Investigation of the Energy Metabolism and Stress Defence Pathways of the Emerging Pathogen Campylobacter Concisus and Other Human Hosted Campylobacter Species

Investigation of the energy metabolism and stress defence pathways of the emerging pathogen Campylobacter concisus and other human hosted Campylobacter species

Melissa Yeow

A thesis submitted in fulfilment of the requirements for the degree of Masters by Research (Biotechnology)

Supervisor: Dr Li Zhang

School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales 2020

ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ……………………………………………......

Date ……………………………5/3/2020.………......

Thesis/Dissertation Sheet

Surname/Family Name : Yeow Given Name/s : Melissa, Wei Bao Abbreviation for degree as given in the University : MSc – Biotechnology (2036) calendar Faculty : Science School : School of Biotechnology and Biomolecular Sciences Investigation of the energy metabolism and stress defence pathways of the Thesis Title : emerging pathogen Campylobacter concisus and other human hosted Campylobacter species

Abstract 350 words maximum: (PLEASE TYPE) Campylobacter species have historically been considered non-saccharyolytic and unable to ferment sugars for energy. Recent evidence refutes this, as several species have been found to use fucose, or glucose via the Entner Doudoroff pathway. Additionally, all human-hosted Campylobacter species have been observed to require hydrogen for growth, but little information is available about their energy metabolisms. Thus, this project examines the pathways of central carbon metabolism and energy metabolism of human-hosted Campylobacter species, using the model organism Escherichia coli, and the most well-studied Campylobacter species, Campylobacter jejuni as references for BLASTp comparison. This project also investigates their oxidative and nitrosative stress mechanisms, which protect bacteria from reactive oxygen and nitrogen species generated by the electron transport chain during respiration and host immune defense.

This study found that key enzymes from glycolytic pathways, the tricarboxylic acid cycle and fucose use pathways were not identified in human-hosted Campylobacter species, providing a molecular basis for their non-saccharyolytic nature. Analysis of human-hosted Campylobacter species found that they had branched electron transport chains similar to C. jejuni, but with fewer possible electron donors and acceptors. Enzymes for use of hydrogen were encoded in all species. Unlike in C. jejuni, enzymes for use of lactate, sulfite and gluconate were not encoded in all species examined, and enzymes for use of fumarate, nitrite and tetrathionate were uncommon. Dimethylsulphoxide and fumarate were proven experimentally to be used as electron acceptors by Campylobacter concisus, as their addition to media significantly increased growth of C. concisus.

Likely fewer possible amino acids are used, as enzymes for use of proline, serine and branched chain amino acids were not encoded. Fewer oxidative and nitrosative stress defence genes were encoded, and the key enzyme catalase was not encoded in most species, suggesting that they are more sensitive to oxidative and nitrosative stress than C. jejuni. However, the nitric oxide reductase NorZ and nitrous oxide reductase NosZ previously reported in C. concisus are present in most species.

In summary, this project provides a molecular basis for the non-saccharolytic and hydrogen-dependent nature of human-hosted Campylobacter species, presenting insights into their growth requirements and pathogenicity.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents a non-exclusive licence to archive and to make available (including to members of the public) my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known. I acknowledge that I retain all intellectual property rights which subsist in my thesis or dissertation, such as copyright and patent rights, subject to applicable law. I also retain the right to use all or part of my thesis or dissertation in future works (such as articles or books).

…………………………………………………………… Signature 5/3/2020 ……….……………………...…….…………………………. Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years can be made when submitting the final copies of your thesis to the UNSW Library. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.

‘I hereby grant the University of New South Wales or its agents a non-exclusive licence to archive and to make available (including to members of the public) my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known. I acknowledge that I retain all intellectual property rights which subsist in my thesis or dissertation, such as copyright and patent rights, subject to applicable law. I also retain the right to use all or part of my thesis or dissertation in future works (such as articles or books).’

‘For any substantial portions of copyright material used in this thesis, written permission for use has been obtained, or the copyright material is removed from the final public version of the thesis.’

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Date …………………………17/06/2020……......

AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis.’

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Date …………………………17/06/2020……......

INCLUSION OF PUBLICATIONS STATEMENT

UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure.

Publications can be used in their thesis in lieu of a Chapter if: • The candidate contributed greater than 50% of the content in the publication and is the “primary author”, ie. the candidate was responsible primarily for the planning, execution and preparation of the work for publication • The candidate has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis

Please indicate whether this thesis contains published material or not: This thesis contains no publications, either published or submitted for publication ☐ (if this box is checked, you may delete all the material on page 2)

Some of the work described in this thesis has been published and it has ☐ been documented in the relevant Chapters with acknowledgement (if this box is checked, you may delete all the material on page 2)

This thesis has publications (either published or submitted for publication) incorporated into it in lieu of a chapter and the details are presented ☒ below

CANDIDATE’S DECLARATION I declare that: • I have complied with the UNSW Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis.

Candidate’s Name Signature Date (dd/mm/yy) Melissa Yeow 06/03/2020

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POSTGRADUATE COORDINATOR’S DECLARATION I declare that: • the information below is accurate • where listed publication(s) have been used in lieu of Chapter(s), their use complies with the UNSW Thesis Examination Procedure • the minimum requirements for the format of the thesis have been met. PGC’s Name PGC’s Signature Date (dd/mm/yy)

Michael Janitz 07/03/2020

For each publication incorporated into the thesis in lieu of a Chapter, provide all of the requested details and signatures required Details of publication #1: Full title: Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity Authors: Melissa Yeow, Fang Liu, Rena Ma, Timothy J. Williams, Stephen M. Riordan and Li Zhang

Journal or book name: Gut Pathogens Volume/page numbers: Volume 12 Article 13 Date accepted/ published:05/03/2020 Status Published X Accepted and In In progress press (submitted) The Candidate’s Contribution to the Work Conducted bioinformatics analysis, analyzed data and wrote manuscript Location of the work in the thesis and/or how the work is incorporated in the thesis: Chapter 2 PRIMARY SUPERVISOR’S DECLARATION I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Primary Supervisor’s name Primary Supervisor’s signature Date (dd/mm/yy) Li Zhang 06/03/2020

UNSW Authorship Declaration

Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity

In the case of the above-mentioned, paper contributions to the work involved the following:

Name Contribution Nature of Contribution (%)

Melissa 70 Conducted bioinformatics Yeow analysis, analyzed data and wrote manuscript

Timothy J. 7.5 Provided guidance on Williams bioinformation analysis, discussed data and co-edited manuscript

Fang Liu 7.5 Performed DMSO experiment, provided help with bioinformatic analysis and co-edited manuscript

Rena Ma 5 Performed fumarate experiment and co-edited manuscript

Stephen M. 5 Conceived the project and co- Riordan edited manuscript

Li Zhang 5 Conceived the project, discussed data and co-wrote manuscript

Declaration by co-authors

The undersigned hereby certify that: • they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field or expertise; • they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication; • there are no other authors of the publication according to these criteria; • potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or publisher of journals or other publications, and (c) the head of the responsible academic unit; and • the original data are stored at the following location(s) and will be held for at least five years from the date indicated below:

Location(s) 1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia 2 3

Name Signature Date

Melissa 19/1/20 Yeow

Timothy 19/1/20 J. Williams

Fang 19/1/20 Liu

Rena 19/1/20 Ma

Stephen 19/1/20 M. Riordan

Li 19/1/20 Zhang

Acknowledgements

I would like to take this opportunity to express my heartfelt gratitude to everyone who has supported me throughout the past two years.

I sincerely thank my supervisor Dr Li Zhang for all her patient guidance, sage advice and support during difficult times while figuring out all the details of this project. She is a great mentor and I am deeply grateful to her for making this experience possible.

I would like to thank Fang for all her helpful advice and guidance, .and for her help with the writing and submission of my first paper.

I would like to thank Tim for all his invaluable advice on bacterial respiratory enzymes and bioinformatics. I deeply appreciate it and couldn’t have done this project without your help.

Thank you, Ada, for being a great friend, for all your encouragement and support, and for sharing all the cute cat memes and snacks.

Thanks Richard, for the moral support and comic relief.

Thanks Peter, Seula, and Charlotte for going out for coffee with me and helping me stay sane when I was stressed about research.

Thanks to Alan and Eden for help with learning R for making all those figures, I couldn’t have done this without you.

I would also like to thank A/Prof Chris Marquis and Prof Lan Ruiting for being on my review panel and for their invaluable insight and advice on my project.

Lastly but definitely not least, I thank my parents for all their love, encouragement and of course, their financial support. Also, thanks to my brother, Mark, for your support and taking care of things while I wasn’t around to help. Thanks also to Aunty Yew Lan and Uncle Kok Kiong for all the love and support.

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Table of Contents

Originality Statement……………………………...…………………………………I Thesis/Dissertation Sheet/Abstract ……………………………..…………………..II Inclusion of Publications Statement……………….………...…………………...... III Authorship Declaration Form……………………………………………………….V Acknowledgements……………………………………………………………..…VII Table of Contents……..………………………………………………...………...VIII List of Tables………………………………………………………………………X List of Figures……………………………………………………………………XII List of Abbreviations………..…………………………………………………....XIV

Chapter 1: Introduction…………………………………..……………….…….……1 1.1 Campylobacter genus……………………………………..….………….1 1.2 Human-hosted Campylobacter species and their disease associations….7 1.2.1 Campylobacter concisus (C. concisus)………………………….…8 1.2.2 Campylobacter curvus (C. curvus)…………………………..…….9 1.2.3 Campylobacter gracilis (C. gracilis)……………………………..10 1.2.4 Campylobacter hominis. (C. hominis)………………………...….11 1.2.5 Campylobacter rectus (C. rectus)………………….……………..12 1.2.6 Campylobacter showae (C. showae)……………………………..13 1.2.7 Campylobacter ureolyticus (C. ureolyticus)……………………...14 1.3 Campylobacter jejuni (C. jejuni)……………...………………………..15 1.4 Central carbon metabolism in Campylobacter species………………....17 1.5 Electron transport chain in C. jejuni……………..……………………..21 1.6 Hydrogenases and their relationship to Campylobacter species….……24 1.7 Oxidative and nitrosative stress………………………………………...27 1.8 Hypothesis and aims………………………………………..…………..28

Chapter 2: Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity..………………………………29 (paper included in lieu of chapter)

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Chapter 3: Bioinformatics investigation of the molecular bases of the energy metabolisms of human-hosted Campylobacter species……………..………...……43 3.1 Introduction………………………………………………………...…..43 3.2 Materials and Methods…………………………………………………45 3.2.1 Bioinformatic methods used to examine the presence of genes encoding enzymes in energy metabolism and stress defence in human-hosted Campylobacter species………………………………………………….….45 3.3 Results………………………………………………………….………48 3.3.1 Glycolytic pathways and fucose metabolism pathways are incomplete in all human-hosted Campylobacter species examined, the TCA cycle is incomplete in most species examined, while acetate metabolism pathways are found in all species examined…………………………..……48 3.3.2 Human-hosted Campylobacter species may use fewer electron donors than C. jejuni………………………………………………………..53 3.3.3 Human-hosted Campylobacter species may use fewer electron acceptors than C. jejuni…………………………………………………….55 3.3.4 Human-hosted Campylobacter species may use amino acids than C. jejuni…………………………………………………………………….57 3.3.5 Human-hosted Campylobacter species have fewer enzymes to deal with oxidative and nitrosative stress than C. jejuni………………………..59 3.4 Discussion …………………………………………………..…………61 3.5 Conclusion……………………………………………………………...65

Chapter 4: General Discussion and Future Directions……………………………..66

References ………………………………………………………………………....70

List of Tables

Table 1-1: Campylobacter species, disease association and their host type………...…..3 Supplementary Table S1: NCBI locus tags for genes involved in central carbon metabolism in C. concisus……………………………………………………...………90 Supplementary Table S2: NCBI locus tags for genes involved in use of electron donors of C. concisus………………………………………………………………………..…94 Supplementary Table S3: Query genes and proteins from E. coli strain K-12 MG1655 for identification of genes and proteins in C. concisus central carbon metabolism pathways………………………………………………………………………………..97 Supplementary Table S4: Query genes and proteins from C. jejuni subsp. jejuni NCTC 11168 for identification of genes and proteins of C. concisus central carbon metabolism pathways………………………………………………………………………………100 Supplementary Table S5: NCBI locus tags for genes involved in use of electron acceptors in C. concisus……………………………………………………………….103 Supplementary Table S6: NCBI locus tags for genes involved in use of amino acids in C. concisus……………………………………………………………….……………105 Supplementary Table S7: Query genes and proteins from C. jejuni subsp. jejuni NCTC 11168, E. coli strain K-12 MG1655, and C. jejuni subsp. jejuni strain NCTC 81116 used to identify genes and proteins for amino acid use in C. concisus…………………..…106 Supplementary Table S8: NCBI locus tags for genes involved in oxidative stress in C. concisus……………………………………………………………………….....……107 Supplementary Table S9: Query genes and proteins from C. jejuni subsp. jejuni NCTC 11168 that were used to identify genes and proteins for oxidative and nitrosative stress defence in C. concisus……………………………………………………...…………108 Supplementary Table S10: C. concisus strains used in BLASTn analysis……………109 Supplementary Table S11: Genes encoding electron donors and acceptors investigated in C. concisus as referenced from C. jejuni subsp jejuni NCTC 11168 and C. jejuni subsp. jejuni strain 81116…………………………………………………………..…115 Supplementary Table S12: Additional references cited in Supplementary Tables 7, 9 and 11…………………………………………………………………………………119 Supplementary Table S13: NCBI locus tags for genes involved in central carbon metabolism in human-hosted Campylobacter species………………………………..122

Supplementary Table S14: NCBI locus tags for genes involved in amino acid use in human-hosted Campylobacter species………………………………………….…….128 Supplementary Table S15: NCBI locus tags for genes involved in electron donors in human-hosted Campylobacter species…………………………………………..……130 Supplementary Table S16: NCBI locus tags for genes involved in electron acceptors in human-hosted Campylobacter species………………………………………………..133 Supplementary Table S17: NCBI locus tags for genes involved in oxidative and nitrosative stress defence in human-hosted Campylobacter species……….…………135

List of Figures

Figure 1-1: Enzymes and their genes of central carbon metabolism pathways identified in E. coli…………………………………………………………………………...……19

Figure 1-2: Enzymes and their genes of central carbon metabolism pathways identified in C. jejuni. The figure illustrates the pathways of central carbon metabolism in E. coli…………………………………………………………………………………..….20

Figure 1-3: Respiratory enzymes and their genes identified in C. jejuni……...……….23

Figure 2-1: Enzymes and their genes of central carbon metabolism pathways identified in C. concisus…………………………………………………………………………...32

Figure 2-2: Respiratory enzymes and their genes identified in C. concisus…………....33

Figure 2-3: Genes for enzymes of amino acid use pathways identified in C. concisus..34

Figure 2-4: Genes for enzymes of oxidative stress defence pathways identified in C. concisus………………………………………………………………………………...34

Figure 2-5: The effects of sodium fumarate on C. concisus growth under anaerobic conditions with and without H2 gas…………………………………………………….35

Figure 2-6: The effects of dimethyl sulphoxide (DMSO) on C. concisus growth……..36

Figure 3-1: Genes for enzymes of the Pentose Phosphate pathway identified in human- hosted Campylobacter species………………………………………………………….48

Figure 3-2: Genes for enzymes of the Entner Doudoroff pathway identified in human- hosted Campylobacter species………………………………………………………….49

Figure 3-3: Genes for enzymes of the Embden Meyerhof pathway identified in human- hosted Campylobacter species………………………………………………………….50

Figure 3-4: Genes for enzymes of the Tricarboxylic Acid Cycle identified in human- hosted Campylobacter species………………………………………………………….51

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Figure 3-5: Genes for enzymes of the acetate metabolism pathways identified in human- hosted Campylobacter species………………………………………………………….52

Figure 3-6: Genes for enzymes of the fucose metabolism pathways identified in human- hosted Campylobacter species………………………………………………………….52

Figure 3-7: Genes for enzymes of the electron donor pathways identified in human- hosted Campylobacter species………………………………………………………….54

Figure 3-8: Genes for enzymes of the electron acceptor pathways identified in human- hosted Campylobacter species………………………………………………………….56

Figure 3-9: Genes for enzymes of the amino acid use pathways identified in human- hosted Campylobacter species………………………………………………………….58

Figure 3-10: Genes for enzymes of the oxidative stress defence pathways identified in human-hosted Campylobacter species…………………………………………………59

Figure 3-11: Genes for enzymes of the nitrosative stress defence identified in human- hosted Campylobacter species………………………………………………………….60

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List of abbreviations

2-KG 2-ketoglutarate

DMSO dimethyl sulphoxide

ED pathway Entner Doudoroff pathway

EMP pathway Embden Meyerhof Parnas pathway

ETC Electron transport chain

H2 hydrogen gas

TCA cycle Tricarboxylic Acid cycle/Krebs cycle

PCR Polymerase Chain Reaction

PP pathway Pentose Phosphate pathway

ROS Reactive Oxygen Species

RNS Reactive Nitrogen Species

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Introduction

1.1 Campylobacter genus

Campylobacter species are Gram negative, curved rod-shaped bacteria which require microaerobic to anaerobic conditions for growth (1). They are generally motile with a single polar flagellum, except for Campylobacter gracilis and Campylobacter hominis which are known to be non-motile and Campylobacter showae which has a unipolar bundle of flagella (2, 3). Currently, the Campylobacter genus is known to contain 32 species and many of which have been discovered in the last decade and are constantly being discovered (4). While most of the Campylobacter species commensally inhabit the gastrointestinal tract of various animals, some use humans as their natural host and are referred to as human hosted Campylobacter species (5). Many Campylobacter species have been associated with various diseases in humans and animals. The species comprising the Campylobacter genus, their hosts, and their disease associations are listed in Table 1-1.

Campylobacter species are generally considered to be non-saccharyolytic, not being able to oxidise or ferment carbohydrates (1). Thus, they are considered to use amino acids and tricarboxylic acid cycle intermediates as main energy sources (1). However, this dogma has been challenged by studies in the last decade, as it has been found that

C. jejuni subsp. doylei 269.79 has the ability to use glucose via an alternative pathway of glycolysis, the Entner Doudoroff (ED) pathway (6), and that certain strains of C. jejuni can utilize fucose via an yet unclear pathway (7, 8). The fucose permease (fucP) gene was found in 30.3% of 710 C. jejuni isolates, suggesting that the ability to utilize l-

fucose is common in C. jejuni (9). Significantly, C. jejuni mutants lacking the FucP permease showed a competitive disadvantage in colonization of both chickens and piglets (8). The ED pathway has also recently been found in some strains of

Campylobacter coli (10), and in Campylobacter hepaticus (11). In light of these findings, further research into use of carbohydrates by Campylobacter species seems warranted.

Table 1-1: Campylobacter species, disease association and their host type Campylobacter species Disease Association Type of host References

Campylobacter avium No case reported Animal: chickens and turkeys (1, 12, 13)

Campylobacter armoricus Gastroenteritis in humans Isolated from humans with (4) gastroenteritis and river water samples Campylobacter blaseri No case reported Animal: seals (14)

Campylobacter canadensis No case reported Animal: whooping cranes (15)

Campylobacter coli Enteritis, abortion and septicaemia in Animal: chickens, ducks, turkeys, (16-28) humans, enteritis in dogs wild birds, dogs, pigs, cattle, marmosets, sheep and ostriches Campylobacter corcagiensis No case reported Animal: lion-tailed macaques (29)

Campylobacter concisus Inflammatory bowel disease, periodontal Human oral cavity (1, 19, 30-34) disease, septicaemia, Barrett’s oesophagitis and foot ulcers in humans, enteritis in dogs Campylobacter cuniculorum No case reported Animal: rabbits (1)

Campylobacter curvus Hepatic abcesses, alveolar abscesses, and Human oral cavity (32, 35-37) gastroenteritis in humans Campylobacter fetus subsp. Septicaemia, meningitis, vascular infection, Animal: cattle, dogs, sheep and (1, 17, 26, 38-42) fetus and abortion in humans turtles Campylobacter fetus subsp. Septicaemia, enteritis and pulmonary Animal: skinks, snakes and turtles (1, 17, 43-45) testudinum oedema in humans

Table 1-1 (continued)

Campylobacter species Disease Association Type of host References

Campylobacter fetus subsp. Septicaemia in humans, infectious infertility Animal: cattle and sheep (1, 26, 46-49) venerealis in cattle Campylobacter geochelonis No case reported Animal: tortoises (50)

Campylobacter gracilis Septicaemia in humans, enteritis in dogs Human oral cavity, Animal: dogs (1, 19, 33, 51)

Campylobacter helveticus No case reported Animal: dogs, cats and pigs (1)

Campylobacter hepaticus Spotty liver disease in chickens Animal: wild birds and rats (52, 53)

Campylobacter hominis No case reported Human intestinal tract (1, 32)

Campylobacter Enteritis, proctitis and septicaemia in Animal: pigs, cattle, sheep, (1, 17, 21, 49, 54-58) hyointestinalis subsp. humans, enteritis in reindeer hamsters, monkeys, elephants and hyointestinalis reindeer Campylobacter Enteritis in humans Animal: pigs, chickens, birds and (1, 59) hyointestinalis subsp. cattle lawsonii Campylobacter iguaniorum No case reported Animal: bearded dragons, iguanas (60) and tortoises Campylobacter Enteritis and septicaemia in humans Animal: seals and porpoises (1, 61) insulaenigrae Campylobacter jejuni subsp. Enteritis and septicaemia in humans Unknown (1, 32, 62, 63) doylei

Table 1-1 (continued)

Campylobacter species Disease Association Type of host References Campylobacter jejuni subsp. Enteritis, septicaemia, abortion, Animal: cattle, sheep, chickens, (1, 16, 18, 20, 21, 26, jejuni appendicitis, colitis, myocarditis, reactive turkeys, dogs, cats, mink, ferrets, 27, 42, 64-76) arthritis, Reiter’s syndrome and Guillain- thrushes, sparrows, crows and Barré syndrome in humans; spontaneous non-human primates abortion in cattle and sheep, gastroenteritis in dogs and cats Campylobacter lanienae No case reported Animal: cattle, pigs (1, 49, 77, 78)

Campylobacter lari subsp. No case reported Animal: shellfish and seagulls (1) concheus Campylobacter lari subsp. Enteritis and septicaemia in humans, Animal: mussels, oysters, dogs (19, 21) lari enteritis in dogs and horses Campylobacter mucosalis Enteritis in dogs Animal: pigs (1, 19)

Campylobacter ornithocola No case reported Animal: wild birds (79)

Campylobacter peloridis No case reported Animal: shellfish (1)

Campylobacter No case reported Animal: seals (80) pinnipediorum Campylobacter rectus Ulcerative colitis, periodontitis in humans Human oral cavity (1, 32, 33, 81) Campylobacter showae Intraorbital abscesses, septicaemia, Human oral cavity (1, 32, 33, 82-84) gingivitis, periodontitis, cholangitis and inflammatory bowel disease in humans Campylobacter sputorum No case reported Cattle and sheep (1, 85) biovar faecalis

Table 1-1 (continued)

Campylobacter species Disease Association Type of host References Campylobacter sputorum Gastroenteritis, abcesses Human gastrointestinal tract, (1, 49, 85-89) biovar sputorum Animals: dogs, cattle Campylobacter sputorum No case reported Animals: cattle (85, 90) biovar paraureolyticus Campylobacter No case reported Animal: penguins and albatrosses (1, 91) subantarticus Campylobacter troglodytis No case reported Animal: chimpanzees (1, 92)

Campylobacter upsaliensis Enteritis, septicaemia, abortion, breast Animal: cats, dogs, ducks and (1, 21) abscesses and Guillain-Barre syndrome in monkeys humans, and gastroenteritis in dogs and cats Campylobacter ureolyticus Ulcerative colitis, genital tract disease and Human oral cavity, Animal: cats, (20, 21, 34, 93-100) gastroenteritis in humans cattle, dogs, pigs Campylobacter volucris Bacteremia in humans Animal: black-headed gulls and (1, 101) crows

1.2 Human-hosted Campylobacter species and their disease associations

There are seven known human hosted Campylobacter species, namely Campylobacter concisus, Campylobacter curvus, Campylobacter gracilis, Campylobacter hominis,

Campylobacter rectus, Campylobacter showae and Campylobacter ureolyticus (5).

Interestingly, all these species have been reported to require hydrogen for growth, and all of these grow at 37◦C under anaerobic conditions but not under aerobic conditions

(80). Fully sequenced genomes are available for only C. concisus, C. curvus, C. gracilis, C. hominis, C. showae and C. ureolyticus of the abovementioned species. Most of the reported diseases related to human-hosted Campylobacter species are inflammatory diseases of the human gastrointestinal tract (Table 1-1). It has also been suggested that the tendency for Campylobacter species to enter viable but nonculturable states under stress conditions (102), as well as the difficulty of isolating hydrogen- requiring bacteria using standard culture techniques currently employed in most routine diagnostic laboratories (103) may have led to failure to detect these Campylobacter species, causing their underreporting. This is supported by studies that have detected higher rates of Campylobacter using non-culture methods such as PCR as compared to culture methods (104, 105), and a study in 2019 which found that culture methods fail to correctly detect Campylobacter in 30% of positive patient stools compared to non- culture methods (106). Despite their observed disease associations and the view of many of them as emerging pathogens, detailed and comprehensive information of their molecular pathways involving energy metabolism and stress defense is unavailable.

1.2.1 C. concisus

Campylobacter concisus (C. concisus) is a fastidious, non-saccharolytic slow-growing

Gram-negative, curved, bacterium of the oral cavity with a single polar flagellum. It requires an anaerobic environment, or a microaerophilic environment enriched with H2 for growth, with an optimum growth temperature of 37◦C. It has also been found to be oxidase positive and catalase negative, which may contribute to its anaerobic nature (30,

107-109). Using the Hugh-Leifson Oxidative-Fermentative test, it was found that 6 out of 6 strains tested of C. concisus produced the end products hydrogen sulfide, succinate and hydrogen, and 3 out of 6 strains produced acetate as detected by gas-liquid chromatography (>4mM more than control) (30).

C. concisus naturally colonizes the human oral cavity with a high prevalence and has been isolated from saliva samples of healthy individuals. Zhang et al. found C. concisus via bacterial culture and 16S rRNA gene polymerase chain reaction (PCR) in saliva samples from 97% of the healthy individuals (57/59) and 100% of the patients with inflammatory bowel disease tested (107), and Petersen et al. detected C. concisus in

100% of saliva samples (11/11) collected from healthy individuals by PCR targeting the

16S rRNA gene (34).

Despite this, C. concisus is not a dominant oral bacterial species (110). Interestingly, in contrast to the high isolation rate of C. concisus from saliva samples, C. concisus had low detection rates from faeces or intestinal biopsies from healthy individuals. Engberg et al. examined the presence of C. concisus by 23S rDNA PCR and found C. concisus

DNA in faecal samples collected in only 3 out of 107 healthy individuals (2.8 %) (111).

Similarly, Nielsen et al. did not isolate C. concisus from any of 108 fecal samples

collected from healthy individuals (112). The low isolation rates of C. concisus from fecal samples suggest that the gastrointestinal tract of healthy individuals is a less optimal site than the oral cavity for C. concisus colonization.

C. concisus has been associated with gingival and diarrheal disease and is often isolated from diarrheal faecal samples (111-113). It has also been associated with IBD, (98, 99,

107, 111, 114, 115) and there is evidence that C. concisus may cause damage to the intestinal epithelial barrier via phospholipase A (116), prophage-encoded zonula occludens toxin (117, 118), and a secreted enterotoxin B homologue, Csep1, which has been associated with active Crohn’s Disease (31). A significantly higher prevalence of

C. concisus was detected using PCR in both intestinal biopsies and faecal samples collected from patients with Inflammatory Bowel Disease as compared to controls (98,

99, 111, 114, 115).

H2 has been found to be a critical requirement for C. concisus growth, acting as an electron donor for energy metabolism (28). Lee et al demonstrated that without H2, C. concisus grew very slowly under anaerobic conditions and did not grow under microaerobic conditions (119). Despite these phenotypic observations, detailed and comprehensive information of the molecular pathways involving energy metabolism and stress defense in C. concisus is unavailable.

1.2.2 C. curvus

C. curvus (formerly Wolinella curva) is a oxidase positive, catalase-negative anaerobic bacterium that displays rapid darting motility and produces formate and acetate during growth (120). It is described as using hydrogen and formate as energy sources, and requiring formate and fumarate for broth culture (120). It is a rarely encountered

Campylobacter species in humans, being only isolated in 0.05% (2/4122) of stool samples from pediatric patients with diarrhea (28). C. curvus has also been associated with an outbreak of bloody gastroenteritis and Brainerd’s diarrhea in Northern

California, and hepatic and alveolar abscesses (35, 36).

1.2.3 C. gracilis

C. gracilis (formerly known as Bacteroides gracilis) is a non-motile oxidase-negative bacterium unlike most Campylobacters, is anaerobic and catalase negative, and has been reported to produce succinate (7 of 7 strains) and acetate (1 of 7 strains) (1, 30,

121). 7 out of 7 strains of C. gracilis were reported to have their growth stimulated by presence of formate and fumarate, and were found to reduce nitrite (30). C. gracilis has been isolated from patients with periodontal and endodontic infections (30, 33).

However, its role in pathogenesis is controversial. Macuch et al. reported that C. gracilis was the most dominant Campylobacter species other than C. rectus isolated from subgingival sites among eight Campylobacter species examined, but also found that similar levels of C. gracilis were detected in healthy and diseased sites (33).

Additionally, a study of primary endodontic infections found that prevalence of C. gracilis was not associated with clinical symptoms (122). The fully sequenced genome of C. gracilis encodes virulence factors such as zonula occludens toxin, haemagglutinins, immunity proteins and other putative pathogenic factors (123). C. gracilis has also been linked to bacteremia (51, 124), however more research is warranted to provide evidence for the virulence of C. gracilis.

1.2.4 C. hominis

C. hominis is a non-motile aflagellate bacterium isolated from fecal samples of healthy humans that achieves optimal growth at 37◦C under anaerobic conditions, and has been reported to be oxidase-positive and catalase-negative (3). C. hominis commensally colonizes the human gastrointestinal tract, unlike the other human-hosted

Campylobacter species which colonize the oral cavity(5). It has also been noted to be bile-resistant and able to grow in 2% bile as is C. jejuni (3). It is non-hemolytic and shows no growth under aerobic conditions at 25◦C or 37◦C (3). It has not been associated with any disease in humans.

1.2.5 C. rectus

C. rectus (formerly named Wollinella recta) is a single polar flagellated bacterium (30) that grows under anaerobic conditions at 30◦C, 35◦C and 42◦C, but did not grow under microaerobic conditions (125). It was also reported to produce formate and acetate during growth (120). It is catalase-negative, oxidase positive and produces hydrogen sulphide in Triple Sugar Iron Agar (2, 80). It has been associated with ulcerative colitis and perionditis in humans (32, 33, 81).

1.2.6 C. showae

C. showae is a catalase-positive motile bacterium with multiple unipolar flagella that was isolated from the human oral cavity (82). It has been reported to prefer an anaerobic environment to a microaerobic environment for growth, and its growth is stimulated by formate and fumarate (82). It has been associated with intraorbital abscesses, septicaemia, gingivitis, periodontitis, cholangitis and inflammatory bowel disease in humans (1, 32, 33, 81-84).

1.2.7 C. ureolyticus

C. ureolyticus (formerly Bacteroides ureolyticus) is an oxidase-positive non-motile bacterium that does not grow microaerobically without hydrogen, and grows anaerobically in media supplemented with formate and fumarate (93). It grows at 30°C and 37°C, but not at 25°C; strain-dependent growth at 42°C, and grows on media containing 0.1 % trimethylamine-N-oxide (93). It has also been reported to have urease which is not found in any other Campylobacter species except for Campylobacter pinnipediorium (80). It is an emerging pathogen (100), that has been detected in unpasteurized milk and bovine feces (96). It has been associated with ulcerative colitis, genital tract disease and gastroenteritis in humans (32, 95, 97, 100).

1.3 C. jejuni

C jejuni colonizes the avian gut commensally but is a known human pathogen that is the leading cause of bacterial gastroenteritis in both developing and developed countries

(126-128). Gastroenteritis as a result of Campylobacter infection (campylobacteriosis) has been reported to mostly be due to consumption of undercooked or contaminated chicken and other meat products (129-131).

C. jejuni contains two subspecies, C. jejuni subsp. jejuni and C. jejuni subsp. doylei which can be differentiated by the inability of subsp. doylei to use nitrate (132). Most cases of campylobacteriosis are caused by C. jejuni subsp. jejuni (133). Isolation of C. jejuni subsp. doylei is infrequent, but it is associated with not only gastroenteritis but also bacteremia (62, 134).

As C. jejuni is a well-known pathogen and is the most well studied member of the

Campylobacter species (135, 136), it is used in this study as a reference for energy metabolism pathways. C. jejuni grows under microaerobic conditions. Although it can utilize hydrogen gas (H2) as an electron donor, H2 is not a necessary requirement for C. jejuni growth unlike for the human-hosted Campylobacters (80), showing that C. jejuni is able to use other electron donors for energy metabolism (28).

C. jejuni has a complex branched respiratory chain, being able to make use of a wide range of electron donors and acceptors (136), more so than the related pathogen H. pylori (137). The details of the respiratory electron transport chain of C. jejuni are further discussed in the Section 1.5 below.

1.4 Central carbon metabolism in Campylobacter species

Campylobacter species in general have been historically considered to be non-

saccharyolytic, as they were not known to oxidise or ferment carbohydrates (2).

Tanner et al. 1981 showed that Campylobacter species (including C. concisus and

C. jejuni) failed to produce acid from a variety of sugars, including glucose, xylose,

mannitol, lactose, sucrose, maltose, fructose, galactose, arabinose, cellobiose,

mannose, , ribose, sorbitol, starch and trehalose (30). They were considered unable

to catabolise various sugars, especially glucose, due to the specific lack of enzymes

such as glucokinase (Glk) and phosphofructokinase (PfkA) of the classical Embden-

Meyerhof-Parnas (EMP) glycolysis pathway (138, 139). The lack of these enzymes

results in Campylobacter species having very limited carbohydrate catabolisms

compared to many other enteric pathogens (135, 136).

However, studies in the past decade have changed this view. C. jejuni subsp doylei

269.79 was found to have the ability to use glucose via an alternative pathway of

glycolysis, the Entner Doudoroff (ED) pathway (6, 140). The ED pathway has also

recently been found in some strains of Campylobacter coli, which is also a zoonotic

enteric pathogen (10, 140). In addition, C. jejuni subsp. jejuni strains with the

genomic island (cj0480-cj0490) were found to have the ability to utilize fucose for

growth (7, 8) and the mutation of cj0486 also demonstrated a competitive

disadvantage when colonizing the piglet model of human disease (8). Thus, it would

seem apt to further investigate the dogma of the non-saccharyolytic nature of

Campylobacter species, given the increasing evidence against it.

In general, carbohydrates are metabolized by a series of integrated pathways of carbon source transport and oxidation referred to as the central carbon metabolism

(CCM). The main pathways of the CCM are the three major interlinked glycolytic pathways: the Embden-Meyerhof-Parnas (EMP) pathway, Entner Doudoroff (ED) pathway, and the Pentose Phosphate (PP) pathway and the tricarboxylic acid cycle

(TCA) with the glyoxylate bypass. The CCM pathways are most well studied in

Escherichia coli, and thus E. coli is used as a reference in this project (141). These pathways in E. coli and their connection with the TCA cycle and glyoxylate cycle are shown in Figure 1-1.

In addition, C. jejuni is the most well-studied Campylobacter species, and thus it is used as an additional reference for comparison with Campylobacter species. These pathways in C. jejuni and their connection with the TCA cycle and glyoxylate cycle are shown in Figure 1-2. Glycolysis is the first step of carbohydrate metabolism, from which pyruvate is generated. Pyruvate undergoes oxidative carboxylation to form acetyl coA, which then enters the Tricarboxylic acid (TCA) cycle or the glyoxylate cycle. The glyoxylate cycle is a modified TCA cycle, in which isocitrate is converted to malate, bypassing 2-ketoglutarate (2-KG) and succinyl-CoA (142).

As a result of these processes, ATP is generated either directly by substrate level phosphorylation, or as a result of electrons being donated to the electron transport chain (ETC) by NADH and FADH2 generated in the TCA cycle or intermediate metabolites in the TCA cycle such as succinate, generating a proton motive force used to synthesize ATP molecules via ATP synthase (141).

Figure 1-1: Enzymes and their genes of central carbon metabolism pathways identified in E. coli. The figure illustrates the pathways of central carbon metabolism in E. coli. The enzymes in this figure and their functions are further described in Table S3. Legend: glk=glucokinase, pgi=phosphoglucoisomerase, pfkAB=phosphofructokinase, fbaA=fructose biphosphate aldolase A, tpiA=triosephosphate isomerase A, gapA=glyceraldehyde-3-phosphate dehydrogenase A, pgk=phosphoglycerate kinase, gpmA=phosphoglycerate mutase, eno=enolase, pykAF=pyruvate kinase, lpd=dihydrolipoyl dehydrogenase, aceEF=Pyruvate dehydrogenase component E1, gltA=citrate synthase, acnB=aconitase, icd=isocitrate dehydrogenase, sucAB, lpdA=2-oxoglutarate dehydrogenase complex, sucCD=succinyl-co-A synthase, sdhABCD=succinate dehydrogenase, fumABC=fumarate hydratases, mdh=malate dehydrogenase, mqo=malate:quinone oxidoreductase, ackA=acetate kinase, pta=phosphate acetyltransferase, acs= acetyl-coenzyme A synthetase, gdh=glucose dehydrogenase, zwf= glucose-6-phosphate dehydrogenase, gnd= 6-phosphogluconate dehydrogenase, rpe= phosphopentose epimerase, rpiB= ribose-5- phosphate isomerase, tktAB= transketolase, talAB= transaldolase, pgl= 6-phosphogluconolactonase, edd= phosphogluconate dehydratase, eda= 2-dehydro-3-deoxy-phosphogluconate aldolase, pckA= phosphoenolpyruvate, G6P = glucose-6-phosphate, F6P = fructose-6-phosphate, F1,6BP = fructose 1,6- bisphosphate, GAP = glyceraldehyde-3-phosphate, 1,3-BPG = 1,3-bisphosphoglycerate, 3PG = 3- phosphoglycerate, 2PG = 2-phosphoglycerate, PEP = phosphoenolpyruvate, ribulose 5P = ribulose 5-phosphate, ribose 5P = ribose 5-phosphate, xylulose 5P = xylulose 5-phosphate, sedoheptulose 7P = sedoheptulose 7- phosphate, erythrose-4P = erythrose 4-phosphate, 6P gluconate = 6-phosphogluconate, OAA = oxaloacetate, 2- KG = 2-oxoglutarate carboxykinase

Figure 1-2: Enzymes and their genes of central carbon metabolism pathways identified in C. jejuni. The figure illustrates the pathways of central carbon metabolism in E. coli. The enzymes in this figure and their functions are further described in Table S4. Legend: glk=glucokinase, pgi=phosphoglucoisomerase, pfk=phosphofructokinase, fbaA=fructose biphosphate aldolase A, tpiA=triosephosphate isomerase A, gapA=glyceraldehyde-3-phosphate dehydrogenase A, pgk=phosphoglycerate kinase, pgm=phosphoglycerate mutase, eno=enolase, pyk=pyruvate kinase, gltA=citrate synthase, acnB=aconitase, icd=isocitrate dehydrogenase, oorABCD=2-oxoglutarate oxidoreductase complex, sucCD=succinyl co-A synthase, frdABC=dual-functioning succinate/fumarate oxidoreductase, mfrABE=methylmenaquinol : fumarate reductase, fumC=fumarate hydratase, mdh=malate dehydrogenase, mqo=malate:quinone oxidoreductase, ackA=acetate kinase, pta=phosphate acetyltransferase, acs= Acetyl- coenzyme A synthetase, gdh=glucose dehydrogenase, zwf= glucose-6-phosphate 1-dehydrogenase, gnd= 6- phosphogluconate dehydrogenase, rpe= phosphopentose epimerase, rpiB= ribose-5-phosphate isomerase, tkt= transketolase, tal= transaldolase, pgl= 6-phosphogluconolactonase, edd= phosphogluconate dehydratase, eda= 2- dehydro-3-deoxy-phosphogluconate aldolase, pckA= phosphoenolpyruvate carboxykinase, G6P = glucose-6- phosphate, F6P = fructose-6-phosphate, F1,6BP = fructose 1,6-bisphosphate, GAP = glyceraldehyde-3-phosphate, 1,3-BPG = 1,3-bisphosphoglycerate, 3PG = 3-phosphoglycerate, 2PG = 2-phosphoglycerate, PEP = phosphoenolpyruvate, ribulose 5P = ribulose 5-phosphate, ribose 5P = ribose 5-phosphate, xylulose 5P = xylulose 5-phosphate, sedoheptulose 7P = sedoheptulose 7-phosphate, erythrose-4P = erythrose 4-phosphate, 6P gluconate = 6-phosphogluconate, OAA = oxaloacetate, 2-KG = 2-oxoglutarate *=gene found in C. jejuni subsp. doylei 269.97

1.5 Electron transport chain (ETC) of C. jejuni

C. jejuni has been observed to make use of a wide range of electron donors and acceptors and to be able to survive in a variety of hosts, commensally colonizing a range of avian hosts, and pathogenically colonizing a range of mammalian hosts, including humans (136,

143). In order to successfully infect and colonize this wide range of hosts, C. jejuni must be able to survive a range of environmental stresses before being ingested by potential hosts via water or food. This suggests that C. jejuni must have a considerable degree of metabolic flexibility to make use of the different metabolites that are available in each environmental niche.

The array of electron donors and acceptors that make up the ETC used in C. jejuni as well as their relevant enzymes are summarized in Figure 1-3. C. jejuni has been found to use eleven electron donors and seven electron acceptors. These electron donors comprise of

2-oxoglutarate (144), flavodoxin (instead of NADH at complex I) (144), formate (145,

146), fumarate (147), gluconate (140, 148), hydrogen (145, 149), lactate (150), malate

(146), pyruvate (151), succinate (147) and sulphite (152).

The electron acceptors used by C. jejuni include fumarate (147, 153) , nitrate (154), nitrite

(155), oxygen (156), SN-oxides such as TMAO (trimethylamine N-oxide) and DMSO

(dimethyl sulfoxide) (157), and tetrathionate (158). The various electron donors, via their respective enzyme complexes, donate electrons to the quinone pool. The electrons from the quinone pool are then transferred to the electron acceptors via their enzymes, generating a proton motive force that is used to synthesize ATP.

In addition to using electron donors as energy sources, C. jejuni has been reported to make use of amino acids and TCA cycle intermediates for energy (135, 136). These pathways

are interlinked as some TCA cycle intermediates such as malate and fumarate function as electron donors or acceptors respectively. In addition, amino acids can be converted into

TCA cycle intermediates eg. aspartate can be converted into fumarate by aspartate ammonia lyase and can enter the TCA cycle and be used in energy metabolism. The wide variety of energy sources available to C. jejuni illustrate its high degree of metabolic flexibility and its adaptability to survive various environments (136).

1.6 Hydrogenases and their relationship to Campylobacter species

Hydrogenases are metalloenzymes that catalyze the reversible conversion of molecular hydrogen to protons and electrons. Hydrogenases are grouped into 3 main classes based on the metal content of their active sites, being [FeFe], [NiFe] and [Fe-only].

Hydrogenases can be hydrogen-producing, hydrogen-oxidizing, or bi-directional (159).

Hydrogenases are generally inhibited by oxygen, though certain NiFe hydrogenases are known to be more oxygen-tolerant (160).

The uptake and oxidation of H2 for use as an energy source is a widespread trait amongst pathogenic bacteria (161, 162). H. pylori has Ni-Fe type uptake hydrogenases that they rely on for energy metabolism, as does Salmonella enterica serovars Typhi and

Typhimurium, Escherichia coli 0157 (E. coli), Shigella (flexneri and sonnei) and

Campylobacter jejuni (161). The hydrogen-utilizing capability of these bacteria is concluded based on their genomes containing structural genes for the membrane-bound hydrogenase and for shuttling of those electrons to quinone or haem binding proteins, accessory proteins for the NiFe hydrogenase enzymes’ maturation, and also the complete respiratory electron transport chain (normally used in common by H2 and other low potential electrons donors), including more than one O2-binding terminal oxidase. (163)

It was found that mutant strains of H. pylori unable to use H2 are deficient in colonizing mice compared with the parent strain (161) and that respiratory use of H2 was necessary for virulence and colonization of the gut by Salmonella enterica serovar Typhimurium

(163, 164). As such, it is theorized that a Ni-Fe hydrogen-uptake type hydrogenase inhibitor would make an effective novel narrow-spectrum antimicrobial agent that

specifically targets certain hydrogen-utilizing pathogenic bacteria in the gut. Indeed, many of the abovementioned bacteria were found in a targeted search in tissues of patients with IBD (165).

In contrast with the abovementioned bacteria, C. concisus has been found to require hydrogen for survival, as a previous study from our lab found that all 63 strains of C. concisus tested were completely unable to grow in microaerobic conditions in the absence of hydrogen (119). This is supported by the reported presence of the Ni-Fe type hydrogen-oxidising hydrogenase (hyd gene family: hydEDCBA) and the required associated accessory proteins (hyp gene family: hypAEDCB, nikR, hypF) in the strains

C. concisus strains 13826 and 51562 (166).

The Ni-Fe type hydrogen-producing hydrogenase hyf (hyf gene family: hyfABCEFGHI, hycH, hycI, focA) has also been reported in C. concisus strains 13826 and 51562 (166).

The possibility that C. concisus is able to produce hydrogen for its own use may help its survival in a low-hydrogen environment such as the oral cavity. This is supported by a recent study being unable to produce successful hypE and hydB mutants, despite being able to produce hyfB mutants which had similar growth rates to wild type though they had no hydrogen production (166). This suggests that hydrogen uptake, but not hydrogen production is essential for C. concisus growth under the conditions tested.

The same study also found that C. concisus strains 13826 and 51562 were unable to grow in microaerobic conditions without hydrogen. This is similar to the previous findings from our lab, where 57 oral strains and 7 enteric strains of C. concisus were

unable to grow in microaerobic conditions without hydrogen, but addition of 5% hydrogen gas increased growth significantly (119).

In addition, all human-hosted Campylobacter species have been reported to require hydrogen gas for growth (80). In light of the importance of hydrogen to human-hosted

Campylobacter species, research appears to be warranted into the use of hydrogen by human-hosted Campylobacter species.

1.7 Oxidative and nitrosative stress

Oxidative and nitrosative stress defence mechanisms play a vital role in the survival of bacteria against host immune defence systems. Macrophages, neutrophils and inflammatory monocytes of the innate immune system phagocytose and expose bacteria to harmful compounds such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) in order to kill pathogenic bacteria (167).

In addition, oxidative stress is generated both exogenously by the environment or host immune defence system eg. through the release of ROS by macrophages to kill bacteria, and endogenously, from the electron transport chain as a result of respiration (168).

Furthermore, aerotolerance, or the ability of bacteria to survive in high oxygen environments, is affected by the ability of a bacteria’s oxidative stress defence mechanisms to neutralize ROS and is important for bacteria to survive transfer between hosts and to cause zoonoses such as campylobacteriosis (169).

It has been previously suggested that species such as C. avium which do not encode specific oxidative and nitrosative stress defence proteins such as MsrAB and Cgb may have lower aerotolerance and be more sensitive to oxidative and nitrosative stress (13).

Thus, investigation of these oxidative and nitrosative stress defence proteins is warranted in order to have a better understanding of factors affecting growth and survival of Campylobacter species.

1.8 Hypothesis and aims

Previous studies have shown that C. concisus is non-saccharolytic and dependent on hydrogen for growth. However, few studies have looked at the energy metabolism of C. concisus, and limited information is available about the energy sources used by C. concisus and the molecular basis of its energy metabolism. There is a similar lack of in- depth research into the molecular basis of the energy metabolisms of other human- hosted Campylobacter species.

The hypothesis of this project is that the energy metabolism pathways of C. concisus and other human-hosted Campylobacter species differ from the well-known human pathogen C. jejuni, which explains their hydrogen-dependent and non-saccharolytic nature.

The specific aims of this project are:

1. To investigate the energy metabolism pathways of C. concisus

2. To investigate the energy metabolism pathways of other human hosted

Campylobacter species

2.1 Analyses of energy metabolism and stress defence provide insights into

Campylobacter concisus growth and pathogenicity

(paper in lieu of chapter)

Chapter 3: Bioinformatics investigation of the molecular basis of the energy metabolism of human-hosted Campylobacter species

3.1 Introduction

While there are many species belonging to the Campylobacter genus, most of these species are animal-hosted rather than human-hosted (5). There are seven currently known human-hosted Campylobacter species, Campylobacter concisus, Campylobacter curvus, Campylobacter hominis, Campylobacter gracilis, Campylobacter rectus,

Campylobacter showae, and Campylobacter ureolyticus, all of which have clinical relevance (5). These species have been linked to various diseases in humans, in particular gastrointestinal diseases (Table 1-1).

Interestingly, all these species are known to require hydrogen for growth, while the majority of Campylobacter species do not require hydrogen (80). Additionally, human- hosted Campylobacter species are more often involved in chronic inflammatory conditions of the gastrointestinal tract, in contrast with animal hosted Campylobacter species (5). Most human-hosted Campylobacter species use the oral cavity as their natural colonization site, and only C. hominis naturally colonizes the human intestinal tract (3). It is possible that the variable availability of energy sources such as hydrogen in these different colonization sites plays a role in determination of their growth and pathogenicity.

The findings in Chapter 2 demonstrated that the dependence of C. concisus on hydrogen for growth is likely due to a scarcity of other sources of energy due to its limited choice of electron donors and non-saccharyolytic nature. There has been scarcity of information about the molecular bases of energy metabolism and stress defence of human-hosted Campylobacter species in general. Thus, this chapter investigates the energy metabolism and stress defence pathways of all the human-hosted Campylobacter species, in addition to C. concisus, to further examine the molecular bases for hydrogen- dependent and non-saccharolytic natures of human-hosted Campylobacter species, and the consequences on their pathogenicity.

3.2 Materials and Methods

3.2.1 Bioinformatic methods used to examine the presence of genes encoding enzymes in energy metabolism and stress defence in human hosted Campylobacter species

The sequences of the reference genes from reference bacterial strains (see the section below) were obtained from the National Center for Biotechnology Information (NCBI) database. Proteins encoded by these genes were used as query sequences to identify similar proteins in the six human-hosted Campylobacter species with fully sequenced genomes from the National Center for Biotechnology Information (NCBI) database (C. curvus 525.92 (NC_009715.2), C. concisus 13826 (NC_009802.2), C. concisus ATCC

33237 (NZ_CP012541.1) (170), C. concisus P2CDO4 (NZ_CP021642.1) (31), C. gracilis ATCC 33236 (NZ_CP012196.1) (123), C. hominis ATCC BAA-

381(NC_009714.10), C. showae B91_SC (NZ_LR535679.1), C. ureolyticus RIGS

9880 (NZ_CP012195.1) (171)) using the BLASTp program (172). C. rectus is excluded from this study as its fully-sequenced genome is unavailable. C. showae B91_SC is used despite it not being the representative genome because it is the only strain of C. showae with a fully sequenced genome. Previously published criteria (more than 30% identity,

E-values <10-10 and bit scores of >50) were used to determine presence of a gene (173).

Protein sequences that were identified as hydrogenases based on catalytic domains were classified further using the hydrogenase classifier HydDB (174).

3.2.2. The reference genes used to identify similar genes and proteins in the human- hosted Campylobacter species genomes

To examine the presence of genes encoding enzymes in the pathways of energy metabolism and stress defence in human-hosted Campylobacter species, a total of 152 reference genes and their encoded proteins from Escherichia coli and C. jejuni were used as query genes and proteins to identify similar genes and proteins in Campylobacter genomes using the methods described above.

Reference genes and their encoded proteins from E. coli strain K-12 MG1655

(NC_000913.3), C. jejuni subsp. jejuni strain NCTC 11168 (NC_002163.1) and C. jejuni subsp. doylei strain 269.79 (NC_009707.1) were used as the query genes and proteins.

The choice of E. coli strain K-12 MG1655 as a reference strain was due to its well-studied metabolic pathways. C. jejuni strain NCTC 11168 (NC_002163.1) was used as a reference because it is a member of the Campylobacter genus with well-studied metabolic pathways (138, 175), except for the ED pathway, which was discovered in C. jejuni subsp. doylei 269.97 (NC_009707.1) and is not found in most C. jejuni subsp. jejuni strains

(140). In addition, the tetrathionate reductase genes tsdA (C8J_0815) and the tsdA paralog

(C8J_0040) were discovered in C. jejuni strain 81116 (158). As such, C. jejuni strain

81116 was used as a reference for the tetrathionate reductase pathway.

The reference genes and encoding proteins that were used as query genes and proteins for identification of similar genes and enzymes in the central carbon metabolic pathways of human-hosted Campylobacter species are listed in Supplementary Table S4 (C. jejuni subsp. jejuni and C. jejuni subsp. doylei reference genes). The reference genes and proteins that were used as query genes or proteins for identification of similar respiratory chain enzymes in C. concisus are listed in Supplementary Table S11 (C. jejuni subsp.

jejuni reference genes). The reference genes and encoded proteins from C. jejuni subsp. jejuni NCTC 11168, C. jejuni strain 81116 (livJKHMGF) and E. coli strain K-12 (gdhA) were used as query genes and proteins for identification of enzymes involved in amino acid use in are listed in Supplementary Table S7. The reference genes and encoded proteins from C. jejuni subsp. jejuni NCTC 11168 were used as query genes and proteins for identification enzymes involved in oxidative and nitrosative stress in C. concisus are listed in Additional Table 11. In addition, the nosZ and norZ genes found in C. concisus but not in C. jejuni were used as an additional reference for nitrosative stress defence genes (Additional Table 16).

3.3 Results

3.3.1 Glycolytic pathways and fucose metabolism pathways are incomplete in all human-hosted Campylobacter species examined, the TCA cycle is incomplete in most species examined, while acetate metabolism pathways are found in all species examined.

All three glycolytic pathways are incomplete in all human-hosted Campylobacter species examined (Figures 3-1, 3-2, and 3-3). The genes glk, zwf and pgl encoding the enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase and 6- phosphogluconate dehydrogenase respectively, for the oxidative phase of the Pentose

Phosphate pathway of glycolysis are missing in all species examined. The genes encoding enzymes for the non-oxidative Pentose Phosphate pathway are present in all species examined except for rpiB encoding ribose-5-phosphate isomerase in C. gracilis

ATCC 33236. All the genes of the Entner Doudoroff Pathway are absent in all species examined except for pgi which encodes glucose-6-phosphate isomerase. The Embden

Meyerhof Parnas pathway is incomplete in all species examined due to absence of the two critical genes glk (encoding glucokinase) and pfk (encoding phosphofructokinase).

Strain ey

Absence Presence

Gene

Figure 3-1. Genes for enzymes of the Pentose Phosphate pathway identified in human- hosted Campylobacter species. The genes for the enzymes of the Pentose Phosphate pathway of glycolysis identified in human-hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S4 and S13.

Strain

Absence Presence

Gene

Figure 3-2. Genes for enzymes of the Entner Doudoroff pathway identified in human- hosted Campylobacter species The genes for the enzymes of Entner Doudoroff pathway of glycolysis identified in human-hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S4 and S13.

Strain

Gene

Figure 3-3. Genes for enzymes of the Embden Meyerhof pathway identified in human- hosted Campylobacter species The genes for the enzymes of Embden Meyerhof pathway of glycolysis identified in human-hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S4 and S13.

Only C. curvus appears to have a complete TCA cycle. The genes sucCD which encode succinyl coA synthetase are absent in C. concisus, C. gracilis, and C. showae. The gene fumC which encodes fumarate hydratase is only present in C. showae and C. curvus.

The gene mqo which encodes malate:quinone oxidoreductase is absent in C. showae and

C. ureolyticus, but its absence may be compensated with malate dehydrogenase (mdh) which similarly oxidises malate to oxaloacetate. (Figure 3-4)

Absence Presence Figure 3-4. Genes for enzymes of the Tricarboxylic Acid Cycle identified in human- hosted Campylobacter species The genes for the enzymes of Tricarboxylic Acid Cycle identified in human-hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S4 and S13.

Though acs which encodes acetyl-coA synthetase is absent, ackA which encodes acetate kinase and pta which encodes phosphate acetyltransferase are present in all human- hosted Campylobacter species and they constitute a full acetate anabolic pathway, allowing the synthesis of acetate and ATP from pyruvate (Figure 3-5). Additionally, the fucose metabolism genes are absent in all human-hosted Campylobacter species except for dapA (Figure 3-6).

Strain

Absence Presence

Figure 3-5. Genes for enzymes of the acetate metabolism pathways identified in human- hosted Campylobacter species The genes for the enzymes of acetate metabolism pathways identified in human-hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S4 and S13.

Absence Presence

Figure 3-6. Genes for enzymes of the fucose metabolism pathways identified in human- hosted Campylobacter species The genes for the enzymes of fucose metabolism pathways identified in human-hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S4 and S13.

3.3.2 Human-hosted Campylobacter species may use fewer electron donors than C. jejuni

The ability of human-hosted Campylobacter species to use electron donors (Figure 3-8) was investigated by comparison of their genomes with known gene sequences for genes for the relevant enzymes from C. jejuni. The genes for use of 2-oxoglutarate

(oorABCD), succinate (frdABC), pyruvate (por) and hydrogen (hydABCD, and hypABCDEF) are present in all human-hosted Campylobacter species. The nikZ gene which encodes a nickel transporter is absent in C. showae, C. hominis, C. gracilis and

C. curvus, but these species may have alternative methods of nickel transport.

All species examined have genes for at least one pathway of malate use (mdh), even though C. ureolyticus and C. showae lack mqo. The genes for use of flavodoxin nuoABCDGHIJKLMN and cj1575c and cj1574c (the genes displacing nuoE and nuoF respectively) are present in all human-hosted Campylobacter species, except C. showae.

The genes for use of formate (fdhABCD) are present in all species examined except that the sulfur carrier protein fdhD was absent in C. hominis and C. curvus.

In contrast, none of the human-hosted Campylobacter species examined had genes for use of gluconate, lactate or sulfite using any of the pathways examined (Figure 3-8).

o oglutarate o

Sulfite

alate

Pyruvate

ydrogen

Formate

Gluconate

Flavodo in Flavodo Succinate

2 Lactate

Absence Presence Figure 3-7. Genes for enzymes of the electron donor pathways identified in human-hosted Campylobacter species The genes for the enzymes of electron donor pathways identified in human-hosted Campylobacter species are shown in this figure. The corresponding electron donor used by the enzymes is shown by the colour coded bar above. The enzymes in this figure and their functions are in Supplementary Tables S11 and S15.

3.3.3. Human-hosted Campylobacter species may use fewer electron acceptors than C. jejuni

The ability of human-hosted Campylobacter species to use electron acceptors (Figure 3-9) was investigated by comparison of their genomes with known gene sequences for genes for the relevant enzymes from C. jejuni.

Genes for use of fumarate as an electron acceptor (mfrABE) are present in all but C. ureolyticus and C. hominis. The genes encoding the main catalytic units of nitrate reductase

(napAGHB) are present in all species examined, though the genes for the accessory subunits napL and napD are not present in C. gracilis and C. hominis, and napD is absent in C. showae and C. ureolyticus. The genes for nitrite reductase (nrfAH) are only present in C. hominis, C. showae and C. ureolyticus.

The genes tsdA and a tsdA paralog from C. jejuni which encode two pathways of tetrathionate use are absent in all species examined except C. curvus and C. gracilis which have the tsdA gene. However, genes for another pathway for tetrathionate use previously reported (ttrDE) was found in all strains of C. concisus examined as well as in C. showae. There are two alternative pathways for use of oxygen as an electron acceptor, via ubiquinol cytochrome C oxidase (CcoNOQP), or cyanide-insensitive quinol oxidase (CioAB). Genes for at least one pathway of use of oxygen as a terminal electron acceptor are present in all species examined, though C. gracilis lacks ccoNOQP. All Campylobacter species investigated have torA and torC (Figure 3-10).

Figure 3-8. Genes for enzymes of the electron acceptor pathways identified in human-hosted Campylobacter species The genes for the enzymes of electron acceptor pathways identified in human-hosted Campylobacter species are shown in this figure. The corresponding electron acceptor used by the enzymes is shown by the colour coded bar above. The enzymes in this figure and their functions are in Supplementary Tables S4 and S14.

3.3.4 Human-hosted Campylobacter species may use fewer amino acids than C. jejuni

The ability of human-hosted Campylobacter species to use amino acids (Figure 3-10) was investigated by comparison of their genomes with known gene sequences for genes for the relevant enzymes from C. jejuni. All species examined have genes for use of aspartate (aspA and at least two transporters dcuAB, though dctA is absent in some strains) and asparagine

(ansB).

However, they all lack the livJKHMGF branched amino acid utilization genes, l-serine dehydratase (sdaA) and all species but C. hominis lack the serine transporter (sdaC). Though the proline transporter (putP) is present in all species examined, the proline dehydrogenase gene putA is missing in all species examined. gdhA is present in all species examined except for C. curvus. but its presence may be substituted by aspB which is present in C. curvus and all other species examined except for C. hominis.

Aspartate

Leucine Isoleucine Valine

Serine

Glutamate Proline Asparagine

c c c c

ut ut

Gene

cu cu s

s s s

s C

c c c c c c ey

Absence

Presence Figure 3-9. Genes for enzymes of the amino acid use pathways identified in human-hosted Campylobacter species The genes for the enzymes of amino acid use pathways identified in C. concisus are shown in this figure. The corresponding amino acid used by the enzymes is shown by the colour coded bar above. The enzymes in this figure and their functions are in Supplementary Tables S7 and S14. 58

3.3.5 Human-hosted Campylobacter species have fewer enzymes to deal with oxidative and nitrosative stress than C. jejuni

Catalase (katA) is absent in most species examined except for C. gracilis and C. ureolyticus. mdaB is present only in C. concisus and C. gracilis, and rrc is absent in C. showae and C. curvus. cgb and ctb were absent in all strains examined and nrfA is present only in C. hominis, C. showae and C. ureolyticus. The genes for nitrous oxide reductase nosZ and nitric oxide reductase norZ were present in in all strains except C. hominis and C. ureolyticus.

Absence

Presence

Figure 3-10. Genes for enzymes of the oxidative stress defence pathways identified in human-hosted Campylobacter species The genes for the enzymes of oxidative stress defence pathways identified in human- hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S9 and S17.

Absence Presence

Figure 3-11. Genes for enzymes of the nitrosative stress defence identified in human- hosted Campylobacter species The genes for the enzymes of nitrosative stress defence pathways identified in human- hosted Campylobacter species are shown in this figure. The enzymes in this figure and their functions are in Supplementary Tables S9 and S16.

3.4 Discussion

In this study, the pathways of energy metabolism and stress defence in human-hosted

Campylobacter species were examined by analysis of the presence of relevant genes in human-hosted Campylobacter species genomes.

This study found that all human-hosted Campylobacter species lack complete glycolytic pathways, and all the species investigated did not have a complete TCA cycle except for

C. curvus. Furthermore, none of the species examined had genes for fucose metabolism pathways. This is consistent with previous characterizations of Campylobacter species as non-saccharyolytic bacteria (1). Interestingly, a complete acetogenesis pathway was found in all species examined, which is significant as the acetogenesis pathway is required for initial commensal colonization of the avian intestinal tract by C. jejuni

(176) and is important for invasion of the intestinal epithelial barrier by Salmonella enterica serovar Typhimurium (177).

This study found that human-hosted Campylobacter species are likely to use a smaller variety of electron donors and acceptors, as well as fewer amino acids compared to C. jejuni. The general trend is that these hydrogen-requiring Campylobacter species tend to lack the genes for the enzymes required to use gluconate, lactate and sulfite, while having the necessary genes for using hydrogen, formate, succinate and 2-oxoglutarate.

It may be possible to use the lack of lactate use in human-hosted Campylobacter species to differentiate them from the closely related and morphologically similar Arcobacter species which are known to use lactate as an electron donor (1).

In C. jejuni, the respiratory complex I (the NADH dehydrogenase complex) is homologously displaced by NADH:ubiquinone oxidoreductase (nuo), and mediates electron flow into the electron transport chain using reduced flavodoxin instead of

NADH (149). The two genes not present in C. jejuni (nuoE and nuoF) encode the

NADH dehydrogenase and in their place in the operon are the novel genes cj1575c and cj1574c (149). It was found that the nuo genes were dispensable as growth can be restored in their mutants with addition of an alternative electron source such as formate, but cj1574c is an essential gene in C. jejuni. This study found that most human-hosted

Campylobacter species had cj1574c suggesting that they have flavodoxin coupled to complex I like C. jejuni, except for C. showae, which may rely on an alternative substance coupled to complex I.

All Campylobacter species investigated have torA or torC, which are needed for use of

DMSO or TMAO as an electron acceptor, and napAB, which are needed for use of nitrate as an electron acceptor. The lack of a nrfH gene suggests that the NapAB complexes in C. concisus, C. curvus and C. gracilis only serve as an electron sink, as they are not coupled to nitrite respiration to result in a proton motive force (178).

The absence of the genes ccoNOQP (encoding uniquinol-cytochrome C oxidoreductase) in only C. gracilis among all the human-hosted Campylobacter species is consistent with previous research that reports that C. gracilis is the only human-hosted

Campylobacter species with a negative Oxidase Test result (80).

In addition, four out of the six human-hosted Campylobacter species examined

(including all three fully sequenced strains of C. concisus), have at least one pathway of tetrathionate usage (ttrDE or tsdA). It has been suggested that the ability to respire

tetrathionate which is produced during inflammation may help survival of these bacteria in the inflamed gut (166), as has been found in Salmonella typhimurium which has a selective advantage over competing microbiota without this ability (179).

This study found that human-hosted Campylobacter species are likely to use fewer amino acids compared to C. jejuni. All species examined have the gene encoding aspartate ammonia lyase (aspA) which allows use of aspartate to generate fumarate, and the asparaginase encoding gene (ansB) which allows use of asparagine to generate aspartate and ammonia. However, all species examined lack the l-serine dehydratase gene sdaA and the proline dehydrogenase gene putA, suggesting that they are unable to make use of serine and proline. This is significant as use of serine and proline have been reported to be important for C. jejuni intestinal colonization of mice and C. jejuni host colonization of chicks and mice, respectively (139, 180). All species examined also lack the genes for uptake of branched chain amino acids, the inactivation of which have caused severe colonization defects in C. jejuni for mouse and chicken intestinal models of infection (143, 181). This suggests the amino acid metabolisms of human-hosted

Campylobacter species differ significantly from C. jejuni.

This study found that human-hosted Campylobacter species have fewer oxidative and nitrosative stress defence genes compared to C. jejuni. mdaB is present only in C. concisus and C. gracilis, and rrc is absent in C. showae and C. curvus. All of the species examined lack catalase except for C. gracilis and C. ureolyticus. C. showae

B91_SC was found to not have catalase. This contradicts the initial characterization of

C. showae as catalase positive (82), but is supported by more recent research that found most strains of C. showae to be catalase negative (182). The relative lack of oxidative

stress defence genes in human-hosted Campylobacter species suggests that they are more sensitive to oxidative stress than C. jejuni, particularly in the light that catalase has been reported to be especially vital for C. jejuni in vitro hydrogen peroxide resistance and intramacrophage survival (183).

Most of the nitrosative stress defence genes found in C. jejuni were not found in the species investigated. cgb and ctb were absent in all strains examined and nrfA is present only in C. hominis, C. showae and C. ureolyticus. Instead, the previously reported nitric oxide reductase NorZ and a nitrous oxide reductase NosZ found in C. concisus strain

13826 (166) and which are not present in C. jejuni (184), are present in most of the strains examined. This study found that these enzymes were present in all species examined except C. hominis and C. ureolyticus. This likely explains the increased growth of C. concisus strain 33237 in response to an increased concentration of nitric oxide donor sodium nitroprusside (185), and suggests that other Campylobacter species with NorZ and NosZ may similarly increase growth with increased nitric oxide concentration.

Findings from this study and previous studies suggest that the pathogenicity of human- hosted Campylobacter species are not only determined by the virulence of individual strains but also the microenvironment of the gastrointestinal tract of individual hosts and particularly the availability of H2 for growth. As the composition of microbiota and diet are the two major factors influencing the production of H2 in the gastrointestinal tract

(186), their impact on Campylobacter enteric pathogenicity warrants future investigation.

3.5 Conclusion

This study found that human-hosted Campylobacter species lack critical genes in the central carbon metabolism pathways and have an incomplete TCA cycle except for C. curvus. Many of the species investigated lack succinyl-CoA synthetase (sucCD) and fumarate hydratase (fumC). Human-hosted Campylobacter species were also found to have fewer genes that encode enzymes for utilizing amino acids, electron donors and acceptors, as well as stress defence compared to C. jejuni, although this study cannot rule out the possibility that they may use other alternative pathways. In conclusion, this study provides a molecular basis for the non-saccharolytic and hydrogen-dependent nature of human-hosted Campylobacter species via their energy metabolism and stress defence pathways, which provide insights into the growth requirements and pathogenicity of these species.

The ability to use a wide range of electron donors and electron acceptors allows flexibility in the respiratory chain, which could be a key factor in organisms being able to survive and grow in different niches, such as human and animal hosts, and different parts of the host gastrointestinal tract. As such, this study sheds light on the nutritional requirements that allow the survival and growth of human-hosted Campylobacter species. As phenotypic tests are commonly used to identify Campylobacter species (1), this study may give insight as to possible future methods of separating different

Campylobacter species based on their nutritional requirements. Future work should involve phenotypic studies with gene knockouts and proteomics-based investigations of the respiratory mechanisms of Campylobacter species.

Chapter 4: General Discussion and Future Works:

The principal findings in this aster’s research project are that it has for the first time reported the molecular bases of central carbon metabolism, energy metabolism and oxidative and nitrosative stress in human-hosted Campylobacter species, and demonstrated the molecular bases for their non-saccharyolytic and hydrogen-dependent natures.

Despite the interesting findings culminating from this research project, the exact usage of energy sources and stress defence mechanisms in human-hosted Campylobacter species is largely yet unsubstantiated by phenotypic experiments. Further investigations need to be carried out using phenotypic studies with gene knockouts and proteomics- based investigations of the respiratory mechanisms of Campylobacter species, to better elucidate the association between nutrition and pathogenicity.

The ability to use a wide range of electron donors and electron acceptors allows flexibility in the respiratory chain, which could be a key factor in organisms being able to survive and grow in different types of hosts, and colonize different niches of the host gastrointestinal tract. Investigating the nutritional requirements that allow the survival and growth of human-hosted Campylobacter species, provides the basis for further investigation of the pathogenic potential of these species, and their adaptations to their colonization niches.

Previous attempts have been made to investigate the pathogenic potential of these emerging pathogens (187-189), but they were found to generally lack similarity to

known pathogens and virulence factors. In addition, previous studies did not examine their energy metabolisms and stress defence mechanisms in detail. The emerging concept of nutritional virulence refers to the accumulating evidence that pathogenic bacteria, in addition to relying on canonical virulence factors, make use of specific metabolic traits to colonize and proliferate within their hosts (143, 190). By adapting to be able to make use of specific nutrients available in their colonization niches, these bacteria are able to outcompete the local microbiota and colonize the said niche. This concept is especially worth exploring in these species which are considered emerging pathogens, but generally lack similarity to known virulence factors. For example, the reliance on hydrogen by C. concisus may help to explain why it is a commensal oral bacterium but has been associated with various gastrointestinal diseases (Table 1-1), as hydrogen is only produced in the intestines by the action of colonic microbiota (191,

192), and is present in the oral cavity only in low amounts (193).

Hydrogen appears to play a key role in energy metabolism of human-hosted

Campylobacter species as all human-hosted Campylobacter species have been observed to require hydrogen for growth (80). In addition to the role of hydrogenases in energy metabolism, hydrogenases may play a protective role in C. concisus against oxidative stress. While various oxidative stress enzymes including superoxide dismutase are present in C. concisus, C. concisus was found to grow better anaerobically than microaerobically at a low (5%) H2 concentration (119). This would suggest that C. concisus has poor capacity to deal with oxidative stress. However, C. concisus has been demonstrated to grow better in microaerobic conditions than in anaerobic conditions when H2 concentration was raised to 20% (166). This suggests that use of hydrogen may play a protective role in C. concisus against oxidative stress.

This may be explained by a similar phenomenon observed in C. jejuni. A study found that presence of hydrogenase significantly improved survival of C. jejuni at 25% O2 and

12% O2 as compared with hydrogenase-deficient mutants and that wild-type C. jejuni was able to grow under high O2 atmospheres (25% O2) supplemented with H2 while little or no growth was seen under high O2 atmospheres without H2 (194). This phenomenon was suggested to be the result of respiratory protection where the cells rapidly oxidize H2 and transport the electrons through the electron transport chain terminating in the reduction of O2 to H2O. The ensuing depletion of O2 lowers O2 to levels supporting the growth of C. jejuni, allowing survival of C. jejuni under high O2 atmospheres with H2. Similarly, oxidation of H2 may result in lowering of oxidative stress in C. concisus, allowing its survival under microaerobic conditions.

As such, further research is warranted to investigate the role that hydrogenases play in growth and survival of human-hosted Campylobacter species. Evidence of the use of hydrogen as an energy source has thus far only been limited to archaea, bacteria and some lower eukaryotes (159). As yet, there is no evidence in favour of Ni-Fe hydrogenases in higher eukaryotes, except that certain subunits of ancestral Ni-Fe hydrogenases may have contributed to the evolution of the mitochondrial complex

(195). As hydrogenases have not been found in human cells, and Ni-Fe enzymes accounted for only 6% of hydrogenases detected in bacteria colonizing the human gut of healthy individuals (192), it is likely that targeted narrow-spectrum therapy with specific hydrogen-uptake NiFe hydrogenase inhibitors may have fewer unwanted side- effects compared to traditional antibiotic therapy in terms of disruption of gut microbiota and resultant gastrointestinal side-effects (196). This suggests that a targeted

hydrogenase inhibitor could be an effective novel therapeutic drug for eradication of hydrogen-dependent bacteria such as human-hosted Campylobacter species. This is an especially promising area of inquiry given the reported increase in antibiotic resistance in clinically relevant Campylobacter species which has been recognized as a problem by the World Health Organization (197-201).

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Supplementary files

Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity

Table S1: NCBI locus tags for genes involved in central carbon metabolism

(Prefixes for locus tags are as follows: : C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain

P2CDO4: CCS77_.)

Table S1a: NCBI locus tags for genes involved in the Embden Meyherhof Pathway of C. concisus EMP (compared with E. coli str. K-12 substr. MG1655) glk pgi pfkA pfkB fbaA fbaB tpiA gapA pgk gpmA gpmM ytjC eno pykA pykF 13826 ------0514 0516 0515 - 0563 - 2093 0964 0964 ATCC ------1485 1487 1486 - 1496 - 1664 1542 1542 33237 P2CDO4 ------1525 1527 1526 - 1555 - 1696 0396 0396 EMP (compared with C. jejuni subsp. jejuni NCTC 11168) glk pgi pfk fba tpiA gapA pgk gpm eno pyk 13826 n/a 0430 n/a 2070 0514 0516 0515 0563 2093 0964 ATCC n/a 1355 n/a 1271 1485 1487 1486 1496 1664 1542 33237 P2CDO4 n/a 0594 n/a 1259 1525 1527 1526 1555 1696 0396

Table S1b: NCBI locus tags for genes involved in the Pentose Phosphate Pathway of C. concisus PPP (compared with E. coli str. K-12 substr. MG1655) glk zwf pgl gnd rpiA rpiB rpe tktA tktB talA talB 13826 - - - - - 2228 1342 1735 1735 - - ATCC - - - - - 0591 0736 1715 1715 - - 33237 P2CDO4 - - - - - 1396 0916 1794 1794 - - PPP (compared with C. jejuni subsp. jejuni NCTC 11168) glk zwf pgl gnd rpiB rpe tkt tal 13826 n/a n/a n/a n/a 2228 1342 1735 1583 ATCC n/a n/a n/a n/a 0591 0736 1715 1442 33237 P2CDO4 n/a n/a n/a n/a 1396 0916 1794 1422

Table S1c: NCBI locus tags for genes involved in the Entner Doudouroff Pathway of C. concisus

ED Pathway (compared with E. coli str. K-12 substr. MG1655) ptsG pgi glk pgl zwf edd eda 13826 ------ATCC ------33237 P2CDO4 ------ED Pathway (compared with C. jejuni subsp. doylei 269.97) glcP pgi glk pgl zwf edd eda 13826 - 0430 - - - - - ATCC - 1355 - - - - - 33237 P2CD04 - 0594 - - - - -

Table S1d: NCBI locus tags for genes involved in the tricarboxylic cycle of C. concisus

TCA cycle (compared with E. coli str. K12 substr. MG1655) gltA acn acn Icd sucA sucB sucC suc sdh sdh sdh sdh mdh mqo fum fum fum lpdA ace ace A B D A B C D A B C A B 13826 228 - 1408 1088 - - - - 0425 0424 - - 2254 ------7 ATCC 164 - 0630 0995 - - - - 1361 1362 - - 0994 ------33237 2 P2CDO 172 - 1110 1019 - - - - 0589 0588 - - 1018 ------4 6 TCA cycle (compared with C. jejuni subsp. jejuni NCTC 11168) gltA acn icd oorA oorB oorC oorD sucC suc frdA frdB frdC mrf mrf mrfE mdh mqo fum B D A B C 13826 228 1408 1088 1294 1293 1292 1295 - - 0425 0424 0426 1283 1282 1281 2254 0434 - 7 ATCC 164 0630 0995 0992 0991 0990 0993 - - 1361 1362 1360 0984 0983 0982 0994 1481 - 33237 2 P2CDO 172 1110 1019 1016 101 1014 1017 - - 0589 0588 0590 1012 1011 1010 1018 1519 - 4 6 5

Table S1e: NCBI locus tags for genes involved in the pyruvate dehydrogenase complex of C. concisus

Pyruvate Dehydrogenase Complex (compared with E. coli str. K-12 substr. MG1655) aceE aceF lpdA pfo/ydbK 13826 - - - 1933 ATCC - - - 1610 33237 P2CDO4 - - - 0237

Pyruvate-Flavodoxin Oxidoreductase (compared with C. jejuni subsp. jejuni NCTC 11168)

por

13826 1933

ATCC 1610 33237 P2CDO4 0237 92

Table S1f: NCBI locus tags for genes involved in acetate metabolism of C. concisus

Acetate metabolism (compared with E. coli str. K-12 substr. MG1655) acka Pta acs 13826 0104 0103 - ATCC 1014 0104 - 33237 P2CDO4 1039 1038 - Acetate metabolism (compared with C. jejuni subsp . jejuni NCTC 11168) cj0689 cj1688 cj1537c 13826 0104 0103 - ATCC 1014 0104 - 33237 P2CDO4 1039 1038 -

Table S1g: NCBI locus tags for genes involved in gluconeogenesis of C. concisus Gluconeogenesis (compared with C. jejuni subsp . jejuni NCTC 11168) pckA Fbp pycA pycB 13826 1509 1544 1812 1508 ATCC 0495 0574 0148 0494 33237 P2CDO4 0496 1457 0067 0495

Table S2: NCBI locus tags for genes involved in use of electron donors of C. concisus Table S2a: NCBI locus tags for genes involved in use of electron donors of C. concisus (2-oxoglutarate, Flavodoxin) (Prefixes for locus tags are as follows: C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain P2CDO4: CCS77_.)

Flavodoxin

glutarate

- oxo

Gene

cj1567c nuoM nuoM cj1567c

cj1572c nuoH nuoH cj1572c

cj1576c nuoD nuoD cj1576c nuoG cj1573c nuoN cj1566c

cj1579c nuoA cj1579c nuoB cj1578c nuoC cj1577c nuoK cj1569c

cj1568c nuoL nuoL cj1568c

cj1570c nuoJ nuoJ cj1570c (with C. nuoI cj1571c

cj1382c fldA cj1382c

cj0535 oorD cj0535

cj0536 oorA cj0536 oorB cj0537 oorC cj0538

jejuni cj1575c cj1574c subsp. jejuni

NCTC

1168 as

reference) C. 1294 1293 1292 1295 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1656 concisus 13826 C. 0992 0991 0990 0993 0216 0215 0214 0213 0212 0211 0210 0209 0208 0207 0206 0205 0204 0203 1606 concisus ATCC 33237 C. 1016 1015 1014 1017 0213 0214 0215 0216 0217 0218 0219 0220 0221 0222 0223 0224 0225 0226 0241 concisus P2CDO4

Table S2b: NCBI locus tags for genes involved in use of electron donors of C. concisus (Formate, Fumarate, Gluconate, Hydrogen)

Formate Fumarate Gluconate Hydrogen

Gene JJD26997_1271 *edd JJD26997_1272 *eda (with C.

cj1364c fumC cj1364c

cj1264c hydD cj1264c

cj1267c hydA cj1267c hydB cj1266c hydC cj1265c

cj1508c fdhD fdhD cj1508c

cj1511c fdhA cj1511c fdhB cj1510c fdhC cj1509c

cj1584c nikZ cj1584c

cj0625 hypD cj0625 jejuni hypA cj0627 hypB cj0623 hypC cj0624 hypE cj0626 hypF cj0622

subsp. cj0414 cj0415 jejuni

NCTC

1168 as

reference)

C. 0207 0762 2138 0211 - - - - - 0101 0100 0099 0098 1092 1096 1095 1094 1093 0356 2116 concisus 13826 C. 0355 0356 0328 0548 - - - - - 1011 1010 1009 1008 0998 1002 1001 1000 0999 1006 0279 concisus ATCC 33237 C. 0310 0311 1653 1566 - - - - - 1036 1035 1034 1033 1022 1026 1025 1024 1023 1030 1674 concisus P2CDO4

Table S2c: NCBI locus tags for genes involved in use of electron donors of C. concisus (Lactate, Malate, Pyruvate, Succinate and Sulphite)

Lactate Malate Pyruvate Succinate Sul

ite

Gene

cj0004c sorA cj0004c sorB cj0005c mqo cj0393c

cj0409 frdA cj0409 frdB cj0410 frdC cj0408

cj0532 mdh cj0532 por cj1476c (with C. lutC cj0073 lutB cj0074 lutA cj0075

jejuni cj1585c subsp. jejuni NCTC

11168 as reference) C. - - - - 0434 2254 1933 0425 0424 0426 - - concisus 13826 C. - - - - 1481 0994 1610 1361 1362 1360 - - concisus ATCC 33237 C. - - - - 1519 1018 0237 0589 0588 0590 - - concisus P2CD04

Table S3: Query genes and proteins from E. coli strain K-12 MG1655 for identification of genes and proteins in C. concisus central carbon metabolism pathways

Gene Locus Relevant pathway Protein Function name tag glk b2388 EMP, PP and ED glucokinase phosphorylates glucose to glucose-6- pathways phosphate pgi b4025 EMP and ED pathways glucose-6-phosphate isomerase converts glucose-6- phosphate to fructose-6-phosphate pfkA b3916 EMP pathway phosphofructokinase phosphorylates fructose-6- phosphate to fructose-1,6-bisphosphate pfkB b1723 EMP pathway phosphofructokinase phosphorylates fructose-6- phosphate to fructose-1,6-bisphosphate fbaA b2925 EMP pathway fructose-bisphosphate aldolase condenses dihydroxyacetone phosphate with glyceraldehyde-3- phosphate to form fructose bisphosphate fbaB b2097 EMP pathway fructose-bisphosphate aldolase condenses dihydroxyacetone phosphate with glyceraldehyde-3- phosphate to form fructose bisphosphate tpiA b3919 EMP pathway triosephosphate isomerase converts dihydroxyacetone phosphate to glyceraldehyde-3- phosphate gapA b1179 EMP pathway glyceraldehyde-3-phosphate dehydrogenase A phosphorylates glyceraldehyde-3-phosphate to 1,3- bisphosphoglycerate pgk b2926 EMP pathway phosphoglucokinase reversibly phosphorylates 3- phosphoglycerate to 1,3-bisphosphoglycerate gpmA b0755 EMP pathway phosphoglycerate mutase interconverts 3- phosphoglycerate and 2-phosphoglycerate gpmM b3612 EMP pathway phosphoglycerate mutase interconverts 3- phosphoglycerate and 2-phosphoglycerate ytjC/ b4395 EMP pathway phosphoglycerate mutase interconverts 3- gpmB phosphoglycerate and 2-phosphoglycerate eno b2779 EMP pathway enolase reversibly converts 2-phosphoglycerate to phosphoenolpyruvate pykA b1854 EMP pathway pyruvate kinase phosphorylates phosphoenolpyruvate to pyruvate pykF b1676 EMP pathway pyruvate kinase phosphorylates phosphoenolpyruvate to pyruvate zwf b1852 PP and ED pathways glucose-6-phosphate 1-dehydrogenase oxidizes glucose-6-phosphate to 6-phosphoglucono-lactone pgl b0767 PP and ED pathways 6-phosphogluconolactonase hydrolyzes 6- phosphogluconolactone to 6-phosphogluconate gnd b2029 PP pathway 6-phosphogluconate dehydrogenase oxidatively decarboxylates 6-phosphogluconate to ribulose 5-phosphate and CO2 rpiA b2914 PP pathway ribose-5-phosphate isomerase reversibly converts ribose-5-phosphate to ribulose 5-phosphate

rpiB b4090 PP pathway ribose-5-phosphate isomerase reversibly converts ribose-5-phosphate to ribulose 5-phosphate rpe b3386 PP pathway ribulose-phosphate 3-epimerase reversibly epimerizes of ribulose-5-phosphate to xylulose 5- phosphate tktA b2935 PP pathway transketolase converts sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to xylulose-5- phosphate and ribose-5-phosphate tktB b2465 PP pathway transketolase converts sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to xylulose-5- phosphate and ribose-5-phosphate talA b2464 PP pathway transladolase converts glyceraldehyde-3- phosphate and sedoheptulose 7-phosphate to fructose-6-phosphate and erythrose 4-phosphate talB b0008 PP pathway transladolase converts glyceraldehyde-3- phosphate and sedoheptulose 7-phosphate to fructose-6-phosphate and erythrose 4-phosphate ptsG b1101 ED pathway glucose permease imports glucose into the cell edd b1851 ED pathway phosphogluconate dehydratase converts 6- phosphogluconate to 2-keto-3-deoxy-6- phosphogluconate eda b1850 ED pathway 2-dehydro-3-deoxy-phosphogluconate aldolase converts 2-dehydro-3-deoxy-D-gluconate 6- phosphate to glyceraldehyde-3-phosphate and pyruvate gltA b0720 TCA cycle citrate synthase condenses acetyl-coA and oxaloacetate to citrate acnA b1276 TCA cycle aconitase isomerizes citrate to isocitrate acnB b0118 TCA cycle aconitase isomerizes citrate to isocitrate ybhJ b0771 TCA cycle aconitase isomerizes citrate to isocitrate icd b1136 TCA cycle isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate, to 2-oxoglutarate and CO2 sucA b0726 TCA cycle 2-oxoglutarate dehydrogenase converts 2- oxoglutarate to succinyl-CoA and CO2 sucB b0727 TCA cycle 2-oxoglutarate dehydrogenase converts 2- oxoglutarate to succinyl-CoA and CO2 sucC b0728 TCA cycle succinyl-CoA synthetase catalyzes the reversible conversion of succinyl-CoA to succinate sucD b0729 TCA cycle succinyl-CoA synthetase catalyzes the reversible conversion of succinyl-CoA to succinate sdhA b0723 TCA cycle succinate dehydrogenase converts succinate to fumarate with the reduction of ubiquinone to ubiquinol sdhB b0724 TCA cycle succinate dehydrogenase converts succinate to fumarate with the reduction of ubiquinone to ubiquinol sdhC b0721 TCA cycle succinate dehydrogenase converts succinate to fumarate with the reduction of ubiquinone to ubiquinol

sdhD b0722 TCA cycle succinate dehydrogenase converts succinate to fumarate with the reduction of ubiquinone to ubiquinol mdh b3236 TCA cycle malate dehydrogenase reversibly catalyzes the oxidation of malate to oxaloacetate mqo b2210 TCA cycle malate quinone oxidoreductase reversibly catalyzes the oxidation of malate to oxaloacetate fumA b1612 TCA cycle fumarate dehydratase reversibly converts fumarate to malate fumB b4122 TCA cycle fumarate dehydratase reversibly converts fumarate to malate fumC b1611 TCA cycle fumarate dehydratase reversibly converts fumarate to malate lpdA b0116 TCA cycle dihydrolipoyl dehydrogenase converts pyruvate to acetyl-CoA and CO2 aceA b4015 Glyoxylate cycle isocitrate lyase catalyzes the cleavage of isocitrate to succinate and glyoxylate aceB b4014 Glyoxylate cycle malate synthase converts acetyl-CoA and glyoxylate to malate and CoA aceE b0114 TCA cycle pyruvate dehydrogenase converts pyruvate to acetyl- CoA and CO2 aceF b0115 TCA cycle pyruvate dehydrogenase converts pyruvate to acetyl- CoA and CO2 ydbk/p b1378 TCA cycle pyruvate-flavodoxin oxidoreductase fo converts pyruvate to acetyl coA ackA b2296 Acetate metabolism acetate kinase phosphorylates acetate to acetyl phosphate pta b2297 Acetate metabolism phosphate acetyltransferase converts acetyl-CoA and phosphate to CoA and acetyl phosphate acs b4069 Acetate metabolism acetyl CoA synthetase reversibly converts acetyl CoA to acetate

Table S4: Query genes and proteins from C. jejuni subsp. jejuni NCTC 11168 for identification of genes and proteins of C. concisus central carbon metabolism pathways

Gene Locus tag Relevant pathway Protein Function name glk* JJD26997_1 EMP, PP and ED glucokinase phosphorylates glucose to glucose-6- 268 pathways phosphate pgi* cj1535c/ EMP and ED glucose-6-phosphate isomerase converts glucose-6- JJD26997_1 pathways phosphate to fructose-6-phosphate 267 fba cj0597 EMP pathway fructose-bisphosphate aldolase condenses dihydroxyacetone phosphate with glyceraldehyde- 3-phosphate to form fructose bisphosphate tpiA cj401c EMP pathway, triosephosphate isomerase converts gluconeogenesis dihydroxyacetone phosphate to glyceraldehyde-3- phosphate gapA cj1403c EMP pathway glyceraldehyde-3-phosphate dehydrogenase A phosphorylates glyceraldehyde-3-phosphate to 1,3- bisphosphoglycerate pgk cj1402c EMP pathway, phosphoglucokinase reversibly phosphorylates 1,3- gluconeogenesis bisphosphoglycerate to 3-phosphoglycerate pgm cj0434 EMP pathway Phosphoglycerate mutase interconverts 3- phosphoglycerate and 2-phosphoglycerate eno cj1672c EMP pathway Enolase reversibly converts 2-phosphoglycerate to phosphoenolpyruvate Pyk cj0392c EMP pathway pyruvate kinase phosphorylates phosphoenolpyruvate to pyruvate zwf* JJD26997_1 PP and ED glucose-6-phosphate 1-dehydrogenase oxidizes 270 pathways glucose-6-phosphate to 6-phosphoglucono-lactone pgl* JJD26997_1 PP and ED 6-phosphogluconolactonase hydrolyzes 6- 269 pathways phosphogluconolactone to 6-phosphogluconate rpiB cj0925 PP pathway ribose-5-phosphate isomerase reversibly converts ribose-5-phosphate to ribulose 5-phosphate rep cj0451 PP pathway ribulose-phosphate 3-epimerase reversibly epimerizes of ribulose 5-phosphate to xylulose 5- phosphate Tkt cj1645 PP pathway transketolase converts sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to xylulose-5- phosphate and ribose-5-phosphate Tal cj0281c PP pathway transladolase converts glyceraldehyde-3- phosphate and sedoheptulose 7-phosphate to fructose-6-phosphate and erythrose 4-phosphate glcP* JJD26997_1 ED pathway glucose permease imports glucose into the cell 266 edd * JJD26997_1 ED pathway phosphogluconate dehydratase converts 6- 271 phosphogluconate to 2-keto-3-deoxy-6- phosphogluconate eda * JJD26997_1 ED pathway 2-dehydro-3-deoxy-phosphogluconate aldolase 272 converts 2-dehydro-3-deoxy-D-gluconate 6- phosphate to glyceraldehyde-3-phosphate and pyruvate

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gltA cj1682 TCA cycle citrate synthase condenses acetyl-coA and oxaloacetate to citrate acnB cj0835c TCA cycle aconitase isomerizes citrate to isocitrate icd cj0531 TCA cycle isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate, to 2-oxoglutarate and CO2 oorA cj0536 TCA cycle 2-oxoglutarate oxidoreductase converts 2-oxoglutarate to succinyl-coA and CO2 oorB cj0537 TCA cycle 2-oxoglutarate oxidoreductase converts 2-oxoglutarate to succinyl-coA and CO2 oorC cj0538 TCA cycle 2-oxoglutarate oxidoreductase converts 2-oxoglutarate to succinyl-coA and CO2 oorD cj0539 TCA cycle 2-oxoglutarate oxidoreductase converts 2-oxoglutarate to succinyl-coA and CO2 sucC cj0533 TCA cycle succinyl-CoA synthetase catalyzes the reversible conversion of succinyl-CoA to succinate sucD cj0534 TCA cycle succinyl-CoA synthetase catalyzes the reversible conversion of succinyl-CoA to succinate mrfA cj0437 TCA cycle methylmenaquinol fumarate reductase converts fumarate to succinate with the oxidation of menaquinol to menaquinone mrfB cj0438 TCA cycle methylmenaquinol fumarate reductase converts fumarate to succinate with the oxidation of menaquinol to menaquinone mrfE cj0439 TCA cycle methylmenaquinol fumarate reductase converts fumarate to succinate with the oxidation of menaquinol to menaquinone frdA cj0409 TCA cycle bidirectional fumarate reductase reversibly converts succinate to fumarate with the reduction of menaquinone to menaquinol frdB cj0410 TCA cycle bidirectional fumarate reductase reversibly converts succinate to fumarate with the reduction of menaquinone to menaquinol frdC cj0408 TCA cycle bidirectional fumarate reductase reversibly converts succinate to fumarate with the reduction of menaquinone to menaquinol mdh cj0532 TCA cycle malate dehydrogenase reversibly catalyzes the oxidation of malate to oxaloacetate mqo cj0393c TCA cycle malate quinone oxidoreductase reversibly catalyzes the oxidation of malate to oxaloacetate fumC cj1364c TCA cycle fumarate hydratase reversibly converts fumarate to malate ackA cj0689 Acetate acetate kinase phosphorylates acetate to acetyl metabolism phosphate pta cj0688 Acetate phosphate acetyltransferase converts acetyl-CoA metabolism and phosphate to CoA and acetyl phosphate acs cj1537c Acetate acetyl CoA synthetase reversibly converts acetyl metabolism CoA to acetate pycA cj1037c Gluconeogenesis pyruvate carboxylase subunit A involved in conversion of pyruvate to oxaloacetate

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pycB cj1038c Gluconeogenesis pyruvate carboxylase subunit B involved in conversion of pyruvate to oxaloacetate pckA cj0932c Gluconeogenesis phosphoenolpyruvate carboxykinase converts oxaloacetate to phosphoenolpyruvate Fbp cj00840c Gluconeogenesis fructose-1,6-bisphosphatase converts fructose- 1,6-bisphosphate to D-fructose 6-phosphate

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Table S5: NCBI locus tags for genes involved in use of electron acceptors

(Prefixes for locus tags are as follows: C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain

P2CDO4: CCS77_.)

Table S5: NCBI locus tags for genes involved in use of electron acceptors of C. concisus (Fumarate, Nitrate, Nitrite)

Fumarate Nitrate Nitrite

Gene (with nrfH cj1358c

cj1357c nrfA cj1357c

cj0782 napH cj0782

cj0781 napG cj0781 napD cj0785

cj0780 napA cj0780 napB cj0783

cj0437 mrfA cj0437 mrfB cj0438 mrfE cj0439 napL cj0784 C. jejuni subsp. jejuni 11168 as

reference)

C. 1283 1282 1281 0868 0867 0866 0865 0863 0862 - - concisus 13826 C. concisus 0984 0983 0982 0641 0642 0643 0644 0646 0647 - - ATCC 33237 C. concisus 1012 1011 1010 0645 0646 0647 0648 0650 0651 - - P2CD04

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Table S5b: NCBI locus tags for genes involved in use of electron acceptors of C. concisus (Oxygen, SN-oxide, Tetrathionate)

Oxygen SN-Oxide Tetrathionate

Gene C8J_0815 tsdA C8J_0815

cj1490c ccoN cj1490c ccoO cj1489c ccoQ cj1488c

cj1487c cj1487c

cj1186c petA cj1186c petB cj1185c petC cj1184c

cj0264c torA cj0264c torC cj0265c

cj0081 cioA cj0081 cioB cj0082

(with C. C8J_0040 jejuni subsp. jejuni ccoP

11168 as

reference) C. 0723 0724 0725 0299 1921 1920 1919 0906 0905 1119 1398 - - concisus 13826 C. 0296 0297 0298 0726 1599 1598 1597 1795 1796 0015 0067 - - concisus ATCC 33237

C. 0272 0273 0274 0275 0249 0250 0251 1880 1881 1098 1099 - - concisus P2CD04

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Table S6: NCBI locus tags for genes involved in amino acid use

(Prefixes for locus tags are as follows: : C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain

P2CDO4: CCS77_.)

Table S6a: NCBI locus tags for genes involved in use of amino acids of C. concisus Amino acid use (compared with C. jejuni subsp. jejuni NCTC 11168) Aspartate Asparagine Glutamate dcuA dcuB dctA aspA ansB cj0919c cj0920c cj0921c cj0922c gdhA paqP paqQ aspB 13826 0390 1000 1512 0391 0029 0415 1927 0664 0414 1815 0661 0663 1274 ATCC 1351 0438 0496 1352 1259 1381 1604 1189 1380 1177 0319 0913 33237 0145 P2CDO4 0598 0430 0499 0597 1244 1435 0243 0762 1434 0064 0765 0763 1003

Table S6b: NCBI locus tags for genes involved in use of amino acids of C. concisus Amino acid use (compared with C. jejuni subsp . jejuni NCTC 11168) Serine Proline Isoleucine, Leucine, Valine sdaA sdaC putA putP livJ livK livH livM livG livF 13826 - - - 0416 ------ATCC - - - 1382 ------33237 P2CDO4 - - - 1436 ------

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Table S7: Query genes and proteins from C. jejuni subsp. jejuni NCTC 11168, E. coli strain K-12 MG1655, and C. jejuni subsp. jejuni strain 81116 used to identify genes and proteins for amino acid use in C. concisus

Gene name Locus tag Function Reference sdaA cj1624c serine dehydratase converts serine to 1 pyruvate sdaC cj1625c transports serine 1 putA cj1503c proline dehydrogenase converts 2 proline to S-1-pyrroline carboxylate putP cj1502c transports proline 2 dcuA cj0088 uptake of fumarate and aspartate 3 dcuB cj0671 uptake of fumarate and aspartate, 3 succinate efflux dctA cj1192 uptake of fumarate, aspartate and 3 succinate aspA cj0087 aspartate ammonia lyase converts 4 aspartate to fumarate and ammonia aspB cj0762c aspartate:glutamate transaminase 4 converts glutamate and oxaloacetate to 2-oxoglutarate and aspartate ansB cj0029 asparaginase deaminates asparagine to 5 aspartate cj0919c ABC-type amino-acid transporter 6 permease cj0920c ABC-type amino-acid transporter 6 permease peb1a cj0921c aspartate/glutamate‐binding protein 6 of ABC transporter pebC cj0922c ABC-type amino-acid transporter ATP- 6 binding protein paqP cj0467 ABC-type glutamate transporter 7 permease paqQ cj0468 ABC-type glutamate transpoter ATP- 7 binding protein gdhA b1761 glutamate dehydrogenase converts 8 glutamate to 2-oxoglutarate livJ CJJ81176_1038 periplasmic binding protein for 9 branched chain amino acid transport livK CJJ81176_1037 periplasmic binding protein for 9 branched chain amino acid transport livH CJJ81176_1036 inner membrane permease for 9 branched chain amino acid transport livM CJJ81176_1035 inner membrane permease for 9 branched chain amino acid transport livG CJJ81176_1034 cytoplasmic ATPase for branched chain 9 amino acid transport livF CJJ81176_1033 cytoplasmic ATPase for branched chain 9 amino acid transport

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Table S8: NCBI locus tags for genes involved in oxidative stress

(Prefixes for locus tags are as follows: C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain

P2CDO4: CCS77_.)

Additional Table S8: NCBI locus tags for genes involved in use oxidative stress defense mechanisms of C. concisus

ahpC bcp tpx docA/cj0358 dps katA mdaB msrA/B rrc sodB

13826 0985 2112 0323 1108 1456 - 1626 1633 1910 0328

ATCC 1523 0275 1761 0161 0695 - 0755 0761 1802 1766 33237 P2CD04 0419 1678 1855 0114 0694 - 0798 0805 1901 1849

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Table S9: Query genes and proteins from C jejuni subsp. jejuni NCTC 11168 that were used to identify genes and proteins for oxidative and nitrosative stress defence in C. concisus

Gene name Locus tag Function Reference docA cj0020c cytochrome C peroxidase with 10 unknown specificity cj0358 cytochrome C peroxidase with 10 unknown specificity ahpC cj0334 alkyl hydroperoxide reductase is 11 involved in oxidative stress defence possibly from an endogenous organic peroxide Bcp cj0271 bacterioferritin comigratory 12 protein acts as a peroxide reductase able to act on a wide variety of compounds including hydrogen peroxide and organic peroxides Dps cj1534c DNA-binding protein involved in 13, 14, 15 sequestration of Fe ions katA cj1385 catalase converts hydrogen peroxide 13 into water and oxygen mdaB cj1545c reduction of soluble quinones in 13, 16 Helicobacter pylori msrA cj0637c S-isomer specific methionine 17 sulphoxide reductase reduces oxidized S-methionine msrB cj1112c R-isomer specific methionine 17 sulphoxide reductase reduces oxidized R-methionine Rrc cj0012c desulforubrerythrin is involved in 13 hydrogen peroxide detoxification sodB cj0169 superoxide dismutase converts 13 superoxide to hydrogen peroxide and oxygen Tpx cj0779 thiol peroxidase reduces hydrogen 12 peroxide Cgb cj1586 single domain hemoglobin involved in 18 nitric oxide detoxification Ctb cj0465c truncated hemoglobin involved in nitric 19 oxide detoxification nrfA cj1357c nitrite reductase reduces nitrite to 20 ammonia, and is involved in nitric oxide detoxification

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Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity Table S10: Campylobacter concisus strains used in BLASTn analysis Strain Name Genome Status 1 13826 Fully sequenced 2 2009-118452 Draft genome 3 2009-119100 Draft genome 4 2009-129008 Draft genome 5 2009-130586 Draft genome 6 2009-158448 Draft genome 7 2009-173039 Draft genome 8 2009-42653 Draft genome 9 2009-75710 Draft genome 10 2009-75775 Draft genome 11 2009-86120 Draft genome 12 2009-91522 Draft genome 13 2010-112100-F Draft genome 14 2010-112100-O Draft genome 15 2010-112708 Draft genome 16 2010-112758 Draft genome 17 2010-112825 Draft genome 18 2010-113332-F Draft genome 19 2010-113332-O Draft genome 20 2010-113862 Draft genome 21 2010-113862-O Draft genome 22 2010-115605-F Draft genome 23 2010-131105 Draft genome 24 2010-16206 Draft genome 25 2010-164712 Draft genome 26 2010-1718 Draft genome 27 2010-25654-F Draft genome 28 2010-25654-O Draft genome 29 2010-30795 Draft genome 30 2010-30800 Draft genome 31 2010-31374 Draft genome 32 2010-33561 Draft genome 33 2010-34330 Draft genome 34 2010-347972 Draft genome 35 2010-36743 Draft genome 36 2010-378007-F Draft genome 37 2010-378007-O Draft genome 38 2010-43100 Draft genome 39 2010-6073 Draft genome 40 2010-8194 Draft genome

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41 2010-88823 Draft genome 42 2012-164712 Draft genome 43 2012-191940 Draft genome 44 2012-37302 Draft genome 45 2013-101463 Draft genome 46 2013-39845 Draft genome 47 2013-42088 Draft genome 48 2013-87946 Draft genome 49 AAUH-10HCce Draft genome 50 AAUH-10HCdes Draft genome 51 AAUH-10HCdes2 Draft genome 52 AAUH-10HCdes3 Draft genome 53 AAUH-10HCdes4 Draft genome 54 AAUH-10HCdes5 Draft genome 55 AAUH-10HCdes6 Draft genome 56 AAUH-10HCdes7 Draft genome 57 AAUH-10HCtra Draft genome 58 AAUH-10UCf2 Draft genome 59 AAUH-10UCil-a Draft genome 60 AAUH-11HCf Draft genome 61 AAUH-11HCo-a Draft genome 62 AAUH-11UCdes-a Draft genome 63 AAUH-11UCo Draft genome 64 AAUH-11UCsig-a Draft genome 65 AAUH-12CDce Draft genome 66 AAUH-12CDdes2 Draft genome 67 AAUH-12CDdes3 Draft genome 68 AAUH-12CDdes4 Draft genome 69 AAUH-12CDo Draft genome 70 AAUH-12CDrec-a Draft genome 71 AAUH-12CDsig Draft genome 72 AAUH-12CDti2-a Draft genome 73 AAUH-12CDti4-a Draft genome 74 AAUH-12CDti5-a Draft genome 75 AAUH-12CDtra2-a Draft genome 76 AAUH-12CDtra-a Draft genome 77 AAUH-12HCf Draft genome 78 AAUH-14HCce Draft genome 79 AAUH-15HCti Draft genome 80 AAUH-15UCdp Draft genome 81 AAUH-15UCdp-a Draft genome 82 AAUH-15UCpp Draft genome 83 AAUH-16UCdp Draft genome 84 AAUH-16UCdp3 Draft genome

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85 AAUH-16UCdp5 Draft genome 86 AAUH-16UCf Draft genome 87 AAUH-16UCf2 Draft genome 88 AAUH-16UCf3 Draft genome 89 AAUH-16UCo-a Draft genome 90 AAUH-19HCf Draft genome 91 AAUH-19HCf2 Draft genome 92 AAUH-1Dasc Draft genome 93 AAUH-1Dce-a Draft genome 94 AAUH-1Dtra Draft genome 95 AAUH-2010376221 Draft genome 96 AAUH-2012179281 Draft genome 97 AAUH-20HCasc Draft genome 98 AAUH-20HCrec-a Draft genome 99 AAUH-20HCsig-a Draft genome 100 AAUH-20UCf Draft genome 101 AAUH-20UCo Draft genome 102 AAUH-22UCpp-a Draft genome 103 AAUH-25Df Draft genome 104 AAUH-25Df3 Draft genome 105 AAUH-2HCtra Draft genome 106 AAUH-35UCdp Draft genome 107 AAUH-35UCf Draft genome 108 AAUH-35UCil2-a Draft genome 109 AAUH-35UCil3-a Draft genome 110 AAUH-35UCil4-a Draft genome 111 AAUH-35UCil-a Draft genome 112 AAUH-35UCpp Draft genome 113 AAUH-37UCf Draft genome 114 AAUH-37UCo-a Draft genome 115 AAUH-39CDf Draft genome 116 AAUH-39CDrec-a Draft genome 117 AAUH-39CDti-a Draft genome 118 AAUH-3HCce2 Draft genome 119 AAUH-3HCo Draft genome 120 AAUH-3UCce Draft genome 121 AAUH-3UCce2 Draft genome 122 AAUH-40UCf Draft genome 123 AAUH-43UCce-a Draft genome 124 AAUH-43UCf Draft genome 125 AAUH-44UCsig6 Draft genome 126 AAUH-47UCil Draft genome 127 AAUH-47UCil-a Draft genome 128 AAUH-48UCdp-a Draft genome

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129 AAUH-48UCil-a Draft genome 130 AAUH-48UCo-a Draft genome 131 AAUH-49UCf Draft genome 132 AAUH-49UCil-a Draft genome 133 AAUH-49UCpp-a Draft genome 134 AAUH-4UCti Draft genome 135 AAUH-4UCti-a Draft genome 136 AAUH-51UCf Draft genome 137 AAUH-55UCtra-a Draft genome 138 AAUH-58UCo Draft genome 139 AAUH-59UCpp-a Draft genome 140 AAUH-5CDo Draft genome 141 AAUH-6HCo-a Draft genome 142 AAUH-7UCil Draft genome 143 AAUH-8HCo Draft genome 144 AAUH-8HCo-a Draft genome 145 AAUH-8UCo Draft genome 146 AAUH-8UCpp Draft genome 147 AAUH-8UCpp-a Draft genome 148 AAUH-9HCasc Draft genome 149 AAUH-9HCce Draft genome 150 AAUH-9UCdp Draft genome 151 AAUH-9UCpp Draft genome 152 ATCC 33237 Fully sequenced 153 ATCC 51561 Draft genome 154 ATCC 51562 Draft genome 155 AUS22-Bd2 Draft genome 156 B124_Slimy-large Draft genome 157 B124_Slimy-small Draft genome 158 B124_Small-clear Draft genome 159 B124_Small-grey Draft genome 160 B38_Tiny-mucoid Draft genome 161 CCUG 19995 Draft genome 162 H10O-S1 Draft genome 163 H11O-S1 Draft genome 164 H11O-S2 Draft genome 165 H12O-S1 Draft genome 166 H14O-S1 Draft genome 167 H15O-S1 Draft genome 168 H16O-S1 Draft genome 169 H17O-S1 Draft genome 170 H19O-S1 Draft genome 171 H1O1 Draft genome 172 H20O-S1 Draft genome

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173 H21O-S1 Draft genome 174 H21O-S2 Draft genome 175 H21O-S3 Draft genome 176 H21O-S5 Draft genome 177 H22O-S1 Draft genome 178 H23O-S1 Draft genome 179 H24O-S1 Draft genome 180 H25O-S1 Draft genome 181 H26O-S1 Draft genome 182 H27O-S1 Draft genome 183 H28O-S1 Draft genome 184 H28O-S2 Draft genome 185 H29O-S1 Draft genome 186 H30O-S1 Draft genome 187 H3O1 Draft genome 188 H7O-S1 Draft genome 189 H9O-S1 Draft genome 190 H9O-S2 Draft genome 191 Lasto127.99 Draft genome 192 Lasto205.94 Draft genome 193 Lasto220.96 Draft genome 194 Lasto28.99 Draft genome 195 Lasto393.96 Draft genome 196 Lasto61.99 Draft genome 197 Lasto64.99 Draft genome 198 P10CDO-S1 Draft genome 199 P10CDO-S2 Draft genome 200 P11CDO-S1 Draft genome 201 P13UCO-S1 Draft genome 202 P13UCO-S3 Draft genome 203 P15UCO-S2 Draft genome 204 P16UCO-S2 Draft genome 205 P18CDO-S1 Draft genome 206 P19CDO-S1 Draft genome 207 P1CDO2 Draft genome 208 P1CDO3 Draft genome 209 P20CDO-S1 Draft genome 210 P20CDO-S2 Draft genome 211 P20CDO-S3 Draft genome 212 P20CDO-S4 Draft genome 213 P21CDO-S1 Draft genome 214 P21CDO-S2 Draft genome 215 P21CDO-S4 Draft genome 216 P24CDO-S2 Draft genome

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217 P24CDO-S3 Draft genome 218 P24CDO-S4 Draft genome 219 P25CDO-S3 Draft genome 220 P26UCO-S1 Draft genome 221 P26UCO-S2 Draft genome 222 P27CDO-S1 Draft genome 223 P27CDO-S2 Draft genome 224 P28CDO-S1 Draft genome 225 P2CDO3 Draft genome 226 P2CDO4 Fully sequenced 227 P2CDO-S6 Draft genome 228 P3UCB1 Draft genome 229 P3UCO1 Draft genome 230 P6CDO1 Draft genome 231 P7UCO-S2 Draft genome 232 RCH 26 Draft genome 233 RMIT-JF1 Draft genome 234 UNSW1 Draft genome 235 UNSW2 Draft genome 236 UNSW3 Draft genome 237 UNSWCD Draft genome 238 UNSWCS Draft genome 239 MGYG-HGUT-01392 Draft genome 240 MGYG-HGUT-02425 Draft genome 241 MGYG-HGUT-02426 Draft genome 242 MGYG-HGUT-02427 Draft genome 243 MGYG-HGUT-02428 Draft genome 244 MGYG-HGUT-02430 Draft genome 245 MGYG-HGUT-02431 Draft genome 246 MGYG-HGUT-02432 Draft genome 247 MGYG-HGUT-02433 Draft genome 248 MGYG-HGUT-02434 Draft genome 249 MGYG-HGUT-02435 Draft genome

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Table S11: Genes encoding electron donors and acceptors investigated in C. concisus as referenced from C. jejuni subsp. jejuni NCTC 11168 and C. jejuni subsp. jejuni strain 81116

Gene name Locus tag Function Reference oorA cj0536 2-oxoglutarate:acceptor 21 oxidoreductase, alpha subunit, involved in conversion of 2- oxoglutarate to succinyl coA oorB cj0537 2-oxoglutarate ferredoxin 21 oxidoreductase, beta subunit, involved in conversion of 2-oxoglutarate to succinyl coA oorC cj0538 2-oxoglutarate oxidoreductase, gamma 21 subunit, involved in conversion of 2- oxoglutarate to succinyl coA oorD cj0535 2-oxoglutarate:acceptor 21 oxidoreductase, delta subunit, involved in conversion of 2-oxoglutarate to succinyl coA nuoA cj1579c integral membrane protein subunit of 21 NADH dehydrogenase/complex I nuoB cj1578c peripheral membrane protein subunit 21 of NADH dehydrogenase/complex I nuoC cj1577c peripheral membrane protein subunit 21 of NADH dehydrogenase/complex I nuoD cj1576c peripheral membrane protein subunit 21 of NADH dehydrogenase/complex I - cj1575c NuoE paralog 21 - cj1574c NuoF paralog 21 nuoG cj1573c peripheral membrane protein subunit 21 of NADH dehydrogenase/complex I nuoH cj1572c integral membrane protein subunit of 21 NADH dehydrogenase/complex I nuoI cj1571c peripheral membrane protein subunit 21 of NADH dehydrogenase/complex I nuoJ cj1570c integral membrane protein subunit of 21 NADH dehydrogenase/complex I nuoK cj1569c integral membrane protein subunit of 21 NADH dehydrogenase/complex I nuoL cj1568c integral membrane protein subunit of 21 NADH dehydrogenase/complex I nuoM cj1567c integral membrane protein subunit of 21 NADH dehydrogenase/complex I nuoN cj1566c integral membrane protein subunit of 21 NADH dehydrogenase/complex I flavodoxin cj1382c electron transfer protein containing 21 flavin mononucleotide (FMN) fdhA cj1511c selenocysteine containing 22 molybdoprotein equivalent to the E. coli 110 kDa FdnG subunit

115

fdhB cj1510c iron-sulphur subunit equivalent to the 22 E. coli 32-kDa FdnH subunit. fdhC cj1509c cytochrome b subunit equivalent to the 22 E. coli 20-kDa FdnI subunit fdhD cj1508c required for activity of the formate 22 dehydrogenase enzyme complex fumC cj1364c fumarate hydratase reversibly converts 23 fumarate to malate - cj0414 gluconate 2-dehydrogenase gamma 24 chain, involved in conversion of gluconate to 2-dehydro-D-gluconate - cj0415 gluconate 2-dehydrogenase alpha 24 chain, involved in conversion of gluconate to 2-dehydro-D-gluconate edd* JJD26997_1271 phosphogluconate dehydratase 25 catalyzes the dehydration of 6- phospho-D-gluconate to 2-dehydro-3- deoxy-6-phospho-D-gluconate. eda* JJD26997_1272 2-keto-3-deoxy-6-phosphogluconate 25 aldolase (KDPG) catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D- glyceraldehyde-3-phosphate hydA cj1267c hydrogenase small subunit Fe–S 26 protein hydB cj1266c hydrogenase large subunit containing 26 the NiFe active site responsible for accepting electrons from hydrogen hydC cj1265c membrane-anchored b-type 26 cytochrome subunit of hydrogenase hydD cj1264c protease involved 26 in enzyme maturation of hydrogenase hypA cj0627 involved in enzyme maturation of 23 hydrogenase hypB cj0623 involved in enzyme maturation of 23 hydrogenase hypC cj0624 involved in enzyme maturation of 23 hydrogenase hypD cj0625 involved in enzyme maturation of 23 hydrogenase hypE cj0626 involved in enzyme maturation of 23 hydrogenase hypF cj0622 involved in enzyme maturation of 23 hydrogenase nikZ cj1484c nickel transporter 26 cj1585 lactate dehydrogenase responsible for 27 oxidation of lactate into pyruvate lutA cj0073 oxidoreductase subunit of lactate 27 oxidase responsible for oxidation of lactate into pyruvate

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lutB cj0074 iron–sulphur subunit which transfers L- 27 lactate-derived electrons to the respiratory chain via the iron–sulphur centres in LutB lutC cj0075 uncertain function 27 Mdh cj0532 malate dehydrogenase reversibly 23 converts malate to oxaloacetate Por cj1476c pyruvate oxidoreductase 28 frdA cj0409 fumarate reductase flavoprotein 29 subunit involved in reversible reduction of fumarate frdB cj0410 fumarate reductase iron-sulphur 29 subunit involved in reversible reduction of fumarate frdC cj0408 fumarate reductase cytochrome b 29 subunit involved in reversible reduction of fumarate sorA cj0004c monohaem cytochrome c 30 oxidoreductase involved in sulphite oxidation to sulphide sorB cj0005c molydopterin oxidoreductase involved 30 in sulphite oxidation to sulphide mfrA cj0437 periplasmic 31 methylmenaquinol:fumarate reductase, MfrA subunit involved in reduction of fumarate to succinate mfrB cj0438 periplasmic 31 methylmenaquinol:fumarate reductase, MfrB subunit involved in reduction of fumarate to succinate mfrE cj0439 periplasmic 31 methylmenaquinol:fumarate reductase, MfrE subunit involved in essential for correct transport of MfrA through the twin-arginine transport system. napA cj0780 catalytic subunit of nitrate reductase 32 involved in reduction of nitrate to ammonia napG cj0781 electron transfer subunit of nitrate 32 reductase involved in reduction of nitrate to nitrite napH cj0782 electron transfer subunit of nitrate 32 reductase involved in reduction of nitrate to nitrite napB cj0783 electron transfer subunit of nitrate 32 reductase involved in reduction of nitrate to nitrite napL cj0784 subunit of nitrate reductase with 32 unknown function

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napD cj0785 ‘Proof-reading’ chaperone required for 32 export of NapA by twin-arginine transport system nrfA cj1357c nitrite reductase catalytic subunit 20 involved in reduction of nitrite to ammonia nrfH cj1358c nitrite reductase electron transfer 20 subunit involved in reduction of nitrite to ammonia ccoN cj1490c cbb3-type cytochrome c oxidase 33 subunit involved in reduction of oxygen to water ccoO cj1489c cbb3-type cytochrome c oxidase 33 subunit involved in reduction of oxygen to water ccoQ cj1488c cbb3-type cytochrome c oxidase 33 subunit involved in reduction of oxygen to water ccoP cj1487c cbb3-type cytochrome c oxidase 33 subunit involved in reduction of oxygen to water petA cj1186c cytochrome bc1 subunit 34 petB cj1185c cbb3-type cytochrome c oxidase 34 subunit involved in reduction of oxygen to water petC cj1184c cbb3-type cytochrome c oxidase 34 subunit involved in reduction of oxygen to water cioA cj0081 cyanide-insensitive oxidase subunit 33 involved in reduction of oxygen to water cioB cj0082 cyanide-insensitive oxidase subunit 33 involved in reduction of oxygen to water torA cj0264c molybdoenzyme that reduces 35 trimethylamine N-oxide or dimethyl sulphoxide to trimethylamine/dimethyl sulphide torC cj0265c monoheme c-type cytochrome 35 involved in reducing trimethylamine N- oxide or dimethyl sulphoxide to trimethylamine/dimethyl sulphide tsdA c8j_0815 tetrathionate reductase reduces 36 tetrathionate to thiosulphate c8j_0040 tetrathionate reductase reduces 36 tetrathionate to thiosulphate Mqo cj0393c malate quinone oxidoreductase 37 oxidises malate to oxaloacetate

118

Table S12 “Additional File 12”

Additional References cited in Supplementary Tables S7, S9 and S10

1. Velayudhan, J., Jones, M. A., Barrow, P. A. & Kelly, D. J. L-serine catabolism via an oxygen-labile L-serine dehydratase is essential for colonization of the avian gut by Campylobacter jejuni. Infection and immunity 72, 260-268 (2004). 2. Hofreuter D, Mohr J, Wensel O, Rademacher S, Schreiber K, Schomburg D, et al. Contribution of amino acid catabolism to the tissue specific persistence of Campylobacter jejuni in a murine colonization model. PLOS ONE. 2012;7(11):e50699-e. 3. Wösten, M. M. S. M., van de Lest, C. H. A., van Dijk, L. & van Putten, J. P. M. Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni. Frontiers in microbiology 8, 174-174 (2017). 4. Guccione E, Del Rocio Leon-Kempis M, Pearson BM, Hitchin E, Mulholland F, Van Diemen PM, et al. Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate. Molecular microbiology. 2008;69(1):77-93. 5. Hofreuter, D., Novik, V. & Galán, J. E. Metabolic Diversity in Campylobacter jejuni Enhances Specific Tissue Colonization. Cell Host & Microbe 4, 425-433 (2008). 6. Del Rocio Leon-Kempis, M., Guccione, E., Mulholland, F., Williamson, M. P. & Kelly, D. J. The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamate- binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids. Molecular microbiology 60, 1262-1275 (2006). 7. Lin, A. E. et al. Atypical Roles for Campylobacter jejuni Amino Acid ATP Binding Cassette Transporter Components PaqP and PaqQ in Bacterial Stress Tolerance and Pathogen-Host Cell Dynamics. Infection and immunity 77, 4912-4924 (2009). 8. Sakamoto, N., Kotre, A. M. & Savageau, M. A. Glutamate dehydrogenase from Escherichia coli: purification and properties. Journal of bacteriology 124, 775-783 (1975). 9. Ribardo, D. A. & Hendrixson, D. R. Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched- chain amino acid transport. Journal of bacteriology 193, 6233-6243 (2011). 10. Bingham-Ramos, L. K. & Hendrixson, D. R. Characterization of Two Putative Cytochrome c Peroxidases of Campylobacter jejuni Involved in Promoting Commensal Colonization of Poultry. Infection and immunity 76, 1105-1114 (2008). 11. Oh, E. & Jeon, B. Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni. PLOS ONE 9, e87312 (2014). 12. Atack, J. M., Harvey, P., Jones, M. A. & Kelly, D. J. The Campylobacter jejuni Thiol Peroxidases Tpx and Bcp Both Contribute to Aerotolerance and Peroxide-Mediated Stress Resistance but Have Distinct Substrate Specificities. Journal of Bacteriology 190, 5279-5290 (2008). 13. Flint A, Sun Y-Q, Butcher J, Stahl M, Huang H, Stintzi A. Phenotypic Screening of a Targeted Mutant Library Reveals Campylobacter jejuni Defenses against Oxidative Stress. Infection and immunity. 2014;82(6):2266-75. 14. Ishikawa, T. et al. The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni. J Bacteriol 185, 1010-1017, doi:10.1128/jb.185.3.1010-1017.2003 (2003).

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15. Huergo, L. F., Rahman, H., Ibrahimovic, A., Day, C. J. & Korolik, V. Campylobacter jejuni Dps protein binds DNA in the presence of iron or hydrogen peroxide. J Bacteriol 195, 1970-1978 (2013). 16. Wang, G. & Maier, R. J. An NADPH quinone reductase of Helicobacter pylori plays an important role in oxidative stress resistance and host colonization. Infection and immunity 72, 1391-1396 (2004). 17. Atack, J. M. & Kelly, D. J. Contribution of the stereospecific methionine sulphoxide reductases MsrA and MsrB to oxidative and nitrosative stress resistance in the foodborne pathogen Campylobacter jejuni. Microbiology 154, 2219-2230 (2008). 18. Elvers, K. T., Wu, G., Gilberthorpe, N. J., Poole, R. K. & Park, S. F. Role of an Inducible Single-Domain Hemoglobin in Mediating Resistance to Nitric Oxide and Nitrosative Stress in Campylobacter jejuni and Campylobacter coli. Journal of Bacteriology 186, 5332-5341 (2004). 19. Wainwright, L. M., Elvers, K. T., Park, S. F. & Poole, R. K. A truncated haemoglobin implicated in oxygen metabolism by the microaerophilic food-borne pathogen Campylobacter jejuni. Microbiology 151, 4079-4091 (2005). 20. Pittman MS, Elvers KT, Lee L, Jones MA, Poole RK, Park SF, et al. Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress. Molecular microbiology. 2007;63(2):575-90. 21. Weerakoon DR, Olson JW. The Campylobacter jejuni NADH:Ubiquinone Oxidoreductase (Complex I) Utilizes Flavodoxin Rather than NADH. Journal of Bacteriology. 2008;190(3):915-25. 22. Hoffman PS, Goodman TG. Respiratory physiology and energy conservation efficiency of Campylobacter jejuni. Journal of bacteriology. 1982;150(1):319-26. 23. Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, et al. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature. 2000;403(6770):665-8. 24. Kelly DJ. Complexity and Versatility in the Physiology and Metabolism of Campylobacter jejuni. Campylobacter , Third Edition: American Society of Microbiology; 2008. 25. Vegge CS, Jansen van Rensburg MJ, Rasmussen JJ, Maiden MCJ, Johnsen LG, Danielsen M, et al. Glucose Metabolism via the Entner-Doudoroff Pathway in Campylobacter: A Rare Trait that Enhances Survival and Promotes Biofilm Formation in Some Isolates. Frontiers in Microbiology. 2016;7(1877). 26. Howlett, R. M., Hughes, B. M., Hitchcock, A. & Kelly, D. J. Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent. Microbiology 158, 1645-1655 (2012). 27. Thomas MT, Shepherd M, Poole RK, van Vliet AHM, Kelly DJ, Pearson BM. Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on l-lactate. Environmental Microbiology. 2011;13(1):48-61. 28. Weerakoon DR, Borden NJ, Goodson CM, Grimes J, Olson JW. The role of respiratory donor enzymes in Campylobacter jejuni host colonization and physiology. Microbial Pathogenesis. 2009;47(1):8-15. 29. Weingarten RA, Taveirne ME, Olson JW. The dual-functioning fumarate reductase is the sole succinate:quinone reductase in Campylobacter jejuni and is required for full host colonization. J Bacteriol. 2009;191(16):5293-300. 30. Myers JD, Kelly DJ. A sulphite respiration system in the chemoheterotrophic human pathogen Campylobacter jejuni. Microbiology. 2005;151(1):233-42.

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31. Guccione, E. et al. Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuni. Environmental Microbiology 12, 576-591 (2010). 32. Pittman MS, Kelly DJ. Electron transport through nitrate and nitrite reductases in Campylobacter jejuni. Biochem Soc Trans. 2005;33(Pt 1):190-2. 33. Jackson RJ, Elvers KT, Lee LJ, Gidley MD, Wainwright LM, Lightfoot J, et al. Oxygen Reactivity of Both Respiratory Oxidases in Campylobacter jejuni: the cydAB Genes Encode a Cyanide-Resistant, Low-Affinity Oxidase That Is Not of the Cytochrome bd Type. Journal of Bacteriology. 2007;189(5):1604-15. 34. Woodall CA, Jones MA, Barrow PA, Hinds J, Marsden GL, Kelly DJ, et al. Campylobacter jejuni gene expression in the chick cecum: evidence for adaptation to a low-oxygen environment. Infection and immunity. 2005;73(8):5278-85. 35. Sellars MJ, Hall SJ, Kelly DJ. Growth of Campylobacter jejuni supported by respiration of fumarate, nitrate, nitrite, trimethylamine-N-oxide, or dimethyl sulfoxide requires oxygen. Journal of bacteriology. 2002;184(15):4187-96. 36. Liu YW, Denkmann K, Kosciow K, Dahl C, Kelly DJ. Tetrathionate stimulated growth of Campylobacter jejuni identifies a new type of bi-functional tetrathionate reductase (TsdA) that is widely distributed in bacteria. Molecular microbiology. 2013;88(1):173-88. 37. Kather, B., Stingl, K., van der Rest, M. E., Altendorf, K. & Molenaar, D. Another unusual type of citric acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase. J Bacteriol 182, 3204-3209 (2000).

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Supplementary Table S13: NCBI locus tags for genes involved in central carbon metabolism of human-hosted Campylobacter species (Prefixes for locus tags are as follows: C. curvus strain 525.92: CCV52592_RS; C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain P2CDO4: CCS77_; C. gracilis strain ATCC 33236: CGRAC_RS; C. hominis strain ATCC BAA-381: CHAB381_RS; C. showae strain B91_SC: E4V70_RS; C. ureolyticus strain RIGS 9880: CUREO_RS)

Supplementary Table S13a: NCBI locus tags for genes involved in the Embden Meyherhof Pathway of human-hosted Campylobacter species glk pgi pfk fba tpiA gapA pgk gpm eno pyk C. curvus strain n/a 07190 n/a 03845 01880 01870 01875 01760 08750 02510 525.92

C. concisus strain n/a 0430 n/a 2070 0514 0516 0515 0563 2093 0964 13826 C. concisus strain n/a 1355 n/a 1271 1485 1487 1486 1496 1664 1542 ATCC 33237 C. concisus strain n/a 0594 n/a 1259 1525 1527 1526 1555 1696 0396 P2CDO4 C. gracilis strain n/a 02870 n/a 04740 07740 07750 07745 07920 00455 04825 ATCC 33236

C. hominis strain n/a 02500 n/a 05695 03690 03680 03685 07445 08320 07635 ATCC BAA-381

C. showae strain n/a 08795 n/a 09650 03150 03160 03155 08070 06940 08175 B91_SC

C. ureolyticus strain n/a 05715 n/a 05750 06900 06910 06905 07725 00985 01605 RIGS 9880

122

Supplementary Table S13b: NCBI locus tags for genes involved in the Pentose Phosphate Pathway of human-hosted Campylobacter species

PPP (compared with C. jejuni subsp. jejuni NCTC 11168) glk zwf pgl gnd rpiB rpe tkt tal C. curvus strain 525.92 n/a n/a n/a n/a 03240 06095 09290 08090

C. concisus strain 13826 n/a n/a n/a n/a 2228 1342 1735 1583 C. concisus strain ATCC n/a n/a n/a n/a 0591 0736 1715 1442 33237 C. concisus strain n/a n/a n/a n/a 1396 0916 1794 1422 P2CDO4 C. gracilis strain ATCC n/a n/a n/a n/a n/a 08710 01270 02145 33236

C. hominis strain ATCC n/a n/a n/a n/a 04870 03640 00995 06115 BAA-381

C. showae strain B91_SC n/a n/a n/a n/a 02025 09285 06625 04650

C. ureolyticus strain n/a n/a n/a n/a 05905 06010 00710 07310 RIGS 9880

123

Supplementary Table S13c: NCBI locus tags for genes involved in the Entner Doudouroff Pathway of human-hosted Campylobacter species

ED Pathway (compared with C. jejuni subsp. doylei 269.97) glcP pgi glk pgl zwf edd eda C. curvus strain - 07190 - - - - - 525.92 C. concisus - 0430 - - - - - strain 13826 C. concisus - 1355 - - - - - strain ATCC 33237 C. concisus - 0594 - - - - - strain P2CDO4 C. gracilis strain - 02870 - - - - - ATCC 33236 C. hominis strain - 02500 - - - - - ATCC BAA-381 C. showae strain - 08795 - - - - - B91_SC C. ureolyticus - 05715 - - - - - strain RIGS 9880

124

Supplementary Table S13d: NCBI locus tags for genes involved in the tricarboxylic cycle of human-hosted Campylobacter species

TCA cycle (compared with C. jejuni subsp. jejuni NCTC 11168) gltA acnB icd oorA oorB oorC oorD sucC sucD frdA frdB frdC mrfA mrfB mrfE mdh mqo fumC C. curvus 08565 04160 04950 04925 04920 04915 04930 04940 04935 07215 07220 07210 04905 04900 04895 04945 01895 - strain 525.92 C. concisus 2287 1408 1088 1294 1293 1292 1295 - - 0425 0424 0426 1283 1282 1281 2254 0434 - strain 13826 C. concisus 1642 0630 0995 0992 0991 0990 0993 - - 1361 1362 1360 0984 0983 0982 0994 1481 - strain ATCC 33237 C. concisus 1726 1110 1019 1016 1015 1014 1017 - - 0589 0588 0590 1012 1011 1010 1018 1519 - strain P2CDO4 C. gracilis - - - 05775 05780 05785 05770 - - 04040 04045 04035 05995 05990 05985 05765 04830 - strain ATCC 33236 C. hominis 07230 06625 05305 05330 05335 05340 05325 05315 05320 04040 04045 04035 - - - 05310 07630 - strain ATCC BAA-381 C. showae 07090 00060 00380 00400 00405 00410 00395 - - 08670 08675 08665 07950 07955 07960 00385 - 00195 strain B91_SC C. 01025 04285 03880 03905 03910 03915 00780 03890 03895 04880 04875 04885 - - - 03885 - - ureolyticus strain RIGS 9880

125

Supplementary Table S13e: NCBI locus tags for genes involved in the pyruvate dehydrogenase complex of human-hosted Campylobacter species

Pyruvate-Flavodoxin Oxidoreductase (compared with C. jejuni subsp. jejuni NCTC 11168) por C. curvus strain 525.92 01445 C. concisus strain 13826 1933 C. concisus strain ATCC 33237 1610 C. concisus strain P2CDO4 0237

C. gracilis strain ATCC 33236 10175

C. hominis strain ATCC BAA-381 07485

C. showae strain B91_SC 07755

C. ureolyticus strain RIGS 9880 02580

Supplementary Table S13f: NCBI locus tags for genes involved in acetate metabolism of human-hosted Campylobacter species

Acetate metabolism (compared with C. jejuni subsp . jejuni NCTC 11168) cj0689 acka cj1688 pta cj1537c acs C. curvus strain 525.92 04630 04635 - C. concisus strain 13826 0104 0103 - C. concisus strain ATCC 33237 1014 0104 - C. concisus strain P2CDO4 1039 1038 - C. gracilis strain ATCC 33236 05485 05490 - C. hominis strain ATCC BAA-381 05405 05410 - C. showae strain B91_SC 00665 00670 - C. ureolyticus strain RIGS 9880 04365 04360 - C. curvus strain 525.92 05485 05490 -

126

Supplementary Table S13f: NCBI locus tags for genes involved in fucose metabolism of human-hosted Campylobacter species

Fucose metabolism (compared with C. jejuni subsp . jejuni NCTC 11168) cj0481 dapA cj0484 cj0485 cj0486 fucP cj0487 C. curvus strain 525.92 04535 - - - - C. concisus strain 13826 - - - - - C. concisus strain ATCC 33237 - - - - - C. concisus strain P2CDO4 - - - - - C. gracilis strain ATCC 33236 06505 - - - - C. hominis strain ATCC BAA-381 02785 - - - - C. showae strain B91_SC 00455 - - - - C. ureolyticus strain RIGS 9880 04465 - - - -

127

Supplementary Table S14: NCBI locus tags for genes involved in amino acid use of human-hosted Campylobacter species (Prefixes for locus tags are as follows: C. curvus strain 525.92: CCV52592_RS; C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain P2CDO4: CCS77_; C. gracilis strain ATCC 33236: CGRAC_RS; C. hominis strain ATCC BAA-381: CHAB381_RS; C. showae strain B91_SC: E4V70_RS; C. ureolyticus strain RIGS 9880: CUREO_RS) Table S14a: NCBI locus tags for genes involved in use of amino acids of human-hosted Campylobacter species Amino acid use (compared with C. jejuni subsp. jejuni NCTC 11168) Aspartate Asparagine Glutamate dcuA dcuB dctA aspA ansB cj0919c cj0920c cj0921c cj0922c gdhA paqP paqQ aspB C. curvus strain 07170 07790 02710 07175 03905 05875 09525 - 02910 01300 05870 05880 04865 525.92 C. concisus 0390 1000 1512 0391 0029 0415 1927 0664 0414 1815 0661 0663 1274 strain 13826 C. concisus 1351 0438 0496 1352 1259 1381 1604 1189 1380 0145 1177 0319 0913 strain ATCC 33237 C. concisus 0598 0430 0499 0597 1244 1435 0243 0762 1434 0064 0765 0763 1003 strain P2CDO4 C. gracilis 05405 10235 - 05410 00545 09630 09380 05270 00225 01480 00235 00225 06250 strain ATCC 33236 C. hominis 07270 05460 - 07265 07745 - 02075 02105 - 01700 - 02080 04355 strain ATCC BAA-381 C. showae 01760 04290 08625 01765 01205 09630 - 09575 08555 07900 09590 09580 00700 strain B91_SC C. ureolyticus 04560 02405 - 04555 00280 06585 - 00275 06590 - 02240 02225 03075 strain RIGS 9880

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Supplementary Table S14b: NCBI locus tags for genes involved in use of amino acids of human-hosted Campylobacter species

Amino acid use (compared with C. jejuni subsp . jejuni NCTC 11168) Serine Proline Isoleucine, Leucine, Valine sdaA sdaC putA putP livJ livK livH livM livG livF C. curvus strain 525.92 - - - 02920 ------C. concisus strain 13826 - - - 0416 ------C. concisus strain ATCC - - - 1382 ------33237 C. concisus strain P2CDO4 - - - 1436 ------C. gracilis strain ATCC - - - 1436 ------33236 C. hominis strain ATCC - - - 09625 ------BAA-381 C. showae strain B91_SC - 04160 - 08090 ------C. ureolyticus strain RIGS - - - 08565 ------9880

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Supplementary Table S15a: NCBI locus tags for genes involved in use of electron donors of human-hosted Campylobacter species (Flavodoxin) (Prefixes for locus tags are as follows: C. curvus strain 525.92: CCV52592_RS; C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain P2CDO4: CCS77_; C. gracilis strain ATCC 33236: CGRAC_RS; C. hominis strain ATCC Flavodoxin Gene cj1579c cj1578c cj1577c cj1576c cj1575c cj1574c cj1573c cj1572c cj1571c cj1570c cj1569c cj1568c cj1567c cj1566c cj1382c nuoA nuoB nuoC nuoD nuoG nuoH nuoI nuoJ nuoK nuoL nuoM nuoN fldA C. curvus 00820 00815 00810 00805 00800 00795 00790 00785 00780 00775 00770 00765 00760 00755 01470 strain 525.92

C. concisus 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1656 strain 13826

C. concisus 0216 0215 0214 0213 0212 0211 0210 0209 0208 0207 0206 0205 0204 0203 1606 strain ATCC 33237

C. concisus 0213 0214 0215 0216 0217 0218 0219 0220 0221 0222 0223 0224 0225 0226 0241 strain P2CDO4 C. gracilis 01545 01550 01555 01560 01565 01570 01575 01580 11290 01590 01595 01600 01605 01610 10155 strain ATCC 33236 C. hominis 00890 00895 00900 00905 00910 00915 00920 00925 00930 00935 00940 00945 00950 00955 05935 strain ATCC BAA-381 C. showae - 03280 - - - 01750 ------07800 strain B91_SC C. 07890 07895 07900 07905 07910 07915 07920 07925 07930 07935 07940 07945 07950 07955 02595 ureolyticus strain RIGS 9880 BAA-381: CHAB381_RS; C. showae strain B91_SC: E4V70_RS; C. ureolyticus strain RIGS 9880: CUREO_RS) 130

131

Supplementary Table S15b: NCBI locus tags for genes involved in use of electron donors of human-hosted Campylobacter species (Formate, Fumarate, Gluconate, Hydrogen)

Formate Fumarate Gluconate Hydrogen

Gene (with C. JJD26997_1271 *edd JJD26997_1272 *eda jejuni subsp.

cj1364c fumC cj1364c

cj1264c hydD cj1264c

cj1267c hydA cj1267c hydB cj1266c hydC cj1265c

cj1508c fdhD fdhD cj1508c

cj1511c fdhA cj1511c fdhB cj1510c fdhC cj1509c

cj1584c nikZ cj1584c

cj0625 hypD cj0625 jejuni NCTC hypA cj0627 hypB cj0623 hypC cj0624 hypE cj0626 hypF cj0622

1168 as cj0414 cj0415 reference)

C. curvus 08375 08370 08205 ------05040 S05035 05030 05025 04970 04990 04985 04980 04975 05015 - strain 525.92 C. concisus 0207 0762 2138 0211 - - - - - 0101 0100 0099 0098 1092 1096 1095 1094 1093 0356 2116 strain 13826 C. concisus 0355 0356 0328 0548 - - - - - 1011 1010 1009 1008 0998 1002 1001 1000 0999 1006 0279 strain ATCC 33237 C. concisus 0310 0311 1653 1566 - - - - - 1036 1035 1034 1033 1022 1026 1025 1024 1023 1030 1674 strain P2CDO4 C. gracilis 05980 04410 04415 05965 11535 05930 05925 05920 05915 05950 - 05980 04410 04415 05965 11535 05930 05925 05920 05915 strain ATCC 33236 C. hominis 04060 05415 05410 04075 04280 04110 04115 04120 04155 08450 - 04060 05415 05410 04075 04280 04110 04115 04120 04155 strain ATCC BAA-381 C. showae 00205 00210 00215 00220 00275 00255 00260 00265 00270 00230 - 00205 00210 00215 00220 00275 00255 00260 00265 00270 strain B91_SC C. ureolyticus 03920 03925 03930 03935 03975 03950 03955 03960 03970 03945 02220 03920 03925 03930 03935 03975 03950 03955 03960 03970 strain RIGS 9880 132

Supplementary Table S15c: NCBI locus tags for genes involved in use of electron donors of human-hosted Campylobacter species (2-oxoglutarate, Lactate, Malate, Pyruvate, Succinate and Sulphite)

2-oxo-glutarate Lactate Malate Pyruvate Succinate Sulphite Gene (with C. cj0536 cj0537 cj0538 cj0535 cj0073 cj0074 cj0075 cj1585c cj0393c cj0532 cj1476c cj0409 cj0410 cj0408 cj0004c cj0005c jejuni subsp. oorA oorB oorC oorD lutC lutB lutA mqo mdh por frdA frdB frdC sorA sorB jejuni NCTC 11168 as reference) C. curvus 04925 04920 04915 04930 - - - - 04945 01895 01445 07215 07220 07210 - - strain 525.92 C. concisus 1294 1293 1292 1295 - - - - 0434 2254 1933 0425 0424 0426 - - strain 13826 C. concisus 0992 0991 0990 0993 - - - - 1481 0994 1610 1361 1362 1360 - - strain ATCC 33237 C. concisus 1016 1015 1014 1017 - - - - 1519 1018 0237 0589 0588 0590 - - strain P2CDO4 C. gracilis 05775 05780 05785 05770 - - - - 05765 04830 10175 04040 04045 04035 - - strain ATCC 33236 C. hominis 05330 05335 05340 05325 - - - - 05310 07630 07485 04040 04045 04035 - - strain ATCC BAA-381 C. showae 00400 00405 00410 00395 - - - - 00385 - 07755 08670 08675 08665 - - strain B91_SC C. ureolyticus 03905 03910 03915 00780 - - - - 03885 - 02580 04880 04875 04885 - - strain RIGS 9880

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Supplementary Table S16a: NCBI locus tags for genes involved in use of electron acceptors of human-hosted Campylobacter species (Fumarate, Nitrate, Nitrite) (Prefixes for locus tags are as follows: C. curvus strain 525.92: CCV52592_RS; C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain P2CDO4: CCS77_; C. gracilis strain ATCC 33236: CGRAC_RS; C. hominis strain ATCC BAA-381: CHAB381_RS; C. showae strain B91_SC: E4V70_RS; C. ureolyticus strain RIGS 9880: CUREO_RS)

Fumarate Nitrate Nitrite Gene (with cj0437 cj0438 cj0439 cj0780 cj0781 cj0782 cj0783 cj0784 cj0785 cj1357c cj1358c C. jejuni mrfA mrfB mrfE napA napG napH napB napL napD nrfA nrfH subsp. jejuni 11168 as reference) C. curvus 04905 04900 04895 07105 07100 07095 07090 07080 07075 - - strain 525.92 C. concisus 1283 1282 1281 0868 0867 0866 0865 0863 0862 - - strain 13826 C. concisus 0984 0983 0982 0641 0642 0643 0644 0646 0647 - - strain ATCC 33237 C. concisus 1012 1011 1010 0645 0646 0647 0648 0650 0651 - - strain P2CDO4 C. gracilis 05995 05990 05985 R7030 07035 07040 07045 - - - - strain ATCC 33236 C. hominis - - - 08030 08035 08040 08045 - - 00645 00650 strain ATCC BAA-381 C. showae 07950 07955 07960 08730 08735 08740 08745 08755 - 04695 04700 strain B91_SC C. - - - 03735 03730 03725 03720 03710 - 01390 01385 ureolyticus strain RIGS 9880 134

Supplementary Table S16b: NCBI locus tags for genes involved in use of electron acceptors of human-hosted Campylobacter species (Oxygen, SN-oxide, Tetrathionate)

Oxygen SN-Oxide Tetrathionate Gene (with C. cj1490c cj1489c cj1488c cj1487c cj1186c cj1185c cj1184c cj0081 cj0082 cj0264c cj0265c C8J_0815 C8J_0040 ttrD ttrE jejuni subsp. ccoN ccoO ccoQ ccoP petA petB petC cioA cioB torA torC tsdA jejuni 11168 as reference) C. curvus 01375 01380 01385 01390 01505 01500 01495 09755 09760 07410 04495 05865 - - - strain 525.92

C. concisus 0723 0724 0725 0299 1921 1920 1919 0906 0905 1119 1398 - - - - strain 13826 C. concisus 0296 0297 0298 0726 1599 1598 1597 1795 1796 0015 0067 - - - - strain ATCC 33237 C. concisus 0272 0273 0274 0275 0249 0250 0251 1880 1881 1098 1099 - - - - strain P2CDO4 C. gracilis - - - - 08460 08465 08470 09515 09510 00075 00120 06265 - - - strain ATCC 33236 C. hominis 06520 06515 06510 06505 05785 05780 05775 02960 02955 07055 07050 - - - - strain ATCC BAA-381 C. showae 07685 07690 07695 07700 03345 03350 03355 06190 06185 10525 10520 - - 00655 00650 strain B91_SC C. ureolyticus 02515 02520 02525 02530 06555 06550 06545 00145 00140 08250 08245 - - - - strain RIGS 9880

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Supplementary Table S17: NCBI locus tags for genes involved in oxidative and nitrosative stress defense mechanisms of human-hosted Campylobacter species (Prefixes for locus tags are as follows: C. curvus strain 525.92: CCV52592_RS; C. concisus strain 13826: CCC13826_; C. concisus strain ATCC 33237: CCON33237_; C. concisus strain P2CDO4: CCS77_; C. gracilis strain ATCC 33236: CGRAC_RS; C. hominis strain ATCC BAA-381: CHAB381_RS; C. showae strain B91_SC: E4V70_RS; C. ureolyticus strain RIGS 9880: CUREO_RS)

ahpC bcp tpx docA/cj0358 dps katA mdaB msrA/B rrc sodB cgb ctb nrfa nosz norz C. curvus strain 07905 01165 08870 00605 06810 - - 05685 N 09565 - - - 09020 03415 525.92 C. concisus strain 0985 2112 0323 1108 1456 - 1626 1633 1910 0328 - - - 1535 1729 13826 C. concisus strain 1523 0275 1761 0161 0695 - 0755 0761 1802 1766 - - - 618 1707 ATCC 33237 C. concisus strain 0419 1678 1855 0114 0694 - 0798 0805 1901 1849 - - - 1788 1466 P2CDO4 C. gracilis strain 09495 01350 08210 11040 08795 04065 05525 07220 05740 09105 - - - 10245 04970 ATCC 33236 C. hominis strain 04725 02490 08305 01830 04645 - - 05085 01580 01800 - - 00645 - - ATCC BAA-381 C. showae strain 04490 05250 06080 05590 08790 - - 08870 - 06745 - - 04695 03665 05575 B91_SC C. ureolyticus strain 00775 00400 07225 02650 05710 00485 - 00495 03150 07360 - - 01390 - - RIGS 9880

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