (2007) showed that the iron-sulfur protein NapG has a major role in electron transfer to the NapAB complex, and an additional protein, NapH, probably acts together with NapG as a quinol dehydrogenase, as in E. coli (Brondijk et al., 2004). It was found, however, that a napG mutant still showed slow nitratedependent growth under oxygen-limited conditions, which was abolished in a nrfH/napG double mutant, suggesting that a low rate of electron transfer from NrfH to NapAB can occur (Pittman et al., 2007). Interestingly, NapG/H are specifically involved in electron transfer from ubiquinol to nitrate in the E. coli Nap system (Brondijk et al., 2002) and are not required for menaquinol-dependent nitrate reduction (Brondijk et al., 2002), indicating a fundamentally different quinol specificity in C. jejuni. The epsilon group of proteobacteria (including W. succinogenes and C. jejuni) are unusual in containing an additional gene of unknown function, designated napL, within their nap operons (Simon et al., 2003). NapL is predicted to be a soluble periplasmic protein with no obvious cofactor binding motifs. A napL mutant possessed -50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor (Pittman et al., 2007). It may possibly be involved in the assembly or maturation of NapA. There is evidence that nitrate respiration may play a significant role in the growth of human and animal pathogens in vivo. In Mycobacterium bovis, membrane-bound nitrate reductase (Nar) activity has been shown to contribute to virulence (Weber et al., 2000). As yet, it is not known if the C. jejuni Nap has any in vivo role; it may also have other functions,

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such as in survival in foods or the environment where nitrate is present, or in nitrogen assimilation in combination with nitrite reductase, which produces ammonia. It should be noted that because of the periplasmic location of the Nap and Nrf enzymes, a proton-motive force can only be generated at the level of the primary dehydrogenases and not from quinol oxidation during electron transfer. A role for the nitrite reductase NrfA in nitric oxide detoxification Cj1357c in strain NCTC 111168 encodes the periplasmic pentaheme cytochrome c nitrite reductase (NrfA), which is the terminal enzyme in the sixelectron dissimilatory reduction of nitrite to ammonia (Simon, 2002; Pittman et al., 2007). High specific activities of nitrite reduction can be detected in intact cells of C. jejuni by using methyl viologen as the electron donor (Sellars et al., 2002); a nrfA mutant lacks this activity and shows no oxygen-limited growth with nitrite (Pittman et al., 2007). NrfA is a wellstudied enzyme, which in most bacteria contains an unusual CXXCK motif at the heme 1 binding site, instead of the CXXCH motif found in the vast majority of heme c-containing enzymes. This requires a dedicated lyase for covalent attachment of the active site heme (Pisa et al., 2002). The C. jejuni NrfA contains a novel ligation site at heme 1, with a “conventional” CXXCH motif instead of CXXCK. This would explain the absence of genes encoding Nrfspecific heme lyases that are found in other bacteria (Einsle et al., 2000), but a site-directed His-for-Lys substitution in the W. succinogenes NrfA results in low catalytic activity (Pisa et al., 2002), so how the enzyme achieves high activity in C. jejuni is unknown. Interestingly, the predicted NrfAs of the closely related C. coli, C. lari, and C. upsuliensis (Fouts et al., 2005) also contain the heme 1 CXXCH motif rather than CXXCK. Cytochrome c nitrite reductases can also carry out the five electron reduction of the nitric oxide radical ( N O ) to NH4+ (Costa et al., 1990). Work in E. coli has shown that this reaction may be physiologically relevant under microaerobic conditions, and it has been proposed that NrfA plays a significant role in nitric oxide detoxification in addition to flavohemoglobin (Hmp) and flavorubredoxin (NorV), the other major E. coli N O detoxification systems (Poock et al., 2002). In C. jejuni, Hmp and NorV are absent, but there is a single domain globin (Cgb), which has been shown to protect against nitrosative stress (Elvers et al., 2004). In keeping with this, Cgb is inducible by nitrosative stress via the regulatory protein NssR (Elvers et al., 2005). nrfA mutants were found

to be hypersensitive to various forms of nitrosative stress, suggesting that NrfA does indeed play a role in nitric oxide metabolism (Pittman et al., 2007). However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite itself causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of a nrfA mutant, in media containing nitrite (Pittman et al., 2007). Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defense against nitrosative stress.

S- or N-oxides as electron acceptors TMAO and DMSO are structurally similar compounds that are widely distributed in many environments, particularly water; TMAO is an excretory product of some fish, and DMSO is produced by some algae (McCrindle et al., 2005). It is therefore likely that campylobacters will come into contact with these electron acceptors in aquatic environments, soil, etc. In fact, a wide range of bacteria possess enzyme systems that catalyze the reduction of TMAO and DMSO (often by the same enzyme), which is a two electron transfer process, yielding trimethylamine or dimethyl sulfide, respectively: (CH,),SO

+ 2H+ + 2e-

-+ -+

(CH,),S (CH,),NO

+ 2H+ + 2e-

(CH,),N

H,O EM, = +160 mV

H,O EM, = +130 mV

As can be seen, the midpoint redox potentials of the DMSO/ dimethyl sulfide and TMAO/ trimethylamine couples are not highly positive, making them thermodynamically poorer electron acceptors compared with, say, oxygen or nitrate, but better than fumarate. Both TMAO and DMSO reductase activities have been demonstrated in intact cells of C. jejuni NCTC 11168 by using methyl viologen as an artificial electron donor, and TMAO- and DMSOdependent growth under oxygen-limited conditions has also been demonstrated (Sellars et al., 2002). Analysis of the C. jejuni NCTC 11168 genome reveals a gene encoding a 93-kDa molybdoprotein TMAO/DMSO reductase homologue (Cj0264) containing conserved residues for the binding of a molybdenum cofactor and an N-terminal “twin arginine” translocase recognition motif for transport to the periplasm. The CjO264c gene was mutated, and the mutant was found to be deficient in both TMAO and DMSO reductase activity, indicating that a single enzyme is responsible for both activities (Sellars et al.,

PHYSIOLOGY AND METABOLISM OF C. TETUNl

CHAPTER 3

2002). In addition, there is an upstream gene encoding a 22-kDa monoheme c type cytochrome (CjO265c) with similarity to the C terminus of the membrane-anchored pentaheme c type cytochrome TorC of E. coli, which transfers electrons to the catalytic TMAO reductase subunit, TorA (Gon et al., 2001; Sellars et al., 2002). An interesting unresolved issue here is how electrons get from the quinol pool to the CjO265/CjO264 complex (Fig. 3 ) . In other bacteria, a family of tetraheme cytochromes (the NapC family; see above) are often involved in connecting terminal reductases in the periplasm with the quinol pool. The additional four hemes of the E. coli TorC protein have this role, but the only NapC homologue in C. jejuni is NrfH (described above), the electron donor to the nitrite reductase. Growth studies with a nrfH mutant clearly showed that it was still capable of oxygen-limited growth with both TMAO and DMSO (Pittman et al., 2007). Because Cj0265 alone is unlikely to act as a quinol oxidase, there appears to be a novel route of electron transfer to these electron acceptors in strain NCTC 11168, which merits further investigation. Of the other genome-sequenced strains of C. jejuni, RM1221 and 81-176 contain a Cj0264 homologue and are thus likely to have a similar type

TMAOiDMSO + 2H+

HCOO - + H.0

He',-+

TMA/DMS

of TMAO/DMSO reductase to that found in NCTC 11168. Given the presence of TMAO and DMSO in water and soil, the physiological role of this enzyme might be assumed to aid survival outside the host. However, strain 81-176 contains, in addition to a Cj0264 homologue, a structurally distinct type of DMSO reductase, which has proven to be interesting with regard to a potential role in vivo (Hofreuter et al., 2006). This system is similar to the DmsABC DMSO reductase complex in E. coli (McCrindle et al., 2005) consisting of a large extrinsic membrane bound molybdoprotein (DmsA), a smaller electron transferring iron-sulfur subunit (DmsB), and an intrinsic membrane anchor and quinol binding subunit (DmsC). Electrons would be transferred from the quinol pool via DmsC then DmsB to DmsA in the periplasm, where the reduction of DMSO would take place (Fig. 3 ) . Like the periplasmic Cj0264/ Cj0265 system, the arrangement of this enzyme across the membrane means that it is not energy conserving, but electron flow through either system can allow energy to be conserved at the level of the primary dehydrogenases (Fig. 3 ) , as with the Nap and Nrf systems described above. Significantly, Hofreuter et al. (2006) showed that a dmsA mutant was less able to colonize a mouse

+ 2H20

TMAODMSO

TMA/DMS

2H+

'&

Cj026

membrane I

2H+

57

cytoplasm

I

2H+

Figure 3. Likely topological organization of two types of TMAO/DMSO reductases found in strains of C. jejuni. In each case, formate is depicted as a typical electron donor to the menaquinone pool (MK), through the action of formate dehydrogenase (Fdh). (a) The Tor/Dor type of reductase system that has been characterized in strain 11168 (Sellars et al., 2002) is illustrated, which is also present in strains RM1221 and 81-176. There is uncertainty over the mechanism of electron transfer from the MK to the monoheme Cj0265 cytochrome (dashed rectangular hypothetical quinol oxidoreductase). In (b), the additional DmsABC-type system shown in strain 81-176 to be needed for optimal colonization of a mouse infection model (Hofreuter et al., 2006) is illustrated. Note that because both of these TMAO/DMSO reductases are predicted to be periplasmic, reduction of the electron acceptor (which consumes protons-in bold) and quinol oxidation (which releases protons-in bold) occur on the same (periplasmic) side of the membrane. Thus, a proton-motive force is only generated at the level of the primary dehydrogenase (binding and releasing protons on opposite sides of the membrane). MGD, molybdenum guanosine dinucleoside cofactor.

58

KELLY

model of infection than the isogenic wild-type parent strain, implying that this enzyme has an important role in the colonization process. It seems unlikely that DMSO is the substrate of this enzyme in an animal host, and it is possible that in vivo, it is reducing a different S oxide-for example, methionine sulfoxide-because it is known that this class of enzyme often has a broad substrate specificity. It is intriguing that this type of DmsABC system has also been shown to be important for virulence in another bacterial pathogen, Actinobacillus pleuropneumoniae (Baltes et al., 2003). The fact that some strains of C. jejuni, a small-genome pathogen, can possess two distinct functional electron transport chains to TMAO/ DMSO underlines its surprising metabolic versatility and indicates a possible role for these electron acceptors in aiding survival outside and inside the host.

Leon-Kempis, John Atack, Marie Thomas, Jon Smart, Chat Phansopa, and Andy Hitchcock.

ADDENDUM IN PROOF Since this chapter was written, several papers have been published which clarify some aspects of the topics covered, particularly growth in vivo. D. Weerakoon and J. Olson (1.Bacteriol, 190: 915-925, 2008) have shown experimentally that reduced flavodoxin is indeed the electron donor to the unusual complex I in C. jejuni. Weingarten et al. (Appl. Environ. Microbiol., in press, 2008) have shown that mutants in napA and nrfA colonize chicks less well than the wild-type parent strain, while cydA and CjO264c mutants show wild-type levels of colonization. This indicates a possible role for nitrateinitrite reduction in growth of C. jejuni in chickens. A ccoN::cm (cb-oxidase) mutant showed no colonization of chickens, although this strain was very oxygen sensitive and had a significant microaerobic growth defect. Bingham-Ramos and Hendrixson (Infect. Immun., in press, 2008) have characterized the cytochrome c peroxidases in strain 81-176 and have shown a potential role in vivo for the Cj0020 homologue but not for the Cj0358 homologue.

CONCLUSIONS REFERENCES

Understanding the metabolism and physiology of C. jejuni and related bacteria has been greatly facilitated by the availability in recent years of several genome sequences and by better experimental techniques for their culture and biochemical analysis. One conclusion that can be drawn is that many campylobacters show an unexpected metabolic versatility, which is particularly reflected in the complexity of the electron transport chains in C. jejuni, as reviewed here. Despite a genome size of only 1.7 Mb, it is clear that this bacterium has retained complex enzymes with a variety of redox cofactors that enable it to be flexible with respect to the variety of electron donors and acceptors it can use. This is surely a function of its ability to occupy so many niches. Published and ongoing colonization studies with metabolic mutants are crucial and are beginning to answer questions about the nature of C. jejuni metabolism during growth in vivo. This information could be use