The Molecular Phylogeny and Ecology of Spiral Bacteria from the Mouse Gastrointestinal Tract

Bronwyn Ruth Robertson

A thesis submitted for the degree of Doctor of Philosophy

School of Microbiology and Immunology The University of New South Wales Sydney, Australia

May, 1998 'Brief rejfection on test-tu.ies

'Ta~ a piece offire, a piece ofwater, a piece of ra66it or a piece of tree, or any piece ofa liuman 6eing, ~ it, slia~ it, stopper it up, k.._eep it wann, in tlie tfarl<:.i in tlie Bglit, refrigerate/, fet it stantf stifffor a wliife - yourselves far from stiff- 6ut that's tlie realjo~.

Jtjter a wliife you wok.._- ~ntf it's growing, a fittfe ocean, a fittle vofcano, a fittfe tree, a fittfe lieart, a fittfe 6rain, so fittfe you don't liear it lamenting as it wants to get out, 6ut that's tlie reafjo~, not liearing it.

'Ift.engo ·antf record it, a[[ tfaslies or a[[ crosses, some witli ~famation-mar/&, a[[ nouglits antf a[[figures, some witli ~famation-marf&, antf that's tlie reafjo~, in effect a test-tu6e is a device for changing nouglits into ~famation mar/&.

'Iliat's tlie reafJo~ wliicli mak.._es you forget for a wliile tliat reaffy you yourself are

In tlie test-tu6e

Mirosfav !Jfo{u6 Poems 'Before arufJtfter Acknowledgements

I extend my grateful thanks to the following people for their assistance and encouragement during my PhD studies.

Professor Adrian Lee for giving me the opportunity to carry out my PhD in his laboratory, for his supervision and for his enthusiasm for the "other helicobacters".

Dr Brett Neilan who has been my mentor and friend from the beginning of my research at l.JNSW, and only more recently my "official" co-supervisor. His encouragement, discussions about phylogeny and everything else were invaluable and made it all more fun.

Jani O'Rourke, for everything she has done to make the birth of a PhD easier, whether in her role as "mouse-murderer", electron microscopist, collage designer or anything else in between. This thesis could not have come together in the way it has without Jani's assistance, experience and advice. Also; Cora DeUngria, for being such an easy person to share a lab with. I think of our lab with happy memories and laugh at how three completely different people could have such a great lab environment.

All of the other members of the A. Lee research group past and present. They are; Stephen Danon, Fiona Buck, Fiona Radcliff, Tassia Kolesnikow, Susie Gekas, Phil Sutton and Kylie Fisher. Particular thanks also to John Wilson for acquiring mice for me from outside facilities and for being "quality". The "other Helicobacter group" are also thanked for their interest and encouragement, in particular, Stuart Hazell, Hazel Mitchell, Brendan Bums and Margaret Jorgensen.

Other scientists who contributed to the work presented in this thesis are; Kristine Boxen and Angela Higgins for automated sequencing sample analysis (UNSW Automated Sequencing Facility), Angelina Enno for sample processing (School of Pathology, UNSW) and Tony Gutierrez for advice on phase contrast photo microscopy (School of. Microbiology and Immunology, UNSW).

All of my friends from the School of Microbiology and Immunology who made the whole experience fun (mostly): In particular; the 10.30 coffee club, Brett, Amanda, Susie, Kylie, Daniel, Carolina, Melanie, Candy and Chuck; my fellow "PhD Fossils" in room 323; and those who got out before me, Beth, Serina, Deb and Slick.

My friends from Innominata for making me think hard about something else, other than my PhD, every Tuesday night. Especially Francis Dorman, David Russell and Moira Thompson for good company, vegetarian delights and tasty treats.

Finally, and most importantly my family. My very best friends; Libby, Hugh and Liz, and my parents who have supported and encouraged me in every way for my whole life.

Dad, you told me to go to Uni, but you never told me when to stop ...... I think I' 11 stop now. Certificate of Originality

I hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by another person, nor material which to a substantial extent has 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.

Bronwyn R. Robertson May 1998 Abstract

This thesis examined the hypothesis that the spiral bacteria of the gastrointestinal tract are a phylogenetically related group of organisms which represent part of the "autochthonous microbiota" of their host species. This hypothesis implies that such microorganisms are highly adapted to colonization of particular ecological niches. For this thesis, the organisms investigated were those having a close association with the mucosa! surface. Many of the previously described mucosa-associated spiral bacteria are found within the genus Helicobacter. The model host species examined was the mouse.

The mice used to investigate this hypothesis were obtained from four different animal facilities in Sydney. Spiral bacteria isolated from the gastrointestinal tract of mice were characterized using morphological appearance, PCR amplification profiles and 16S rRNA gene sequence comparisons. Results showed that there was wide variation in the number and presence of different types of spiral bacteria isolated from animals originating from different facilities. Spiral bacteria were cultured from three different phylogenetic groups including, helicobacters, desulfovibrio and a previously undescribed bacterial group.

A number of the helicobacters cultured were concluded to be from previously uncharacterized species. A new species of Helicobacter was isolated and described. One group of spiral bacteria isolated was shown represent a new genus based 16S rRNA gene sequence comparisons. This group was without a close phylogenetic affiliation to . any of the 11 recognized bacterial phyla. It also had only moderate levels of 16S rDNA sequence similarity to its closest phylogenetic relatives which were three bacterial species isolated from diverse environments.

Oligonucleotide primers specific for one species of Helicobacter and two different groups of non-helicobacters were designed and PCRs developed which allowed for rapid identification of members of these groups. A preliminary study to detect different types spiral bacteria in vivo was presented using a nested PCR approach involving universal bacterial 16S rDNA amplification followed by specific PCR.

Results obtained from both culture and in vivo detection studies presented evidence against the working hypothesis of this thesis. A new hypothesis was put forward that the spiral bacteria of the mammalian gastrointestinal tract are found in phylogenetically unrelated (or distantly related) clusters of organisms which have evolved with their host to colonize specialized ecological niches. Table of Contents

Chapter 1

Introduction and Literature Review ...... 1

1.1 Microbial Ecology of the Mammalian Gastrointestinal Tract ...... I 1.1.1 Microbial Colonization of Intestinal Surfaces ...... 2 1.1.2 Colonization of Intestinal Surfaces by Spiral Bacteria ...... 2 1.1.3 Intestinal Spiral Bacteria in Rodents ...... 3 1.1.4 The First Helicobacter species: a Phylogenetically Related Group of Morphologically and Ecologically Similar Bacteria ...... 8 1.2 Microbial Phylogeny...... 9 1.2.1 Molecular Chronometers ...... I 0 1.2.2 Ribosomal RNA ...... 12 1.2.3 Inferring Phylogenetic Relationships from Molecular Sequences ...... 16 1.2.4 Application of Phylogenetic Analysis to Molecular Ecology ...... 17 1.3 Helicobacter Phylogeny, Taxonomy and Ecology ...... 18 1.3.1 Gastric Helicobacter species ...... 19 1.3.2 Lower Bowel Helicobacter species ...... 19 1.4 Murine Helicobacter species...... 20 1.4.1 Helicobacter muridarum ...... 20 1.4.2 "Flexispira rappinf' ...... 22 1.4.3 Helicobacter hepaticus ...... 22 1.4.4 Helicobacter bilis ...... 23 1.4.5 Helicobacter trogontum ...... 23 1.5 Hypotheses and Aims ...... 25

Chapter 2

Materials and Methods...... 2 6

2.1 Culture media ...... 26 2.1.1 Horse blood agar (HBA) ...... 26 2.1.2 Campylobacter selective agar (CSA) ...... 26 2.1.3 Brain Heart Infusion-Glycerol medium (BHIG) ...... 26 2.2 Helicobacter strains and culture ...... 27 2.2.1 Helicobacter strains ...... 27 2.2.2 Helicobacter cultivation ...... 27 2.2.3 Cryopreservation ...... 27 2.3 Isolation of spiral bacteria ...... 27 2.3.1 Direct culture ...... 27 2.3.2 Selective filtration culture ...... 28 2.4 Mouse tissue fixation ...... 28 2.4. l Fixative ...... 28 2.5 Phase contrast photomicroscopy ...... 28 2.6 Electron microscopy...... 28 2. 7 Biochemical testing of bacterial isolates ...... 29 2. 7. l Rapid urease ...... 29 2. 7 .2 Catalase ...... 29 2. 7.3 Oxidase ...... 29 2.7.4 API-Campy ...... 29 2. 7 .5 Susceptibility to antimicrobial agents ...... 30 2.8 Preparation of PCR template ...... 30 2.8. l Genomic DNA extraction ...... 30 2.8.2 Cell lysis using Xanthogenate ...... 31 2.9 O/igonuc/eotide synthesis and purification ...... 31 2.10 DNA amplification by Polymerase Chain Reaction (PCR) ...... 34 2.11 Agarose gel electrophoresis and DNA visualization ...... 34 2.12 Purification of amplified DNA...... 35 2.12.l Prep-A-Gene® ...... 35 2.12.2 Wizard™ PCR preps ...... 35 2.13 DNA sequencing ...... 36 2.13. l Cycle sequencing of amplified DNA ...... 36 2.13.2 Automated DNA sequencing ...... 37 2.14 Phylogenetic inferences from 16S rDNA sequences...... 39 2.15 Preparation of PCR template from paraffin embedded tissue ...... 39 2.15. l Sectioning and deparaffinization ...... 39 2.15.2 DNA extraction from fixed sections ...... 40

Chapter 3

Development and Validation of PCRs for Detection of Helicobacter species ...... 41

3.1 Background...... 41 3.2 Genus specific PCR ...... 41 3.2. l Results ...... 42 3.2.2 Discussion ...... 43 3.3 Helicobacter muridarum specific PCR...... 46 3.3. l Results ...... 47 3.3.2 Discussion ...... 50 3.4 Helicobacter bilis, Helicobacter hepatic us and Helicobacter trogontum specific PCRs ...... 50 3 .4.1 Results ...... 51 3 .4.2 Discussion ...... 51 3.5 Summary ...... 53

Chapter 4

Isolation and Molecular Characterization of Mouse Spiral Bacteria ...... 5 5

4.1 Background...... 55 4.2 Experimental design ...... 56 4.3 Results ...... 58 4.4 Discussion and Summary ...... 72 Chapter 5

Ribosomal RNA Gene Sequences and Inferred Phylogenetic Relationships Among Helicobacter Species ...... 7 5

5.1 Background...... 75

5.2 Experimental design ...... 75 5.3 Results ...... 76 5.4 Discussion and Summary ...... 89

Chapter 6

Description of a new Helicobacter species...... 9 2

6.1 Background...... 92 6.2 Experimental design ...... 92 6.3 Results ...... 92 6.3. l Isolation and Growth Characteristics ...... 92 6.3.2 Ultrastructure ...... 93 6.3.3 Biochemical and Physiological Characteristics ...... 93 6.3.4 Phylogenetic Analysis ...... 93 6.4 Discussion and Summary ...... 97 6.5 Comparison to Helicobacter rodentium ...... 97 6.5.1 H. rodentium specific PCR ...... 98 6.5.2 Ultrastructural, Physiological and Biochemical Comparisons ...... 103

Ribosomal RNA Gene Sequences, Inferred Phylogenies and PCR Identification of Non-Helicobacter, Spiral Bacteria

Chapters 7 and 8 ...... 105 Background ...... 105 Experimental Design ...... 105

Chapter 7

Phylogenetic Identification of Bacterial Group J: "fat S" ...... 10 7

7.1 Experimental Design ...... 107 7.2 Results ...... 107 7.2.1 16S rDNA sequence and phylogenetic analysis ...... 107 7.2.2 PCR design ...... 112 7.3 Discussion and Summary ...... 115 Chapter 8

Phylogenetic Identification of Bacterial Group I: a new Genus Within the Bacterial Domain with an Unusual Phylogenetic Affiliation ...... 118

8.1 Experimental Design ...... 118 8.2 Results ...... 118 8.2.1 16S rDNA sequence and phylogenetic analysis ...... 118 8.2.2 PCR design ...... 125 8.4 Discussion and Summary ...... 129

Chapter 9

Analysis of the Distribution of Spiral Bacterial in vivo using Molecular Techniques! 3 0

9.1 Background...... 130 9.2 Experimental Design ...... 131 9.2.l Samples ...... 131 9.2.2 PCR design ...... 131 9.3 Results ...... 132 9.4 Discussion and Summary ...... 140

Chapter 10

General Discussion...... 14 2

JO. I Phylogeny of spiral shaped bacteria ...... 142 10. 1.1 Helicobacter species diversity ...... 142 10.1.2 Homology vs. Morphology: implications for Helicobacter taxonomy ...... 143 10.1.3 Helicobacters with polymorphic rRNA operons ...... 150 10.1.4 Identification of a novel bacterial genus ...... 151 10.2 Microbial Ecology: culture vs. nested PCR detection ...... 152 10.3 Spiral Morphology: a new hypothesis ...... 153

Summary of Major Findings ...... 15 5

Future Work ...... 15 6

References...... 158

Appendix...... 175 List of Abbreviations

ABHU The UNSW Animal Breeding and Holding Unit ANGIS Australian National Genomic Information Service bp base pairs CMRI The Children's Medical Research Institute CSA Campylobacter selective agar DNA deoxyribonucleic acid EMBL European Molecular Biology Laboratory ddNTP dideoxynucleotide triphosphate dNTP deoxynucleotide triphosphate GIT gastrointestinal tract HBA horse blood agar HRI The Heart Research Institute IBD Inflammatory Bowel Disease IVS intervening sequence LSU large subunit mol mole NCBI National Center for Biotechnology Information PCR Polymerase Chain Reaction Q/S Quackenbush/Swiss RDP Ribosomal Database Project rDNA ribosomal DNA RNA ribonucleic acid rRNA ribosomal RNA SPF Specific Pathogen Free ssu small subunit Tm theoretical melting temperature of oligonucleotide primers UNSW The University of New South Wales UNSW The UNSW Microbiology Animal Facility UV ultraviolet Chapter 1 1

Chapter 1

Introduction and Literature Review

1. 1 Microbial Ecology of the Mammalian Gastrointestinal Tract The mammalian gastrointestinal tract (GIT) is one of the most complex environments in terms of ecological and microbial diversity (83, 85). In recent years there has been a renewed 1nterest in the study of microbial diversity, in a wide range of environments, from an ecological viewpoint. This can be mainly attributed to improved methods of detecting and identifying microorganisms without the ne~d to culture (1). The study of so-called "molecular ecology" has meant that microorganisms can be recognized and their functions studied, at a DNA or RNA level, based on the detection of specific nucleic acid sequences usually within the ribosomal RNA (rRNA) or its corresponding gene sequence (4, 115, 122). It is perhaps surprising, therefore, that there has been so little research focused on the molecular ecology of the mammalian gastrointestinal tract.

Most of the early studies in microbial ecology of the gastrointestinal tract focused on those species which could be cultured from faeces. These studies recognized, therefore, those microorganisms which originated in the lumen contents of the large bowel (24 ). Even using conventional culture-based methods, human faeces are estimated to contain 10 11 bacteria per gram belonging to upwards of 400 different species ( 107), and these species are only representative of those inhabiting the lower bowel (83). Other studies, using animal models, examined different regions of the gastrointestinal tract combining histological examination with culture of homogenates from corresponding regions of the GIT (143). The types of bacterial species recognized during these early studies included lactobacilli, coliforms, enterococci and anaerobes and resulted in advances being made in culture media and anaerobic incubation techniques.

From the late 1960s through to the early 1980s the microbial ecology of the intestinal tract of man and animals (particularly mice) was examined in detail. Dubos et al. showed that in mice the whole gastrointestinal tract harbors, throughout life, an abundant bacterial flora, the composition of which is characteristic for each section of the tract, and that some bacterial species are intimately associated with the wall of the various organs (24 ). A distinction was also made between what were generally called "normal microbiota" and the new concept of "autochthonous microbiota". Together these two groups of organisms made up what was termed the "indigenous microbiota". The "autochthonous microbiota" were those microorganisms which had achieved, through a long period of evolutionary Chapter 1 2

relationship and adaptation, an almost symbiotic relationship with the host, and persisted at a constant high level throughout the life of the host. On the other hand, the "normal microbiota" were those microorganisms which were ubiquitous within a population but were not necessarily present in another population of the same species (24 ). It was shown that the "normal microbiota" differed qualitatively from one animal colony to another regardless of the genetic makeup of the animals (143).

1.1.1 Microbial Colonization of Intestinal Surfaces . One of the environments within the mammalian gastrointestinal tract which was widely ignored was the intestinal surfaces which are colonized by large numbers of microorganisms, further contributing to species diversity (83). For bacteria to survive as a stable population in this competitive environment, they must evolve strategies which give them a selective advantage over other species. Also, they must develop mechanisms to resist the rapid flow rate of intestinal contents and turnover of epithelial cells (82). Many such specific adaptations have been observed and described in bacteria colonizing rodent intestinal surfaces (Figure 1.1). Such adaptations include specific attachment to epithelial cells and motility (83).

The lubricating mucus lining of the mammalian gastrointestinal tract and the epithelial crypts containing the mucus secreting goblet cells provide a further example of an ecological niche which requires microorganisms to develop specialized mechanisms in order for them to colonize (82). Bacteria colonizing this niche must be able to overcome the flow of mucus out of the crypt onto the epithelial surface. A universal feature of organisms found to colonize the intestinal crypts is a spiral morphology (85). As early as 1966 Brock hypothesized that a helical movement would enable organisms to move through a viscous medium which impeded bacteria with different motilities (12).

1.1.2 Colonization of Intestinal Surfaces by Spiral Bacteria

The presence of gastric spiral bacteria in animals was recorded by Rappin in 1881 (133), Bizzozero in 1893 (10) and Salomon in 1898 (141). Attempts by Salomon to culture these organisms on artificial media were unsuccessful. However, numerous studies throughout the first half of this century reported the presence of spiral bacteria in the stomachs of both animals and man, often in close association with particular cell types (Figure 1.2). Over the last 100 years, therefore, mucus-associated spiral bacteria have been observed to be widespread throughout the animal kingdom, and Doenges saw spiral bacteria in almost 50% of human stomachs from accident victims in 1939 (22). The true significance of these bacteria remained, however, unknown until the discovery and culture of Helicobacter pylori from human gastric tissue in 1984 ( 101, 179). Chapter 1 3

these bacteria remained, however, unknown until the discovery and culture of Helicobacter pylori from human gastric tissue in 1984 (101, 179).

1.1.3 Intestinal Spiral Bacteria in Rodents From the 1960s up until the discovery of H. pylori, spiral microorganisms were also observed, and studied in some detail, in the mucus layer of the rodent intestinal tract (19, 29, 58, 61, 65, 89, 142, 144).

Savage et al. described 3 types of spiral bacteria using electron microscopy on negatively stained preparations of mucosal epithelial washings ( 144). Two of these types possessed single, monopolar or bipolar flagella, while the third had bipolar tufts of flagella and periplasmic fibres. Many of these bacteria localized in the layers of mucin on the epithelium of the caecal and colonic mucosa. Davis et al. observed Borellia-like organisms and flagellated spiral shaped organisms, both with and without axial or periplasmic fibres, when examination was made of the caecal mucosa of normal rats ( 19).

In 1977-197 8 Leach, Phillips and Lee made a survey of the spiral bacteria found to colonize the intestinal tract of mice and rats (80, 130). Although culture success was limited, phase contrast microscopy enabled differentiation of eight different morphology types. The distribution of each type, at six different locations along the intestine, was carefully mapped using phase microscopy examination of mucosal scrapings. It was found that different organisms were associated with different regions of the intestinal tract.

Differences in the distribution of the mucosa-associated bacteria naturally divided the small intestine of rats into three regions ( 130). These regions were, more than 50 cm from the ileal-caecal junction, within 50 cm of the ileal-caecal junction and within 10 cm of the ileal-caecal junction (Figure 1.3). Similarly, the mucosa-associated bacteria of the caecum and large intestine were also characterized (Figure 1.4). Light and electron microscopy revealed that these organisms were often localized in the crypts of the epithelium with the general conclusion that spiral organisms were found at the base of the crypts almost to the exclusion of any other bacteria, in mixtures with other organisms higher up in the crypts and in lower numbers in the surrounding mucus (80, 81, 130). Figure 1.1 Diagrammatic representation of the surface-associated microbiota of the gastrointestinal tract. (A) stomach lining covered with lactobacilli, (B) Mucosa of terminal ileum with filamentous bacteria inserted into the tissue, (C) crypts of the ileal mucosa packed with spiral bacteria, (D) caecal crypts packed with spiral bacteria of a different morphology to ileal organisms, (E)° mucus secreting goblet cells lined with spiral bacteria inserted into cell surface. Figure reproduced from reference (83). Chapter 1 4

C Figure 1.2 Collage of early illustrations of non-Helicobacter pylori spiral bacteria in the stomachs of a number of animals. (A) Salomon's drawings of bacteria associated with the mucosa of individual cells in the stomachs of mice which were colonized with spiral bacteria from a dog stomach (141), (B) Salomon's hanging drop preparation of mucus from the stomach of a cat ( 141 ), (C) Bacteria in the dog stomach (76), (D) section of the pylorus of a cat showing bacteria in the lumen of a duct (96). Photograph reproduced from reference (84). Chapter 1 5

,;..'- ..~ ... .-\

I- - ... ,. I ... • '-::-, "I ... -, ... .., ' ' .. --­

., ,.. .'}. , \ d . ,~/.,/., ,1 /f/! I ,, .' 'i.

lo, .·1i, ·if , b ,, . ,,,_ 0 l~ i · ~ Iii•, // Ji //.• ft. . ' ,_--) ., ,..,I~,. ""~ '1 . .' t'Z;f // kf[ fI • ..- •1 "' 1y1ll)' · ~ ,.. ,I J ".~f» .. ,,,, . /,' I~ 'J{jf . ' ., .,. /ff: (I. ' •J I .;. ' ' ...

!'> ' .... \ ... '- --· A 1:· " Figure 1.3 (Top) Diagram of the intestine of the rat, showing the location of mucosa-associated microorganisms, with descriptive names given to the different morphology types. St, short, fat spiral; Cr, small, crescent shape; Fil, filamentous organism; Rs, rigid spiral; B, Borrelia-like; Fu, fusiform; Bi, Borrelia-like, smaller than B and Ss, small S shape. (Bottom) mucosal scrapings from the rat small intestine showing different types of mucosa associated microorganisms. (A, B) St, (C) Cr, (D, E, F) Fil. Figure and photographs reproduced from reference (130). Chapter 1 6

small intestine large intestine

I I..__ 1 : -,,I .. 10cm

Key

.,..,, B ~ St Cr ... Fu Fil n Bi ---- Rs ---- Ss -

B Figure 1.4 (Top) Diagram of the intestine of the rat, showing the location of mucosa-associated microorganisms, with descriptive names given to the different morphology types. St, short, fat spiral; Cr, small, crescent shape; Fil, filamentous organism; Rs, rigid spiral; B, Borrelia-like; Fu, fusiform; Bi, Borrelia-like, smaller than Band Ss, small S shape. (Bottom) mucosal scrapings from the rat caecum and large intestine showing different types of mucosa associated microorganisms. (A) Rs, (B) B, (C) Ss, (D) Fu, (E) Bi. Figure and photographs reproduced from reference (130). Chapter 1 7 small intestine large intestine

I I I..._ I 1 I I -.·- Key

St ,,.,,, B ~

Cr w Fu ~ Fil n Bi -- Rs --- Ss - A ,,

..... '

c l

... • ,

. '(, • >'\ ' ;• -- .... • .. . ~

. ~..,~ .. .. . -...... " ~ Chapter 1 8

This study and others led Lee to take the hypothesis of Brock a step further and suggest that the spiral bacteria found in the gastrointestinal tract of mammals have evolved with their hosts and have a selective advantage over other morphology types (83). The suggestion was that these microorganisms were evolutionarily adapted to colonization of the mucus lining of the gastrointestinal which covers the epithelial layer and represented true "autochthonous microbiota".

By this time a number of these mucosa-associated spiral bacteria had been cultured in the laboratory enabling the motility hypothesis to be tested in vitro. Experiments showed that a spiral or cork-screw motility was, in fact, more effective in viscous environments than the motility displayed by rod shaped organisms (34, 64).

1.1.4 The First Helicobacter species: a Phylogenetically Related Group of Morphologically and Ecologically Similar Bacteria The discovery and culture in 1984 (101) of the bacterium which is known today as H. pylori reinvigorated the field of gastrointestinal microbiology and led to the establishment of a new genus of bacteria with the transfer of H. pylori and Helicobacter mustelae from the genus Campylobacter in 1989 (57). Both of these bacteria were gastric mucosa­ associated species and their impact lay in their upset of the dogma that the stomach was an essentially sterile environment, and in the case of H. pylori, that common gastric and duodenal ulcers were caused by a bacterial infection and not, as was widely held, by a stressful lifestyle.

These bacteria were not, however, the first Helicobacter species to be cultured. In 1980 an unidentified bacterium was cultured from the intestinal crypts of rodents ( 131 ). This organism was also described in 1972 using electron microscopic examination of fixed rat intestinal specimens (29). Subsequently, the name Helicobacter muridarum was given to this organism after it was shown to be closely related phylogenetically to H. pylori and H. mustelae (94 ). This finding gave further support to the working hypothesis that the spiral bacteria of the gastrointestinal tract have evolved with their host and also that these bacteria represented a phylogenetically related cluster of bacteria. The diversity within the genus was hypothesized to be the result of bacterial divergence in different environments, that is different hosts and different sites of colonization within a host, since the original spiral bacteria colonized the original gastrointestinal tract.

Since the establishment of this genus less than 10 years ago, the number of recognized species has risen dramatically, standing at around 19 formally named species plus a number of organisms which may belong to new, or are strains of already named, species. Chapter 1 9

The normal site of colonization for all of these bacteria is the gastrointestinal tract; with species being divided between gastric and intestinal colonizers. Species have been isolated from a wide variety of mammals and birds with the intestinal colonizers making up well over half of the species. The rapid expansion of this genus, containing phylogenetically related bacteria all showing a specialized adaptation to colonization of gastrointestinal mucus, again gave support to the hypothesis which is the working hypothesis of this thesis that; • the spiral bacteria of the gastrointestinal tract are phylogenetically related, having evolved with their host to colonize a particular ecological niche.

In order to have a fuller understanding of the group of bacteria under study, from a phylogenetic and ecological viewpoint, and the concepts involved in the elucidation of evolutionary relationships between bacteria, these two areas are reviewed below.

1. 2 Microbial Phylogeny The working hypothesis of this thesis is within an evolutionary or phylogenetic framework. Studies in microbial diversity and ecology are, almost universally, also concerned with bacterial classification and taxonomy. The taxonomic classification of organisms should reflect their phylogenetic or natural evolutionary relationships. This situation is slowly being realized in microbiology which for many years had a taxonomy based on evolutionarily uninformative characteristics.

Phylogenetic classification is that which aims to group organisms according to their ancestral lineage, that is, their evolutionary relationships. This is in contrast to phenetic classification which is based on observable '(phenotypic and genotypic) characteristics of existing organisms regardless of their ancestral lineage (151). Study of natural (phylogenetic) relationships between living things is one of the most fundamental in all biology. Traditionally, microbiologists have unfortunately had to rely on comparisons between a limited number of morphological characteristics or the slightly more abundant physiological traits which led to a classification system grounded firmly in phenotypic comparison (155, 190). In contrast with higher organisms, plants and animals, which are rich in such characteristics, the determination of phylogenetic relationships between prokaryotes based on such characteristics was impossible. Microbial morphologies and other characteristics are too simple or uninterpretable to serve as a basis for a phylogenetically valid classification ( 191 ).

Phenotypic classification, based on gross morphological and cytological observations, even failed to reveal that the division of life at the highest taxonomic level was into three Chapter 1 10

primary domains, Eucarya, Bacteria andArchaea (189, 190, 196, 197). These divisions were immediately evident from molecular sequence data once it became available ( 195). Both of the conventionally accepted earlier views, the five-kingdom taxonomy or the prokaryote-eukaryote dichotomy, failed to represent this primary division of life into three domains with differences of a more fundamental kind than were reflected in either of these views (197).

1.2.1 Molecular Chronometers _The division of life into three primary domains was revealed by nucleic acid sequence comparisons from particular types of molecules which have been called "molecular chronometers". The concept of a "molecular evolutionary clock" was first suggested by Zuckerkandl and Pauling (202) and comes from the observation in molecular evolution that, for a particular protein or DNA sequence, the rate of amino acid or nucleotide substitution is constant among diverse lineages over time. The sequences of such molecules are, therefore, an indicator time and evolutionarily significant. Random changes in DNA sequences are occurring constantly and the vast majority of these are not reflected by any visible phenotypic or functional change since for every definable phenotypic function there are tens to thousands of evolutionary independent sequence residues ( 100, 118). These neutral genotypic changes are therefore an indicator of time (190, 191).

Not all DNA sequences are equally useful, however, as molecular chronometers. Molecules must fulfill several criteria before being considered useful chronometers and therefore phylogenetic indicators. They must perform an identical function in all organisms which results in a high level of conservation at the level of DNA sequence (functional constancy), be universally distributed within the organisms of study and the size of the molecule must be such that it is able to provide an adequate amount of information, that is, it can accumulate enough random or neutral changes in its DNA sequence without altering function over the time period of study (100, 190, 191). Measured differences in the DNA sequence of such molecules in different organisms are a relative measure of time since the two shared a common ancestor. Within such molecules, however, rates of substitution at different positions usually vary. As such, there is not a linear relationship between sequence dissimilarity and time since two particular organisms diverged. This, although it makes analysis more difficult, can be an advantage since the highly conserved regions reveal distant relationships while the more variable regions give information at the lower phylogenetic levels ( 100). Figure 1.5 Rooted universal phylogenetic tree showing the three domains, Bacteria, Archaea and Eucarya based on rRNA sequence comparisons. The position of the root was determined by comparing sequences of pairs of paralogous genes that diverged from each other before the three primary lineages emerged from their common ancestral condition. Figure reproduced from reference (192). Chapter 1 11

Bacteria Archaea Eucarya

Animals

Euryarchaeota Methanosarcina Methano- Halophiles Purple bacterium bacteria Chapter 1 12

Determination of a nucleic acid sequence from a particular molecule within an organism in order to infer phylogenetic relationships carries with it some additional assumptions. One such assumption is that the phylogeny of the molecule is the same as the phylogeny of the organism. A molecule which satisfies the first criterion for a molecular chronometer, functional constancy, as well as not being exchanged between organisms can be assumed to reflect whole organism phylogeny (122).

The difficulty in finding useful molecular chronometers is three fold. Most DNA sequences are changing too quickly for deep evolutionary relationships to be determined. Few molecules retain strict functional constancy. Additionally, few functions are truly universally distributed and can be used, therefore, only on limited numbers of organisms (191).

Comparison of gene sequences from common essential proteins has provided much useful information regarding the phylogeny of all living things. Such molecules include the c-type cytochromes, A TPases and globins (20, 190). The molecule which has become the champion of the microbial phylogenist and ecologist however, is the ribosomal RNA (rRNA). In addition to revealing the 3 domain structure of life, analysis of small subunit rRNA sequences has shown the domain Bacteria to be divided into at least 11 subgroups or "phyla" ( 118, 190, 191 ).

1.2.2 Ribosomal RNA The ribosome is an essential component of any living cell, being involved in the translation process of protein synthesis. The bacterial ribosome (70S) contains one molecule of each of three types; 5S, 16S and 23S, plus some 55 accessory proteins which are contained in two subunits (30S and 50S). The 16S rRNA occurs in the small subunit (SSU) while the 5S and 23S rRNA occur in the large subunit (LSU). rRNA molecules can adopt complex secondary structures with extensive intramolecular base-pairing resulting in the formation of "hairpins" and "stem-and-loop structures" (176, 190). Certain regions of rRNA sequences have been highly conserved during evolution, and sequence homology studies are widely used to indicate phylogenetic relationships. rRNA sequences, particularly of the SSU are the most widely analyzed molecular chronometer.

The usefulness of rRNA sequence comparisons for inferring phylogenetic relationships lies in the fulfillment of the criteria for a molecular chronometer; functional constancy, universal distribution and size (information content) (190). The function of all rRNAs is identical since they are involved in translation and protein synthesis. As a result, the 3- dimensional structure of rRNAs show only minor, but highly significant, variation. Figure 1.6 Phylogenetic tree of the bacterial domain based on 16S rRNA sequence comparisons of one representative sequence from each of the 11 bacterial phyla. Sequence depth of some groups was separately calculated and are indicated by shaded wedges. Figure reproduced from reference ( 190). 1 Chapter 13

bocteroides­ f lovobacterio spirochetes deinococc; ond relatives and Planctomyces relatives

bacteria Gram'1>0sitive

Purple - ...... ··' ',""'., ' ~ . bacteria Chapter 1 14

Selectively neutral changes are, however, continuously occurring at the level of the primary sequence, while secondary and tertiary structure are maintained with function (62). Furthermore, rRNAs are large molecules consisting of many discrete domains. Nucleotide positions within the molecule change at very different rates allowing for determination of a very wide range of phylogenetic relationships from the species level to the most distant interdomain relationships (155, 190). Within the nucleotide sequence there are regions which are termed universally conserved, semi-conserved and hypervariable ( 176).

In terms of the gene being laterally transferred between organisms, since every viable organism must already have a translation system there can exist no selective pressure to pick up a new one, and there is probably some disadvantage in replacing an essential cellular mechanism with one from a different organism (122). The nucleotide sequence of these molecules, therefore, is assumed to reflect whole organism phylogeny.

Initially the process of oligonucleotide cataloguing was used to group organisms phylogenetically. This technique was based on comparisons of complex patterns obtained from digestion of the 16S rRNAs with T1 ribonuclease into oligonucleotides of up to approximately 20 bases (38, 195). Patterns were characteristic of a particular bacterial species and could be used to select signature sequences for a particular taxon or roughly to estimate evolutionary distances. Later studies involved direct sequencing of isolated rRNA through the use of the enzyme reverse transcriptase (78).

Today 16S rRNA sequences are usually inferred from the sequence of their corresponding gene, the 16S rDNA, which can be readily amplified using polymerase chain reaction (PCR) technology (28, 181). The amplified product can be directly sequenced when the starting DNA is from a pure culture, or, in the case of a complex mixture of organisms or uncultured material, cloned prior to sequencing (17, 56, 153, 188). Figure 1.7 Schematic representation of the Escherichia coli 16S rRNA secondary structure. The primary structure has been highlighted to show the relative positions of phylogenetically significant sequence domains. Universally conserved (thick lines), intermediate c0nservation (normal lines) and hypervariable domains (dashed lines) are indicated. Numbers indicate the positions of nucleotides from the 5' end of the 16S rRNA molecule, according to the E.coli sequence (14). Figure reproduced from reference (176). Chapter 1 15

, ' \ ,' ,, .._ __ -----·~J ", '

, ... - ... ____ ..,J : __ ... --- - ""I, ... _,,,-- --510---- ..... _,, ..-- --

' ' ' '

1300

1400 5'

1500

...... _, 3'

' ,J'

'', r I I ',_, '

,-, ,-- __ _, ...... __ J .. - ... _. , ____ ..,--, ' .. -1-, ,;---, ...... ,, ,,r-----.._.,' 200\ , I r I' ' .... / Chapter 1 16

1.2.3 lnferring Phylogenetic Relationships from Molecular Sequences Phylogenetic trees provide a schematic representation of phylogenetic relationships which can be inferred from molecular sequence data (115). There are two main steps involved in the reconstruction of a phylogenetic tree which are summarized below.

1.2.3.1 Homologous Sequence Alignment The first and critical step in molecular phylogenetic analysis is the proper alignment of the sequences (122). For sequences to be properly aligned, only homologous characters that have arisen from a common position within the ancestor gene are arranged in columns. The alignment which maximizes sequence similarity is not necessarily the same as the one which is comparing homologous residues arising from a common ancestor. Here, it is important to note the difference between homology, which implies a common ancestry, and similarity, which may be either the result of common ancestry or a chance occurrence (118). Where there is uncertainty regarding sequence homology at a position, then the position should be removed from the analysis. The result being that, as nearly as possible, only positions with a common ancestry are compared. The extent to which assumptions of sequence homology are incorrect will reduce the accuracy of the conclusions drawn. Since a number of almost completely conserved regions exist within the 16S rRNA sequence, the task of alignment is made easier (176).

The variable and hypervariable regions of the 16S rRNA sequence are more difficult to align based purely on primary sequence structure. Here secondary structure of the molecule can assist in obtaining reliable alignments (100). Furthermore, the rapidly growing number of 16S rRNA sequences available through computer databases has been the catalyst for the formation of pre-aligned sequences with which it is possible to align new sequences. Such alignments are readily obtained through the Ribosomal Database Project (RDP) ( 116, 117) and rRNA at Antwerp ( 171 ).

1.2.3.2 Tree Inferring methods There are three main types of tree-inferring methods currently being widely used. These are distance matrix (37), maximum parsimony (36) and maximum likelihood (30).

In the distance matrix method, pairs of DNA sequences are compared for all lineages of a tree in order to estimate the "evolutionary" or "genetic" distance separating sequence pairs (37). This distance is usually the average number of point mutations per sequence position and can be corrected for multiple changes at a single position by a number of different methods, the most popular of which is that described by Jukes and Cantor (73). Chapter 1 17

The tree is then drawn which most closely represents the calculated pairwise distance estimates (115).

Maximum parsimony methods search for the tree topology which requires the fewest mutational events. The underlying assumption here is that sequences were derived from their ancestors by acquiring a minimal number of changes. From the aligned sequences the sum of changes which must have occurred to produce these sequences is determined and the tree with the shortest overall length (the minimal number of changes required) is the most parsimonious. If the amount of divergence is very different in different lineages then this method tends to misplace organisms (36).

Maximum likelihood methods take into account a model of sequence evolution and analyzes sequences on a site by site basis which can be derived from the data (30). The derived tree is that which maximizes congruence between the evolutionary model and the observed data. The advantage of this method is that the frequency and type of changes at individual positions are considered, which is particularly useful for rRNA sequence analysis. This method, however, is extremely computationally intensive and therefore not frequently used.

1.2.4 Application of Phylogenetic Analysis to Molecular Ecology Phylogenetic studies based on rRNA gene sequence comparisons have significantly altered the study of microbial ecology and resulted in the birth of the field of molecular ecology. Specific detection of nucleic acid sequences requires the application of either hybridization probes or amplification primers based on a knowledge of their complementary target sequences (56). The approach adopted by microbiologists has been the selection of a singe target gene, the 16S rDNA, for both phylogenetic classification and identification or detection of all bacteria ( 190). The unique combination of universally conserved sequences interspersed with regions of great sequence variation allows for the design of hybridization probes or amplification primers of any specificity ranging from species specific to universal (55, 56, 78). The selection of a single gene has also meant a rapidly increasing database of near complete sequences (GenBank, EMBL, RDP) which in tum facilitates probe and primer design.

The polymerase chain reaction (PCR) provides a rapid method for the direct amplification of specific DNA sequences for further analysis (110). DNA from small quantities of environmental samples can be selectively amplified yielding enough genetic material for Chapter 1 18

direct detection, hybridization, restriction endonuclease digestion and DNA sequencing (159).

The application of universal, 16S rRNA gene, amplification primers ( 181) has facilitated the detection and identification of uncultured organisms (99, 135, 153) and analysis of mixed microbial communities (54). Conserved regions at the 5' and 3' ends of the gene allow for in vitro amplification of any bacterial 16S rRNA gene (181). Direct DNA sequencing of amplified products provides easy access to almost complete sequences (78, 183).

Analysis of amplified rRNA gene sequences has given us the ability to study complex mixtures of microorganisms without the need to culture (56, 115, 122, 176, 188). Current estimates predict that we are only able to culture between 0.001 and 15% of microorganisms in a particular environment compared with numbers for total cell counts, with most estimates for culturability falling between 0.1 and 1% (5). From an ecological point of view, the estimation of bacterial diversity based on culture is, therefore, completely unsatisfactory.

Amplification of all 16S rRNA genes in complex mixtures can then be further analyzed to determine the different bacterial types present (56). Various molecular techniques are adopted for post 16S rDNA amplification of mixed microbial communities, including cloning of products followed by DNA sequencing or restriction endonuclease digestion, direct hybridization with oligonucleotide probes designed to detect specific DNA sequences, and nested PCR amplification.

1. 3 Helicobacter Phylogeny, Taxonomy and Ecology Members of the genus Helicobacter are found within the epsilon subdivision of the class Proteobacteria (111). This separate bacterial lineage has also been defined as rRNA superfamily VI (173) keeping in line with the distinction ofrRNA superfamilies within the proteobacteria (20). Other genera found within this subdivision are Campylobacter and Arcobacter the two of which together make up the family Campylobacteraceae ( 172). In addition to these three genera, the epsilon subdivision also contains Wolinella succinogenes, "Flexispira rappini'', "Gastrospirillum hominis" and two generically misnamed Bacteroides spp. Both "F. rappini" and "G. hominis" are very closely related to other species within the genus Helicobacter but require further investigation before they can renamed as Helicobacter spp. Chapter 1 19

Broadly speaking, each of the 3 genera, Campylobacter, Arcobacter and Helicobacter, form a separate rRNA homology group within the superfamily VI (119, 165, 173). The taxonomy of this group of organisms is complex and rapidly evolving, primarily due to the rapid increase in the number of species ( 119). This is particularly true for the Helicobacter genus which has increased to having almost 20 members since its establishment 9 years ago.

H. pylori is the type species of the genus Helicobacter (57). Members of this genus are Gram negative, non spore-forming, helical, curved or straight rods. They are . microaerophilic and motile by flagella which can be single or multiple, sheathed or unsheathed, polar, bipolar or lateral. Helicobacter species are known to colonize the gastrointestinal tract of a wide range of mammals and birds (46). Identification is made difficult due to the scarcity of useful biochemical tests for distinguishing species ( 104 ).

1.3.1 Gastric Helicobacter species The human stomach, with low gastric pH, was considered, until the discovery and isolation of H. pylori, to be a predominantly sterile organ. This organism is now known to infect between 20 to 80% of adult human populations worldwide (105, 162). H. pylori plays a causal role in persistent, active, chronic gastritis and peptic ulcer disease (59, 88, 102) and is linked with the development of gastric adenocarcinoma and gastric mucosal associated lymphoma (40, 124, 199). Since 1984, new gastric Helicobacter species have been isolated from the stomachs of various animal species including non­ human primates, ferrets, cats, dogs and cheetahs (13, 26, 45, 63, 72, 126).

1.3.2 Lower Bowel Helicobacter species In addition to the discovery of H. pylori and other gastric helicobacters, an increasing number of Helicobacter species have been isolated from the intestinal tract of mammals and birds (21, 33, 42, 48, 51, 94, 104, 147, 149, 156, 157, 168, 178). Clinically important species include Helicobacter cinaedi and Helicobacter fennelliae, which have been linked to proctitis and colitis in immunocompromised individuals (33, 166), and Helicobacter westmeadii which was isolated from blood culture of AIDS patients ( 168). Helicobacter canis and Helicobacter pullorum have been isolated from the faeces of humans with gastroenteritis (15, 156, 160). H. canis has also been isolated from dogs with diarrhoea and a dog liver with multifocal necrotizing hepatitis (43). H. pullorum is also capable of infecting the livers of chickens, causing hepatitis (156). Helicobacter hepaticus and Helicobacter bilis have been isolated from livers of mice with hepatitis as well as from intestines of asymptomatic mice (42, 48). Furthermore, long term infection Chapter 1 20

with H. hepaticus in one strain of inbred mice has been linked to hepatic adenocarcinoma and hepatic adenomas (42, 178).

The large number of Helicobacter species being isolated from similar ecological niches from a wide variety of animal species supports the working hypothesis of this thesis that this phylogenetically related group of organisms represent "autochthonous microbiota" which have been evolving for a long period of time with their particular host species which gives rise to the diversity within the genus. However, under the definition of "autochthonous microbiota" as presented by Dubos (24), such organisms should have an almost symbiotic relationship with their host and not cause any adverse effects to their host. Under this definition many Helicobacter species would not be considered as "autochthonous microbiota".

In the case of the lower bowel helicobacters, disease appears to occur when the organisms are found to colonize areas which are not considered "normal", that is, outside the intestinal tract, particularly liver colonization. Whether Helicobacter spp. represent true "autochthonous microbiota" in a strict sense is questionable. A more loosely interpreted meaning would imply a long standing evolutionary relationship between bacteria and host allowing for continued colonization of a particular ecological niche which is basically uninhabitable to, and therefore protected from, many other types of potential competing bacterial species. Table 1.1 lists the Helicobacter and closely related species, their main host(s) and sites of colonization.

1. 4 Murine Helicobacter species Work presented in this thesis will focus on spiral bacteria isolated from laboratory mice. Therefore, more detailed description of some Helicobacter species found naturally to infect laboratory rodents is presented below.

1.4.1 Helicobacter muridarum As discussed above, H. muridarum was probably the first Helicobacter species to be cultured in the laboratory (131). H. muridarum normally colonizes the intestinal mucosa of rats and mice. However, in older animals or during experimental co-infection with Helicobacter felis, it has been observed to colonize the gastric mucosa (85, 86, 132). It is urease, catalase and oxidase positive. Ultrastructurally, H. muridarum is 3.5-5 µm in length and 0.5-0.6 µm wide with 2-3 spiral turns. It possesses bipolar tufts of sheathed flagella and is.entwined with 9-11 periplasmic fibres coiled around the protoplasmic cylinder (94). (") Table 1.1 ::r Helicobacter (and related) species and their known hosts. ~.... -0..,

Species Main Host Primary Other Reference Site Sites

Gastric helicobacters Helicobacter pylori human stomach (102) Helicobacter mustelae ferret stomach (45) Helicobacter felis cat, dog stomach (90, 126) Helicobacter nemestrinae macaque stomach (13) Helicobacter acinonyx cheetah stomach (26) "Gastrospirillum hominis" human stomach (153) Helicobacter bizz.ozeronii dog stomach (63) Helicobacter salomonis dog stomach (72) Intestinal helicobacters Helicobacter muridarum mouse, rat intestine (94, 131) Helicobacter fennelliae human intestine (33) "Flexispira rappini" sheep, dog, human, mouse intestine liver (sheep), stomach (6, 145) Helicobacter cinaedi human, hamster intestine (51, 161) Helicobacter canis dog, human intestine liver (dog) (43, 157) Helicobacter hepaticus mouse intestine liver (42, 178) Helicobacter pullorum chicken, human intestine liver (chicken) (156) Helicobacter pametensis bird, swine intestine (21, 147) Helicobacter bilis mouse, dog intestine liver, stomach (dog) (25, 48) Helicobacter trogontum rat intestine (104) Helicobacter cholecystus hamster liver (49) Helicobacter rodentium mouse intestine (149) Helicobacter westmeadii human blood (168) -N Chapter 1 22

1.4.2 "Flexispira rappini" "Flexispira rappini'' is the provisional name used to describe a microaerophilic, urease positive bacterium with a fusiform rod shape, tufts of 5-9 bipolar, sheathed flagella and periplasmic fibres (145). "F. rappini" has been isolated from aborted sheep fetuses (75), faeces of a human patient suffering from diarrhoea as well as faeces from an asymptomatic human and dog (6, 138), and the intestinal mucosa of normal laboratory 11)..ice (145). Some "F. rappini'' isolates are catalase positive (sheep isolate) while others are negative (human and mouse).

"F. rappini" has been shown to be phylogenetically related to Helicobacter species based on 16S rRNA sequence comparisons. 16S rRNA gene sequence comparisons among different isolates of "F. rappini'' and other Helicobacter species have, however, shown that members of this tentatively named species are not monophyletic within the genus Helicobacter. The 16S rDNA sequence from one "F. rappini'' isolate was found to be closely related to H. cinaedi, H. bilis and H. canis, while the sequence obtained from another "F. rappini" isolate clustered with H. pullorum within a different clade in the Helicobacter phylogenetic tree (149). The 16S rDNA of some "F. rappini" isolates possess an intervening sequence (IVS) inserted at position 210 (E.coli numbering) (48). No detailed phylogenetic or taxonomic study has been made of "F. rappini''. Such studies are required to clarify the taxonomic position of this organism.

1.4.3 Helicobacter hepaticus The discovery of this organism in 1994 has had far reaching effects in terms of mouse experimentation in the United States. Helicobacter hepaticus was first observed in mice suffering from multifocal necrotic hepatitis. These animals were part of a long term toxicological study where both control and experimental animals presented with chronic, active hepatitis and hepatic tumors (177, 178). H. hepaticus has since been found to be widespread among commercially available mice and colonizes the caecal and colonic mucosa and livers (42, 137, 148). Mice infected with H. hepaticus develop chronic liver inflammation, hepatomegaly and bile duct proliferation (46). After long term infection, some strains of inbred mice proceed to develop proliferative hepatitis and hepatocellular carcinoma (46, 178). In addition, H. hepaticus can cause inflammatory bowel disease (IBD) when infected into germfree mice (47).

H. hepaticus is urease, catalase and oxidase positive. Cells are slender, curved to spiral rods with 1-3 t~rns. Cells are motile by single, bipolar, sheathed flagella (42). Chapter 1 23

1.4.4 Helicobacter bilis Helicobacter bilis was first characterized in 1995. It was isolated from bile, livers and intestines of aged, inbred mice with chronic hepatitis during routine examination for H. hepatic us infection (48). H. bilis was found to be urease, catalase and oxidase positive. Morphologically, H. bilis cells resemble those of "F. rappini''. · Cells are fusiform, slightly curved rods with 3-14 bipolar, sheathed flagella and are entwined in periplasmic fibres (48). Based on 16S rRNA gene sequence comparisons, H. bilis is phylogenetically closely related to "F. rappini", H. cinaedi and H. canis. The 16S rDNA of H bilis also contains an IVS inserted at position 210 (E. coli numbering) which is identical to the IVS found in some "F. rappini'' isolates (48).

H. bilis has been associated with hepatic lesions in aged, inbred mice, but has not been experimentally shown be the cause of liver disease (46). H. bilis has recently been shown to cause proliferative typhlitis and diarrhoea in severe combined immunodeficient (scid) mice, leading to the suggestion that this may be a good model for investigating bacterial related IBD (150).

1.4.5 Helicobacter trogontum Helicobacter trogontum was isolated from the colonic mucosa of rats (104). Like "F. rappini'' and H. bilis, H. trogontum cells are rod shaped, motile by bipolar tufts of sheathed flagella, and the protoplasmic cylinder is entwined with periplasmic fibres. It is urease, catalase and oxidase positive. The pathogenic potential of this species is unknown. Based on 16S rRNA gene sequence comparison, H. trogontum is most closely related to H. hepaticus (104).

A new Helicobacter species isolated from laboratory rodents in the United States was described by other researchers ( 149) during the course of the work presented in this thesis and, as such, is not described here but will be discussed in more detail in the following chapters in conjunction with results obtained as part of this study. Figure 1.8 Collage of electron micrographs of characterized murine Helicobacter species. (A and B) "Flexispira rappini", a rod-shaped bacterium possessing periplasmic fibres and bipolar, sheathed flagella. Bar= 1 µm. Micrograph reproduced from reference (145). (C) Helicobacter muridarum, a spiral bacterium with periplasmic fibres and multiple bipolar sheathed flagella. Bar= 0.4 µm. Micrograph courtesy J. O'Rourke. (D) Helicobacter hepaticus, a spiral bacterium with single, bipolar, sheathed flagella. Bar = 0.5 µm. Micrograph reproduced from reference (42). (E) Helicobacter trogontum, a rod-shaped bacterium with periplasmic fibres and multiple, bipolar, sheathed flagella. Bar= 0.4 µm. Micrograph reproduced from reference (104). (F) Helicobacter bilis, a rod-shaped bacterium with periplasmic fibres and multiple, bipolar, sheathed flagella. Bar= 0.5 µm. Micrograph reproduced from reference (48). A - C

B ----·

D

- Chapter 1 25

1. 5 Hypotheses and Aims As discussed in the above introduction and literature review, one major working hypothesis has been adopted for this thesis which is concerned with the phylogenetic relatedness of a morphologically and ecologically similar group of bacteria. This hypothesis is that; • the spiral bacteria of the gastrointestinal tract are phylogenetically related, having evolved with their host to colonize a particular ecological niche.

The experimental work presented in the following chapters investigated this hypothesis in laboratory mice. Two additional hypotheses specific to the ecosystem investigated are that; • there exists an "autochthonous microbiota" of curved to spiral bacteria closely associated with the mucosa of laboratory mice. • different members of the "autochthonous microbiota" are adapted to different regions of the gastrointestinal tract representing specific ecological niches.

An additional practical reason for investigating the above hypotheses relates specifically to Helicobacter infection in laboratory mice. Prior to the work presented in this thesis, no investigation had been made with the intention to determine the presence and prevalence of Helicobacter spp. in laboratory mice from Australian animal facilities. To investigate the above hypotheses, and with the view to elucidate the question of natural Helicobacter infection in Australian laboratory mice, the specific aims and objectives of the thesis presented here were, to • culture spiral shaped bacteria from specific regions of the gastrointestinal tract of mice, of a single strain, originating from different animal facilities. • determine, using molecular techniques, the identity of spiral isolates. • determine 16S rRNA gene sequences, and reconstruct molecular the phylogeny of spiral isolates. • develop methods for molecular identification of novel isolates. • develop methods for analysis of the distribution of spiral bacteria in vivo, using molecular techniques. Chapter 2 26

Chapter 2

Materials and Methods

2. 1 Culture media

2.1.1 Horse blood agar (HBA)

Blood Agar Base No. 2 (Oxoid, Basingstoke, UK) 18 g sterile defibrinated horse blood 25 ml amphoteri~in (Fungizone®, E.R. Squibb & Sons, Princeton, NJ) 2.5 µg/ml distilled water 500ml

2.1.2 Campylobacter selective agar (CSA) HBA -525 ml Skirrow's selective supplement - polymyxin B (Sigma, St. Lois, MO) 2.5 µg/ml

- vancomycin (Eli Lilly & Co., Australia) 10 µg/ml

- trimethoprim (Sigma) 5 µg/ml

HBA and CSA were prepared by suspending Blood Agar Base No. 2 in water and sterilizing in an autoclave at 121 ·c for 15 minutes. The agar was then allowed to cool to 47°C before the addition of blood, amphotericin and, in the case of CSA, Skirrow's selective supplement. The media was then mixed and approximately 25 ml dispersed into petri dishes. After setting the plates were wrapped in polyethylene food wrap to prevent moisture loss and stored upright at 4 °C for up to 2 weeks.

2.1.3 Brain Heart Infusion-Glycerol medium (BHIG)

Brain heart infusion (BHI) powder (Oxoid) 3.7 g distilled water 100ml glycerol 31 g

The BHI powder and water were prepared and sterilized in an autoclave at 121 ·c for 15 minutes. The glycerol was prepared and sterilized separately. Once cooled the BHI broth was aseptically added to the glycerol and mixed. The medium was stored at 4°C. Chapter 2 27

2. 2 Helicobacter strains and culture

2.2.1 Helicobacter strains Helicobacter pylori NCTC 11639 the Sydney strain, SS 1 Helicobacter felis A TCC 49179, strain CS 1 Helicobacter mustelae strain HM180 H elicobacter muridarum ATCC 49282, strain STl Helicobacter hepaticus ATCC 51450, strain Hh-3 Helicobacter bilis A TCC 51630, strain Hb 1 Helicobactertrogontum ATCC 700117, strain 95-5368 H. mustelae, H. bilis, H_. hepaticus and H. trogontum were kindly supplied by Dr. J.G. Fox, Massachusetts Institute of Technology, Cambridge, Massachusetts.

2.2.2 Helicobacter cultivation Helicobacter species were inoculated onto moist CSA and incubated, lid uppermost, in an anaerobic jar (HP I I, Oxoid) with a microaerophilic gas-generating kit (BR 56, Oxoid) at 37°C for 48-72 hours. Cultures were routinely checked for purity by phase contrast IIllCroscopy.

2.2.3 Cryopreservation Stock cultures were maintained frozen in BHIG at either -70°C or in liquid nitrogen (-196 °C). Where cultures were maintained at -70°C they were first snap frozen by immersing the tube in liquid nitrogen. Revival of frozen cultures was achieved by thawing the vials and inoculating the culture directly onto moist CSA as described above.

2. 3 Isolation of spiral bacteria

2.3.1 Direct culture Intestinal mucus scrapings or homogenized livers were inoculated onto moist HBA and moist CSA. Plates were incubated, lid uppermost, in an anaerobic jar (HP I I, Oxoid) with a microaerophilic gas-generating kit (BR 56, Oxoid) or with an anaerobic gas-generating kit (BR 38, Oxoid) at 37°C for 72-96 hours. Chapter 2 28

2.3.2 Selective filtration culture

Filters with a pore size of 0.65 µm (Millipore, Bedford, MA) were placed onto the surface of moist HBA and CSA plates. Mucus scrapings or homogenized livers were inoculated

onto the centre of the filters and plates placed in a 37°C incubator set at 10% C02 and 95% humidity for 2 hours. After this time the filters were removed and the plates incubated either microaerophilically or anaerobically as described above.

2. 4 Mouse tissue fixation

2.4.1 Fixative

2.26% NaH2P04 41.5 ml 2.52% NaOH 8.5 ml paraformaldehyde 2g

The NaH2P04 and NaOH were combined and heated to 60-80°C. The paraformaldehyde was added and the solution stirred until clear. After cooling on ice the pH was adjusted to 7.2-7.4 by the addition of 10 M NaOH (approximately 2 drops).

Tissue specimens were place in fixative overnight and then transferred into 70% ethanol for storage until paraffin embedding was carried out. Tissue was dehydrated and embedded in paraffin wax using standard techniques. Paraffin embedding was carried out by Angelina Enno, School of Pathology, UNSW.

2. 5 Phase contrast photomicroscopy Fresh bacterial cultures were fixed in a solution of 1% gluteraldehyde. Wet mounts of bacterial suspensions were made using capillary forces to draw the culture between a cover slip placed onto a glass microscope slide. Fixed cultures were viewed using a BH- 2 light microscope (Olympus, Japan) and photographed using Kodak technical pan film, ESTAR-AH Base (TP 135-36) (Eastman Kodak Company, Rochester, NY).

2. 6 Electron microscopy Fresh bacterial cultures were fixed in 1% gluteraldehyde for 1 minute then transferred to cacodylate buffer (0.1 M sodium cacodylate-HCl pH 7.4). Bacteria were negatively stained with phosphotungstic acid ( 1% ) and viewed with an H7000 transmission electron microscope (Hitachi, Japan). Electron microscopy was performed by Jani O'Rourke, School of Microbiology and Immunology, UNSW. Chapter 2 29

2. 7 Biochemical testing of bacterial isolates

2.7.1 Rapid urease 2.7.1.1 Urea media urea 2g phenol red (0.5% w/v) 10ml

Na2HP04.12H20 0.157 g

NaH2P04.2H20 0.08 g

NaN3 (0.02% w/v) 0.02 g distilled water to 100ml adjust to pH 6.3-6.5

Two drops (approximately 50 µl) of urea medium was placed into a well of a microtitre tray. A loopful of bacteria from a culture plate was inoculated into the medium. Urea hydrolysis and liberation of ammonia was indicated by a colour change from orange to dark red.

2.7.2 Catalase Bacteria were harvested from HBA or CSA and washed twice in distilled water to remove any catalase carried over in the medium. A turbid suspension was prepared in distilled

water and 1 drop was added to 10% (v/v) H20 2 on a glass slide. Rapid formation of

bubbles indicated liberation of 0 2 and the presence of the catalase enzyme. 2. 7 .3 Oxidase

A filter paper strip, moistened with a drop of 1% (w/v) tetra-methyl-p-phenylene-diamine dihydrochloride was inoculated with a loopful of bacteria and examined for the development of a deep purple colour within 10 seconds.

2. 7.4 API-Campy

The commercial kit API Campy (bioMerieux, Marcy-l'Etoile, France) for identification of Campylobacter species was used according to the manufacturer's protocol for biochemical testing of unknown isolates. The assays comprising this commercial kit included those testing for urease production, nitrate reduction, alkaline phosphatase hydrolysis, presence of gamma-2-glutamyl transpeptidase and indoxyl acetate hydrolysis. Briefly, a suspension (McFarland 6 turbidity) was made by harvesting bacteria from one or more plates in the supplied medium. The suspension was then inoculated into ampoules on the test strips and incubated either aerobically (part 1 of strip) or in the preferred culture atmosphere for the isolate (microaerophilically or anaerobically) (part 2 of strip). This testing was done to determine the presence of a range of enzymes and various growth characteristics. It was not performed for identification purposes. Chapter 2 30

range of enzymes and various growth characteristics. It was not performed for . identification purposes.

2.7.5 Susceptibility to antimicrobial agents The surface of HBA was spread with a standardized inoculum of bacteria and any excess liquid allowed to diffuse into the agar. Susceptibility disks containing nalidixic acid (30 µg) (NA 30, Oxoid), cephalothin (30 µg) (KF 30, Oxoid) and metronidazole (5 µg) (MTZ 5, Oxoid) were placed onto the agar surfaces. Resistance was determined by the absence of a . clear zone of inhibition after 72 and 96 hours incubation.

2. 8 Preparation of PCR template DNA was obtained from bacterial cultures using either of the two methods described below. All solutions were sterilized by autoclaving at 121 °C for 15 minutes or by filtering through a 0.22 µm sterile filter. Plasticware (disposable pipette tips and microcentrifuge tubes) were autoclave sterilized.

2.8.1 Genomic DNA extraction A loopful of cells was scraped from the surface of an agar plate and suspended in 500 µI TE buffer, pH 8.0 (10 mM Tris-HCI, 1 mM EDTA) and lysozyme added to a final concentration of 1 mg/ml. The solution was incubated at 37°C for 15 minutes. Cell lysis was then promoted by the addition of 5 µl proteinase K (20 mg/ml) and 5 µl SOS and a further incubation at 55°C for 15 minutes. The cell lysate was then extracted 3 times with equal volumes of phenol, phenol:chloroform:isoamylalcohol (25:24: 1) and chloroform. The DNA was then precipitated for 10 minutes at room temperature with 1 ml of isopropanol after the addition of 250 µl 7 .5M ammonium acetate. The DNA was pelletted by centrifugation at 12 000 rpm for 20 minutes and the supernatant carefully removed. The pellet was washed with 70% ethanol then lyophilized using a DNA Speed Vac® vacuum centrifuge (Savant Instruments Inc., Farmingdale, NY). The dried DNA was dissolved in 40 µl TE buffer pH 8.0 (10 mM Tris-HCl, 1 mM EDTA). An aliquot of DNA was diluted 1/10 in TE buffer for use in PCR applications. Chapter 2 31

2.8.2 Cell lysis using Xanthogenate 2.8.2.1 XS buffer potassium ethyl xanthogenate 0.5 g (Fluka Chemika, Buchs, Switzerland) 4 M ammonium acetate 10ml 1 M Tris-HCI pH 8.0 5ml 0.5 M EDTA pH 8.0 2ml 20% (w/v) SDS 2.5 ml distilled water to 50ml

A loopful of cells was scraped from the surface of an agar plate and suspended in a minimal volume of TE buffer (approximately 50 µl) in a 2 ml microcentrifuge tube. One ml XS buffer was added to the cells and mixed by vortexing for 10 seconds. The solution was incubated at 70°C for 30 minutes, vortexed for 10 seconds and incubated on ice for 30 minutes. The tube was then centrifuged at 12 000 rpm for 10 minutes and the supernatant carefully removed into a new 2 ml microcentrifuge tube and the pellet discarded. The supernatant containing genomic DNA was precipitated by the addition of 1 ml isopropanol and incubated at room temperature for 10 minutes. The DNA was pelletted by centrifugation at 12 000 rpm for 20 minutes and the supernatant carefully removed. The pellet was washed with 70% ethanol then lyophilized using a vacuum centrifuge (Savant Instruments Inc.). The dried DNA was dissolved in 200 µl TE buffer, pH 8.0. An aliquot of DNA was diluted 1/10 or 1/100 in TE buffer for use in PCR applications.

2. 9 O/igonucleotide synthesis and purification Oligonucleotide primers were synthesized both for DNA amplification and DNA sequencing. A number of oligonucleotide primers were designed specifically for this study while others were taken from the literature.

Certain amplification primers were synthesized with extra 5' sequence to enable universal sequencing primer recognition. These chimeric oligonucleotides were composed of a 3' bacterial specific sequence and a 5' forward or reverse universal sequencing primer sequence identical to that found in multiple cloning sites and polylinkers of many plasmid vectors.

Oligonucleotides were synthesized using the Oligo 1000 DNA synthesis system (Beckman, Fullerton, CA) at either 30 nmol or 200 nmol scale. Primers were removed from the Chapter 2 32

synthesis column by incubation in 25% ammonium hydroxide for 60 minutes at room temperature. Following cleavage from the column, deprotection of the oligonucleotide was achieved by incubation in the same ammonium hydroxide solution at 70°C for 90 minutes (or at 55°C overnight). The solution was cooled on ice and a 100 µl aliquot ( 1/10 of the total volume) of oligonucleotide precipitated from solution by addition of nine volumes (900 µl) of n-butanol. The mixture was vortexed and centrifuged at 12 000 rpm for 30 minutes at 4 °C. The oligonucleotide pellet was dried using a vacuum centrifuge (Savant Instruments Inc.) and the primer resuspended in 100 µl of sterile distilled water. Oligonucleotide concentrations were measured at a wavelength of 260 nm using a DU® 640 spectrophotometer (Beckman), with yields consistently being greater than 80%. Stocks of primers were diluted to provide a working solution of approximately 10 pmol/µl for the PCRs described.

Oligonucleotides were also synthesized with the trityl group retained on the 5' phosphoramidite. Cleavage, deprotection, and precipitation were performed as described. An oligonucleotide purification column (OPC) (Applied Biosystems Inc., Foster City, CA) was used to separate full length primer from detritylated failure sequences. The column was washed with 5 ml acetonitrile (HPLC-grade) followed by 5 ml 2 M triethylamine acetate. Oligonucleotide (1 ml in water) was loaded onto the column at a rate of 2 drops/second. Eluant was then reloaded. The OPC was washed in triplicate with 5 ml 1.5 M ammonium hydroxide followed by two washes with 5 ml distilled water. The OPC was then treated with 5 ml 2% trifluoracetic acid (TFA) to detritylate the OPC bound oligonucleotide, taking approximately 1 minute to elute this volume. TFA was again passed through the column prior to two 5 ml washes with distilled water. Oligonucleotide was eluted from the OPC by flushing with 1 ml 20% acetonitrile. An aliquot of oligonucleotide was then lyophilized in a vacuum centrifuge and resuspended in sterile distilled water. Oligonucleotide concentrations were measured as described and dilutions made to give a working concentration of 10 pmol/µl. For PCR applications, the quality of unpurified, desalted (by butanol precipitation) oligonucleotides was adequate, while purified oligonucleotides were used for sequencing applications.

The theoretical melting temperature (Tm) of oligonucleotide primers was calculated using the formula; Tm = 2(A+T) + 4(G+C). This value is the theoretical temperature for primer/template disassociation.

Table 2.1 lists the names and sequences of PCR primers used in this study with their target region (E.coli numbering), Tm and reference (where appropriate) where the primer was first described. Table 2.1 n::,- 16S rRNA gene amplification primers. 1 N

Name Sequencea Locush Tmc Reference

F27(UFP) TAGTGTAAAACGACGGCCAGTAGAGTITGATCCTGGCTCAG 8-27 60 (113) F27 AGAGTITGATCCTGGCTCAG . 8-27 60 (181) R1492(URP) TAGCAGGAAACAGCTATGACACGGTTACCTTGTTACGACTT 1492-1512 60 (113) R1492 ACGGTTACCTTGTTACGACTT 1492-1512 60 (181) R274 TCTCAGGCCGGATACCCGTCATAGCCT 274-300 86 (44) H276f CTATGACGGGTATCCGGC 276-293 58 (137) H676r ATTCCACCTACCTCTCCCA 658-676 58 (137) HmurR ACAGAAGTGGCACTCCCA 1019-1032 56 this study Hbr TCTCCCATACTCTAGAAAAGT · 644-664 58 (137) C62 AGAACTGCATITGAAACTACTIT 627-649 60 (48) C12 GGTATTGCATCTCTITGTATGT 1244-1265 60 (48) B38 GCATITGAAACTGTTACTCTG 633-653 58 (148) B39 CTGTTTTCAAGCTCCCC 1031-1047 62 (148, 104) B72 CATAGGTAACATGCCCCA 123-139 54 (104) . D86 GTCCTTAGTTGCTAACTATT 1117-1136 54 (149) D87 AGATITGCTCCATITCACAA 1264-1283 54 (149) DfairF AGGATGAGTCCGCGTCCC 225-242 60 this study DfairR ATACCGGTCCAGGTGGCC 733-750 60 this study GreenF TTTTAGACTGGAACAACTTACC 137-158 60 this study GreenR CCGTTAGCAACTGGAAATAG 1115-1134 58 this study

a Oligonucleotids seuqences are written in 5' to 3' orientation. Underlined sequences are those of the linked universal sequencing primers which were attached to assist in successive sequencing of PCR products. b Position of primers are based on the numbering of the E. coli 16S tRNA. c Theoretical temperature for primer/template disassociation. w w Chapter 2 34

2. 1 O DNA amplification by Polymerase Chain Reaction (PCR) All PCRs were performed in a reaction volume of 50 µl. Thermal cycling was carried out in a FfS 320 Thermal Sequencer (Corbett Research, Sydney, Australia) using 0.5 ml microcentrifuge tubes and the reaction overlaid with 30 µl sterile mineral oil (Sigma). Alternatively, cycling was carried out using the GeneAmp® PCR System 2400 (Perkin Elmer, Emeryville, CA) using 0.2 ml microcentrifuge tubes without the addition of mineral oil due to the presence of a heated cover on the thermal cycler preventing sample evaporation.

The PCR amplification mixture contained reaction buffer (67 mM Tris-HCl, pH 8.8, 16 mM (NH4) 2SO4, 0.45% Triton X-100 and 0.2% gelatin), 2 mM or 2.5 mM MgC1 2, 1 unit Taq polymerase (Biotech International, Perth, Australia) 200 µM deoxynucleotide triphosphates (dNTPs) (Boehringer Mannheim, Germany), 10 pmol of each oligonucleotide primer and 20- 100 ng DNA. The final volume of 50 µl was made up with sterile distilled water.

Reactions underwent an initial denaturation period at 94°C for 3-4 minutes before being cycled 30-35 times through denaturation, annealing and extension temperatures specific for a particular reaction. Specific PCR cycling profiles will be described in the following chapters.

All PCR experiments included a no DNA negative control reaction to test for the presence of contaminating DNA in amplifications. Reactions were set up in a laminar flow cabinet with pipettes dedicated to PCR set up with aerosol resistant barrier tips used for the transfer of solutions containing DNA. Typically a bulk reaction mix was made containing common solutions present in equal volumes for all reactions in an experiment to reduce the error introduced by pipetting of small volumes.

2. 11 Agarose gel electrophoresis and DNA visualization Typically, 10 µl of product from a 50 µl PCR was added to a l0X heavy loading buffer (25% Ficoll (Type 400), 0.4% bromophenol blue, 0.4% xylene cyanol) prior to electrophoresis. Agarose gels in TAE buffer (40 mM Tris-acetate, 1 mM EDTA) were subjected to approximately 70 V, the time for separation depending on the particular application. Concentrations of agarose in gels varied between applications and were between 1-3%. Agarose gels were stained with ethidium bromide (20 µg/ml) after electrophoresis and the DNA was visualized by UV transillumination and photographed using a "Gel Cam" documentation system (Bresatec, Adelaide, Australia). Chapter 2 35

2. 12 Purification of amplified DNA Commercially available kits were used for purification of PCR products prior to DNA sequencing applications. PCR amplified products from several identical reactions (about 6X50 µl reactions) were combined by removing the aqueous layer from beneath the mineral oil overlay and placing into a fresh 1.5 ml microcentrifuge tube. The pooled reactions were then extracted with an equal volume of chloroform to remove traces of oil which were carried over. The extracted product was then purified using one of two methods described below.

2.12.1 Prep-A-Gene®

The Prep-A-Gene® DNA Purification System (Bio-Rad, Hercules, CA) employed the binding of DNA to a silica matrix in a chaotropic buffer. Briefly, the chloroform extracted PCR amplified DNA was added to the buffer and mixed before a suitable volume of silica matrix was added (based on the amount of DNA estimated to be present in the pooled reactions). The DNA was then allowed to bind to the matrix at room temperature. A series of steps involving centrifugation, removal of supernatant and resuspension of the silica pellet in buffer (either for binding or washing) was followed by elution of bound DNA back into a small volume the liquid phase. The quality and concentration of DNA obtained using this procedure was suitable for all sequencing procedures although the procedure suffered from being labour and time intensive.

2.12.2 Wizard-TM PCR preps

The Wizard™ PCR Preps DNA Purification System (Promega, Madison, WI) employed a system of a DNA binding matrix immobilized within a spin column. Briefly the chloroform extracted PCR amplified DNA was added to the supplied purification buffer and mixed prior to the addition of purification resin. The solution was then mixed by vortexing and loaded onto a purification column using a syringe. The column-bound resin was washed by loading 2 ml 80% isopropanol onto the column and the column spun for 10 seconds to remove residual isopropanol. DNA was eluted in 40 µl sterile TE buffer by a 10 second centrifugation through the column. Repeating the elution step by applying the eluant back onto the column increased recovery of amplified DNA without increasing the eluant volume. Again, the quality and concentration of DNA obtained using this procedure was suitable for all sequencing procedures. Chapter 2 36

2. 13 DNA sequencing

2.13.1 Cycle sequencing of amplified DNA 2.13.1.1 Cycle sequencing reactions The commercial kit Cyclist™ Exo·Pju (Stratagene, La Jolla, CA) was used for all cycle sequencing performed during this project. Reactions were carried out according to the manufacturers instructions. Briefly, approximately 200 fmol (equivalent to 200 ng of a 1.5 kbp fragment) purified PCR product was added to approximately 3 pmol sequencing primer in a reaction mixture containing reaction buffer (final concentrations; 20 mM Tris­

HCl, 10 mM KCl, 2 mM MgSO4, 10 µM (NH4) 2SO4, 0.1% BSA, 2 µM dATP, 5 µM dCTP, dGTP and dTTP), 10 µCi a.- 35S dATP, 2.5 units Exo·Pju DNA polymerase and 4 µl DMSO in a total reaction volume of 30 µI. The reaction mixture was then distributed into 0.5 ml microcentrifuge tubes containing a 3 µl mixture of dNTPs and one of the 4 dideoxynucleotide triphosphates (ddNTPs). The reactions were then overlaid with approximately 15 µl sterile mineral oil and subjected to thermocycling in an FTS 320 Thermal Sequencer (Corbett Research) with the following conditions. An initial denaturation period at 94 °C for 3 minutes followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 50°C for 45 seconds and extension at 72°C for 1 minute. After cycling 5 µl of a stop solution (80% formamide, 50 mM Tris-HCl, 1 mM EDTA, 0.1 % bromophenol blue, 0.1 % xylene cyanol) was added prior to reactions being separated using polyacrylamide gel electrophoresis.

2.13.1.2 Polyacrylamide gel electrophoresis and autoradiography Denaturing (with urea) polyacrylamide gel electrophoresis was used for the separation of cycle sequencing reactions. Pre-mixed 6% acrylamide (19:1, acrylamide:bis-acrylamide) containing 8 M urea in TBE buffer (90 mM Tris-borate, 2 mM EDTA) (Novex, San Diego, CA) was used to make gels 55 cm in length and 0.1 mm thick using the Macrophor system (Pharmacia, Uppsala, Sweden). Gels were typically run at 2000 V (40 W) and each reaction loaded and electrophoresed twice for differing lengths of time to separate fragments either immediately 3' of the priming site or further downstream in the PCR product. After electrophoresis gels were washed for 30 minutes in a solution of 10% acetic acid, 10% methanol and dried in an oven at 80°C. Gels were exposed to X-ray film (Fuji Photo Film Co., Japan) for 48-96 hours.

Gels were read using a digitiser and the MacVector™ Sequence Analysis Software then analyzed and assembled using AssemblyLIGN™ (International Biotechnologies Inc., New Haven, CT). Chapter 2 37

2.13.2 Automated DNA sequencing During the course of this study a great deal of advancement was made on the application to large sequencing projects of the recently introduced automated DNA sequencing protocols. The PRISMTM Ready Reaction DyeDeoxy™ and DyeRhodamine™ Terminator Cycle Sequencing kits (Applied Biosystems Inc.) were used for automated sequencing applications. The PRISM™ system is similar to manual cycle sequencing protocols except that fluorescently labeled ddNTPs replace the radiolabeled nucleotide enabling a single tube per reaction (rather than four, one for each of the ddNTPs).

Reactions were carried out according to the manufacturers protocol. Briefly, 8 µl of reaction mix (DyeDeoxy™ or DyeRhodamine™) were added to 100-200 ng DNA template (purified PCR product) and 5-10 pmol primer with a final reaction volume of 20 µl in a 0.2 µl microcentrifuge tube. Reactions were subjected to thermocycling in using the GeneAmp® PCR System 2400 (Perkin Elmer) with the following conditions. An initial denaturation period at 96°C for 1 minute followed by 25 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds and extension at 60°C for 4 minutes. Eighty microlitres of water were added to completed reactions before purification by a single extraction with 100 µl phenol/chlorofonn/water (Applied Biosystems Inc.) and precipitation with 15 µl 2 M sodium acetate and 300 µl 100% ethanol at -20°C for 1 hour. The DNA fragments were collected by centrifugation at 12 000 rpm for 20 minutes at 4°C, washed with 70% ethanol and dried in a vacuum centrifuge.

Sequencing products were separated on model 377 DNA sequencer machines and analyzed using programs contained in the INHERIT™ package (Applied Biosystems Inc.). Facilities for electrophoresis of automated sequencing reactions and analysis of gel data were provided by The Automated DNA Sequencing Facility, School of Biochemistry and Molecular Genetics, UNSW.

Table 2.2 lists the names and sequences of primers used for 16S rDNA sequencing in this study with their target region (E. coli numbering), Tm and reference for where the primer was first described. N

n

0

"'1

::,- ...

w

00

.g

113) 113)

113) 113)

113) 113) 113)

113) 113)

(183) (183,

(181, (183) (183, (183, (183, Reference (183)

(183, (183) (183, (183, (181,

58 58 58-60 52 62 58 62

54 60 60 58-60 60-62 56

Tmc

102-119

1096-1113 1100-1115 1221-1240 1222-1241

1494-1513

341-357 8-27 Locush

515-530 685-704 929-946 927-942

786-803

rRNA.

16S

TG

E.coli

the

orientation.

of

3'

disassociation.

to

5'

TIT(T/C)ACCGCTAC

in

numbering

the

written

on

A)CCGCTTGTGCGGGG

primer/template

primers

are

for

based

TTGTAG(T/C)ACGTGTGTAGCC

AGAGTITGATCCTGGCTCAG

GTGGCGGACGGGTGAGTA CTGCTGCCTCCCGTAGG

Sequences

GTGCCAGCAGCCGCGG TCTACG(G/C)A CTACCAGGGTATCTAATC TCC(T/ A GGGCCCGCACAAGCGG GGTTGCGCTCGTTGCGG CAACGAGCGCAACCCT

GCTACACACGT(NG)CTACAA TACGGCTACCTTGTTACGAC

are

seuqences

sequencing

temperature

primers

gene

of

2.2

rRNA

Position

Oligonucleotids Theoretical

16S

119F

1096R

1115F 1221R 1241F 1494R

Table Name

27F

341R

530F

685R 786R 929R 942F

b

c

a Chapter 2 39

2. 14 Phylogenetic inferences from 16S rDNA sequences 16S rDNA sequences were compared to sequences in the GenBank and EMBL data bases using both BLASTN (at NCBI using the BLAST network) (3) and FastA (52) algorithms. Sequences were aligned using the GCG program pileup (52) and the multiple-sequence alignment tool in the Clustal W package where percentage sequence identities were also calculated (164). Aligned sequences were manually checked and nucleotide positions which contained ambiguities (insertion, deletion or unidentified base) were removed from further analysis. Genetic distances (D) corrected for multiple base changes by the method of Jukes and Cantor (73) were calculated using the DNADIST program in the PHYLIP package, where D = -3/4 logeCl - 413d); d being the level of sequence dissimilarity. The phylogenetic tree was reconstructed by the neighbor-joining method of Saitou and Nei . . (140). Bootstrap values for the phylogenetic tree were obtained from analysis of one hundred replica data sets of the multiple alignment using the programs SEQBOOT (31) and CONSENSE. The phylogenetic inference programs were supplied by the PHYLIP package (version 3.57c) (32). All programs used in the sequence manipulation and phylogenetic analyses were made available by the Australian National Genomic Information Service (ANGIS, Sydney University, Australia).

2. 15 Preparation of PCR template from paraffin embedded tissue

2.15.1 Sectioning and deparaffinization Twenty 5 µm sections were cut from each block and placed in a 2 ml microcentrifuge tube using a Spencer '820' microtome (American Optical Co., Buffalo, NY). As DNA was to be extracted from sections, care was taken to avoid contamination from one block to the next. The microtome was wiped down thoroughly with Histoclear™ (National diagnostics, Manville, NJ) and 70% ethanol between each block and a new blade was used for each block.

To deparaffinize the cut sections 1 ml of Histoclear™ was added to the tube which was then vortexed for 10 seconds and incubated at room temperature for 10 minutes. The tube was centrifuged at 12 000 rpm for 10 minutes and the supernatant carefully removed using a sterile 1 ml pipette tip. A further 1 ml of Histoclear™ was added to the tube and the above procedure repeated. One millilitre of 100% ethanol was then added to the tube and the incubation and centrifugation steps above repeated. After removal of the ethanol supernatant the deparaffinized sections were allowed to air dry for approximately 20 minutes. Chapter 2 40

2.15.2 DNA extraction from fixed sections 2.15.2.1 DNA extraction buffer 5 MNaCl 1 ml 1 M Tris-HCL pH 8.0 0.5 ml 0.5 M EDTA pH 8.0 2.5 ml 20% (w/v) SDS 1.25 ml distilled water to 50ml

The DNA extraction procedure was similar to that described by Isola et al. (71). One millilitre DNA extraction buffer and 15 µl proteinase K (20 mg/ml) were added to tubes containing deparaffinized tissue sections and incubated at 55°C for 24 hours. A further 15 µl proteinase K (20 mg/ml) was added to the tubes and incubated at 55°C for a further 24 hours. The addition of proteinase K and incubation were repeated so that tubes were incubated for a total of 72 hours.

Sample was divided into two aliquots of approximately 500 µl each. Both aliquots were extracted with an equal volume of phenol, phenol:chloroform:isoamylalcohol (25:24:1) and chloroform. The DNA was precipitated for 1 hour at -20°C with 1 ml of 100% ethanol after the addition of 250 µl 7 .5 M ammonium acetate. The DNA was pelletted by centrifugation at 12 000 rpm for 20 minutes and the supernatant carefully removed. The pellet was washed with 70% ethanol then dried in a vacuum centrifuge. The DNA was dissolved in 40 µl TE buffer, pH 8.0 overnight and then the 2 samples originating from the same tube combined. Two microlitres were used in PCR applications. Chapter 3 41

Chapter 3

Development and Validation of PCRs for Detection of Helicobacter species

3. 1 Background Prior to this study many spiral bacteria had been observed and cultured from mouse intestinal tissue in the laboratory of Professor Adrian Lee, UNSW, where the presented study was undertaken. The identity of most of these bacteria remained unclear although it was suspected that many may belong to the genus Helicobacter. In order to test if these bacteria were in fact Helicobacter spp. and to investigate the working hypothesis of this thesis, that the intestinal spiral bacteria represent a phylogenetically related group of organisms which have evolved specialized mechanisms for colonization the GIT, it was necessary to develop methods for the rapid identification of isolates.

The development of PCR in 1987, including the application of a thermostable DNA polymerase (110) has led to the widespread availability and use of this technique. DNA sequencing has also become a more accessible technique, resulting in an increasing database of nucleotide sequences, greatly facilitating primer design. The combination of these two technologies has led to widespread use of PCR for the detection and identification of specific organ.isms. The presented study aimed to take advantage of the explosion which has been occurring over the last 10-15 years in the application and availability of these molecular biological techniques, to develop methods for the identification of helicobacters at both the genus and species level.

3. 2 Genus specific PCR A Helicobacter genus specific hybridization probe targeting the region 274-300 (E.coli numbering) of the 16S rRNA was described by Fox et al. in 1992 (44 ). In 1993 Solnick et al. published a PCR which used this probe sequence in the reverse orientation (R274, Table 2.1) in conjunction with the universally conserved bacterial forward primer (F27, Table 2.1) near the 5' end of the 16S rDNA (153 ). This reaction was used to amplify a product of 292 bp from DNA extracted from stomachs of Specific Pathogen Free (SPF) mice which had been colonized with the bacterium "Gastrospirillum hominis". No product was obtained when this procedure was applied to DNA extracted from uninfected control animals. The aim of this study was to validate this genus specific PCR for use in Chapter 3 42

our laboratory in order to identify Helicobacter species from our collection of spiral bacteria.

3.2.1 Results Amplification reactions were prepared as described (Chapter 2) using primer R274 in conjunction with either primer F27 or primer F27(UFP) (Table 2.1). Cycling conditions initially used for amplification of helicobacter DNA were those described by Solnick et al. and included a denaturation at 95cc for 3 minutes followed by 35 cycles of 94cc for 1 . minute, 55CC for 1 minute and 72CC for 2 minutes, followed by a further extension step at 72cC for 10 minutes (FfS 320, Corbett Research) (153). These conditions were shown to be highly non specific, with DNA from a wide range of bacterial species being amplified. Organisms from which template DNA was amplified using these cycling conditions included species from the genera Arcobacter, Aeromonas, Vibrio, Rhizabium and Microcystis. The annealing temperature of this reaction was then increased until either no product was obtained from any template or only helicobacter DNA was amplified.

The PCR cycling conditions which resulted in specific amplification of helicobacter DNA was when the annealing temperature of the reaction had been raised to 72CC (a 2 step reaction) and including primers F27(UFP) and R274. The cycling conditions required an initial denaturation at 94 CC for 3 minutes followed by 35 cycles of 94 CC for 1 minute and 72CC for 2 minutes. This was followed by a final extension at 72CC for 10 minutes. Non helicobacter template DNA ceased to be amplified at various temperatures below 72CC, with some Campylobacter species remaining positive at temperatures as high as 68°C. The expected product size for this reaction (310 bp) was slightly larger than for the reaction presented by Solnick et al. (292 bp) owing to the addition of the linked universal sequencing primer (UFP) onto the forward primer. PCR products obtained when DNA from Helicobacter or Campylobacter species was used as template in this reaction are presented (Figure 3.1). The reaction including primer F27 was unsuccessful in amplifying only helicobacter DNA at any of the annealing temperatures examined between 55cc and 72CC, being either non-specific at lower temperatures, or failing to amplify even helicobacter DNA at the elevated temperatures required for specificity.

Subsequent to development of the PCR presented above, a Helicobacter genus specific PCR was published by Riley et al. utilizing primers H276f and H676r (Table 2.1) (137). This reaction needed only minor modifications from the published conditions to be workable using the GeneAmp® PCR System 2400 (Perkin Elmer). Specific amplification of helicobacter DNA was achieved with amplification reactions prepared as described (Chapter 2) and the following thermocycling profile. An initial denaturation at 94cc for 5 Chapter 3 43

minutes followed by 35 cycles of 94°C for 5 seconds, 53°C for 5 seconds and 72°C for 30 seconds. This was followed by an extension step at 72°C for 2 minutes (GeneAmp® 2400, Perkin Elmer). This varied from the published cycling profile, performed in a 9600 thermocycler (Perkin Elmer), only in denaturation and annealing times which were both for 2 seconds. Using the GeneAmp® PCR System 2400 (Perkin Elmer) these times were insufficient to amplify helicobacter DNA. PCR products resulting from amplification of DNA from Helicobacter and Campylobacter species under these, slightly modified, conditions are presented (Figure 3.2).

3.2.2 Discussion· A Helicobacter genus specific PCR was developed using primers targeting regions of the 16S rDNA which were either conserved among all bacteria (nucleotide positions 8-27, E. coli numbering) or specific for the genus Helicobacter (nucleotide positions 274-300, E. coli numbering). Both of these oligonucleotides had been described previously and were combined here in a PCR with a predicted product size of approximately 310 bp. The modified consensus primer F27(UFP) was required in this reaction rather than shorter F27 primer. While both of these primers shared an identical target sequence and initial Tm the extended length of the F27(UFP) primer assisted in primer binding at the elevated temperatures required for Helicobacter genus specificity. Once the PCR primers were incorporated into the amplified product during the first rounds of cycling they became template for further and more efficient binding at elevated temperatures. This was due to the increased Tm of the lengthened primers when binding to previously amplified fragments. The previously described PCR utilizing primers F27 and R274 was found to be highly non-specific resulting in successful amplification of all bacterial templates examined which came from a number of diverse phylogenetic groups.

The PCR described by Riley et al. required only slight modification from the published conditions for specific amplification of helicobacter DNA. The modifications involved an increase in the denaturation and annealing times and are attributable to the use of a different model thermocycler. The forward primer used in this PCR (H276f) targeted a site within the same region of the 16S rRN A gene as the reverse primer used in the 2 step PCR, at nucleotide positions 276-293 for H276f compared to 274-300 for R274 (E.coli numbering). Chapter 3 44

Ml 2 3 4 5 6 7 8 91011

Figure 3.1 PCR products from the Helicobacter genus specific reaction using primers F27(UFP) and R274 (Table 2.1). DNA separated in 2% agarose/TAE.

lane M X174/Hae III (200 ng) 1 Helicobacter pylori 2 Helicobacter felis 3 Helicobacter mustelae 4 Helicobacter muridarum 5 Helicobacter bilis 6 Helicobacter hepaticus 7 Helicobacter trogontum 8 Campylobacter jejuni 9 Campylobacter fetus subsp. fetus 10 Campylobacter fetus subsp. veneralis 11 "no DNA" control Chapter 3 45

Ml 2 3 4 5 6 7 8 91011

Figure 3.2 PCR products from the Helicobacter genus specific reaction using primers H276f and H676r (Table 2.1). DNA separated in 2% agarose/TAE.

lane M Xl 74/Hae III (200 ng) 1 Helicobacter pylori 2 Helicobacter felis 3 Helicobacter mustelae 4 Helicobacter muridarum 5 Helicobacter bi/is 6 Helicobacter hepaticus 7 Helicobacter trogontum 8 Campylobacter jejuni 9 Campylobacter fetus subsp. fetus 10 Campylobacter fetus subsp. veneralis 11 "no DNA" control Chapter 3 46

Both of these reactions were found to have unique advantages. The 2 step PCR had one distinct advantage over the 3 step PCR. A number of Helicobacter species, including Helicobacter bilis but not Helicobacter muridarum, Helicobacter hepaticus or Helicobacter trogontum, possess an intervening sequence (IVS) of approximately 190 bp within the 16S rDNA inserted at nucleotide position 210 (E.coli numbering) of the 16S rRNA gene (48). The 2 step PCR amplified the region between nucleotide positions 8-300 and, therefore, those species which have an inserted IVS were clearly distinguished from those without, the product size being approximately 500 bp compared to 300 bp (Figure 3.1, lane 5). This gave additional information about unidentified isolates. The 3 step PCR had the advantage of being more robust and transferable between different laboratories. This was evidenced by its successful use here with different PCR reagents and thermocycler to those who developed the reaction, and is partly due to the fact that both primers in this reaction were specific for Helicobacter species. In the following chapters both of these reactions were used extensively for identification of isolates.

3. 3 Helicobacter muridarum specific PCR The organism which was eventually to be named Helicobacter muridarum in 1992 (94) was cultured in 1980 by M. Phillips and A. Lee (131). Prior to this study only the type strain of this species (strain ST 1, ATCC 49282) had had the sequence of its 16S rDNA examined.

The 16S rDNA from two candidate H. muridarum isolates (from the collection of A. Lee) was sequenced. DNA was isolated from cultures and the 16S rDNA amplified by PCR using the modified consensus primers F27(UFP) and Rl492(URP) (Table 2.1) complementary to the conserved 5' and 3' ends of the 16S rRNA gene respectively. DNA amplification reactions were prepared as described (Chapter 2) with a thermocycling profile as follows. An initial denaturation step at 94 °C for 3 minutes was followed by 5 cycles of 94 °C for 45 seconds, 53°C for 45 seconds and 72°C for 1 minute, then 28 cycles of 94 °C for 45 seconds, 48°C for 45 seconds and 72°C for 1 minute. This was followed by a hold at 72°C for 10 minutes (FfS 320, Corbett Research). The product from several amplification reactions was combined and purified before being used as template DNA in sequencing reactions as described (Chapter 2). Thirteen oligonucleotide primers were used in the sequencing reactions to enable sequencing of both strands of the 16S rDNA with contiguous overlaps (Table 2.2). These sequences and the previously determined H. muridarum 16S rDNA sequence were used to design a H. muridarum specific PCR. Chapter 3 47

3.3.1 Results

The two candidate H. muridarum isolates were designated UNSW1.6cae and UNSW1.7st. Both were isolated by Jani O'Rourke in 1995 from conventional Quackenbush/Swiss (Q/S) mice housed at the UNSW Microbiology animal facility. Strain UNSW1.6cae was cultured from a caecal mucus scraping and strain UNSW1.7st was cultured from a homogenized liver sample. Near complete sequences for both isolates were obtained and have been deposited in the GenBank database under the accession numbers AF013464 (UNSW1.6cae) and AF010140 (UNSW1.7st). Comparisons between these sequences revealed they had 99.6% sequence identity. Comparison with sequences available in GenBank showed they had highest sequence identity with the H. muridarum type strain (STl, accession number M08205) with sequence identities of 99.2% (UNSW1.6cae) and 99.6% (UNSW1.7st). The level of sequence identity between these three organisms, as well as previous descriptions by the isolator (J. O'Rourke, personal communication), and observations made in this study, of their growth pattern, characteristic darting motility and rapid hydrolysis of urea indicated that strains UNSW1.6cae and UNSW1.7st were likely to be strains of H. muridarum ..

A multiple sequence alignment of the three H. muridarum sequences with 11 other 16S rRNA gene sequences from murine helicobacters revealed a single candidate primer for a H. muridarum specific PCR at nucleotide positions 1019-1032 (E.coli numbering). Comparison of the primer target sequence with sequences contained in GenBank revealed no significant homologies with sequences other than to the H. muridarum 16S rRNA gene. The target region for the primer and the nucleotide mismatches present in other Helicobacter species is presented (Table 3.1). This primer was synthesized in the reverse orientation (HmurR) (Table 2.1) and used in conjunction with the Helicobacter genus specific forward primer (H276f) to amplify a PCR product of 750 bp. The theoretical Tm of H276f and HmurR were 58°C and 56°C respectively. Specific amplification of H. muridarum DNA was achieved using the following thermocycling profile. An initial denaturation at 94 °C for 3 minutes followed by 35 cycles of 94 °C for 1 minute, 58°C for 1 minute, 30 seconds and 72°C for 2 minutes. This was followed by an extension step at 72°C for 10 minutes (FTS 320, Corbett Research). PCR products obtained when the three H. muridarum isolates (STl, UNSW1.6cae and UNSW1.7st) and other Helicobacter species were used as template DNA under these conditions are presented (Figure 3.3). Chapter 3 48

Table 3.1 Comparison of Helicobacter muridarum specific primer target site with sequences from various Helicobacter spp.

Organism Sequences

Helicobacter muridarum TGGGAGTG-CCA-CTTCTGT UNSW1.6cae TGGGAGTG-CCA-CTTCTGT UNSW1.7st TGGGAGTG-CCA-CTTCTGT

H elicobacter hepaticus GTGGAGTG-CC--CTTCGGG Helicobacter trogontum GCGGAGTG-CC--CTTCGGG Helicobacter bi/is GTGGAGTG-CTGGCTTGCCA Helicobacter cinaedi GTGGAGTGT-TGGCTTGCCA "Flexispira rappini" GTGGAGTG-CTGGCTTGCCA Helicobacter fennelliae GCGGAGTG-CTGGCTTGCCA Helicobacter rodentium GTGGAGTG-CTAGCTTGCTA Helicobacter pullorum GCGGAGTG-CTGGCTTGCCA Helicobacter sp. CL03 GTGGAGTGTCTAGCTTGCTA Helicobacter sp. BirdB GCGGAGTGTCTAGTTTACTA Helicobacter sp. BirdC TGGGAGTGTCTAGTTTACTA Helicobacter pametensis GCGGAGTG-CTGGCTTGCCA Helicobacter pylori GTGGAGTGTCTAGCTTGCTA Helicobacter felis GTGGAGTGTCTAGCTTGCTA Helicobacter mustelae GCGGAGTGTCTAGTTTACTA ****** **

a Sequences are written in 5' to 3' orientation and represent nucleotide positions 1019- 1032, according to E. coli numbering. Gaps (-) have been introduced into sequences to represent a true sequence alignment. Asterisks(*) indicate nucleotide positions which are conserved across all species listed. Chapter 3 49

M 1 2 3 4 5 6 7 8 9 10

Figure 3.3 PCR products from the Helicobacter muridarum specific reaction with primers H276f and HmurR (Table 2.1). DNA separated in 1% agaroseffAE.

lane M Sppl/Eca RI (200 ng) l Helicobacter pylori 2 Helicobacter felis 3 Helicobacter mustelae 4 Helicobacter muridarum (strain STl) 5 Helicobacter muridarum (strain UNSW l.6cae) 6 Helicobacter muridarum (strain UNSW1.7st) 7 Helicobacter bilis 8 Helicobacter hepaticus 9 Helicobacter trogontum 10 "no DNA" control Chapter 3 50

3.3.2 Discussion The development of a H. muridarum specific PCR was important for this study of murine helicobacters. Although H. muridarum was the first helicobacter to be isolated and described in rodents, only a single isolate had had its 16S rRNA gene sequence determined. The sequencing of a further two isolates and the development of a species specific PCR allowed for the rapid identification of isolates which was not previously possible. The difficulty in finding 2 primers specific for this species illustrated the high sequence homology shared by Helicobacter species and made necessary the use of the genus specific forward primer (H276f) in the reaction.

An additional benefit arising from the development of this PCR is in the detection of H. muridarum infected mice used as models of human Helicobacter infection. The presence of H. muridarum in experimentally infected, with either Helicobacter felis or Helicobacter pylori, mice could lead to misleading results and difficulties in interpretation for two reasons. Firstly, a rapid urease test on a piece of mouse stomach tissue is often used to indicate successful experimental colonization of mice with either H. felis or H. pylori. Since mice are coprophagous, and H. muridarum also expresses high levels of the urease enzyme~ a positive urease result may be obtained if the stomach tissue is not washed well. Secondly, in older mice or after infection with gastric helicobacters, H. muridarum will also colonize the stomach in very large numbers (86). Protocols to screen for H. muridarum in mucus scrapings have been developed (92). These, however, rely on microscopic examination which can be difficult for persons inexperienced with observing H. muridarum morphology and characteristic motility. This is particularly true when screening for a mixed infection of H. pylori and H. muridarum which are morphologically difficult to distinguish using phase contrast microscopy. The recently developed mouse model of H. pylori infection is now being widely used (93), and hence a definitive test for the presence of H. muridarum is even more important. The results presented here provide an opportunity for a PCR-based test.

3. 4 Helicobacter bi/is, Helicobacter hepaticus and Helicobacter trogontum specific PCRs A number of PCRs relevant to this study have been published in the literature and these were evaluated for use in the presented study. These reactions were specific for Helicobacter bi/is (primers H276f and Hbr) (137), (and primers C62 and C12) (48), Helicobacter hepaticus (primers B38 and B39) (148) and Helicobacter trogontum (primers B72 and B39) (104) (Table 2.1). Chapter 3 51

3.4.1 Results Two H. bilis specific PCRs were evaluated for use under the conditions described in the literature. The reaction utilizing primers H276f and Hbr was modified in the same way as the reaction involving H276f and H676r described above, by increasing the denaturation and annealing times to 5 seconds. This reaction, while amplifying H. bilis DNA and not DNA from the other murine helicobacters, was also found to amplify DNA from Helicobacter pylori (Figure 3.4, panel A). Eight clinical isolates of H. pylori were tested and all amplified in this reaction (results not shown). The reaction utilizing primers C62 and C12 was found to be specific for H. bilis with the following thermocycling profile. An initial denaturation at 94 °C for 4 minutes followed by 33 cycles of 94 °C for 1 minute, 56°C for 1 minute, 45 seconds and 72°C for 2 minutes, 30 seconds. This was followed by an extension step at 72°C for 10 minutes (FfS 320, Corbett Research) (Figure 3.4, panel B). This cycling profile was as published.

The H. hepaticus (primers B38 and B39) and H. trogontum (primers B72 and B39) PCRs were evaluated using the following thermocycling profile. An initial denaturation at 94°C for 4 minutes followed by 35 cycles of 94°C for 1 minute, 61 °C for 2 minutes, 15 seconds and 72°C for 2 minutes, 30 seconds. This was followed by an extension step at 72°C for 10 minutes (FfS 320, Corbett Research). This cycling profile was as published for H. hepaticus (148) but varied significantly to that described for H. trogontum (104). Using these conditions, primers B38 and B39 amplified both H. hepaticus and H. trogontum DNA but not any of the other helicobacters tested (Figure 3.4, panel C), while primers B72 and B39 were specific for H. trogontum (Figure 3.4, panel D).

3.4.2 Discussion There were shortcomings with two of the published PCRs evaluated. Firstly, the reaction with primers H276f and Hbr not only amplified H. bilis DNA but also H. pylori DNA. The purpose of this primer set as described when published was to distinguish between H. bilis and "Flexispira rappini'' (137). This study required the ability to determine the identity and presence of unknown isolates, and since there was an alternative H. bilis specific PCR (primers C62 and C12) the reaction with primers H276f and Hbr was not used further. Secondly, the H. hepaticus reaction (primers B38 and B39) also amplified H. trogontum DNA. H. trogontum was not described until 1996 ( 104 ), after the description of H. hepaticus in 1994 (42) and development of the PCR assay in 1995 ( 148). While this lack of specificity was noted the reaction was still judged as being a valuable tool although not providing a definitive classification of isolates to the species level. ).

).

2.1

2.1).

2.1

(Table

2.1).

(Table

(Table

Hbr

B39

agaroseffAE.

B39

(Table

and

D)

and

and

C12

B72

or

H276f

B38

Band

and

reactions.

C62

ng)

primers

(panels

primers

primers

(200ng),

specific

1 % 1

(200

C)

with

with

with

primers

or

D)

and

species

C)

A

PCR with

specific

Band

specific

and

A

(panels

specific specific

III

trogontum mustelae muridarum

bilis hepaticus

pylori

(panels

felis

Helicobacter

trogontum

control

hep~ticus

bilis bilis

(panels

RI

the

2%

in

DNA"

from

X114/Hae

"no

Sppl/Eco

Helicobacter

Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter

Helicobacter

Helicobacter

Helicobacter

Helicobacter Helicobacter

3.4

D.

C.

B.

1

A.

separated

8 3 M 6 5

2

7

4

products

PCR

Figure lane Panel Panel Panel Panel DNA

N N

Vl Vl

w w

~ ~

""I ""I

......

('") ('")

::r ::r

..§ ..§

8 8

8 8

5 5 6 7

1234 1234

M M 1 2 3 4 5 6 7

M M

D D

B B

M M 1 2 3 4 5 6 7 8

M M 1 2 3 4 5 6 7 8

C C A A Chapter 3 53

3.5 Summary The oligonucleotide primers and thermocycling profiles for PCRs described in this chapter are summarized in Table 3.2. The reactions developed and evaluated in the presented study allowed for the identification to species level using PCR, the four recognized murine helicobacters, H. muridarum, H. bilis, H. hepaticus and H. trogontum. The detection of helicobacters, other than these four species, was via PCR amplification using either of the genus specific reactions described. The 2 step helicobacter genus specific reaction was capable of identifying bacterial templates which contained an IVS within the first 300 bp of the 16S rDNA. This information was used to assist in the classification of isolates described in the following chapters which were not found to be members of the four described Helicobacter species.

The case of Helicobacter rodentium, the most recently identified murine helicobacter (149) will be discussed in detail in Chapter 6. Table 3.2 ("} Oligonucleotide primer designations and thermocycling conditions for amplification of Helicobacter (genus and species) DNA. :::,-' .§ Target DNA Primer Set Cycling conditionsa @".., w Forward Reverse Temperature Time Cycles Thermocycler

Helicobacter spp. F27(UFP) R274 94°c 1 minute 35 FTS 320 72°C 2 minutes (Corbett Research)

Helicobacter spp. H276f H676r 94°C 5 seconds 2400 53°C 5 seconds 35 (Perkin Elmer) 72°C 30 seconds

H elicobacter muridarum H276f HmurR 94°c 1 minute FTS 320 58°C 1 minute 35 (Corbett Research) 72°C 2 minutes

Helicobacter bi/is H276f Hbr 94°c 5 seconds 2400 53°c 5 seconds 35 (Perkin Elmer) 72°C 30 seconds

Helicobacter bi/is C62 C12 94°c 1 minute FTS 320 56°C 1 minute, 45 seconds 33 (Corbett Research) 72°C 2 minutes, 30 seconds

Helicobacter hepaticus B38 B39 94°c 1 minute FTS 320 6l°C 2 minutes, 15 seconds 35 (Corbett Research) 72°C 2 minutes, 30 seconds

Helicobacter trogontum B72 B39 94°C 1 minute FTS 320 61 °C 2 minutes, 15 seconds 35 (Corbett Research) 72°C 2 minutes, 30 seconds a All cycles included an initial denaturation period at 94 °C prior to thermocycling as indicated and a final extension period VI at 72°C after thermocycling was completed. .i:,. Chapter4 55

Chapter 4

Isolation and Molecular Characterization of Mouse Spiral Bacteria

4. 1 Background The recent discovery of several murine helicobacters, in particular Helicobacter hepaticus, has led to uncertainties about the suitability of some laboratory mice for helicobacter vaccine studies and the validity of a number of past studies in other areas of biomedical research. In helicobacter research, the question of prior antigen exposure is important in terms of host immune response and the use of mouse models in which animals have been, or are still, naturally infected with other Helicobacter species in the intestinal tract is significant.

Mice infected with H. hepaticus develop chronic liver inflammation and, in some strains of mice, hepatocellular carcinoma develops after long term infection (42, 46). H. hepaticus was first recognized in mice in a long-term toxicology study where both experimental and control animals presented with chronic active hepatitis and hepatic tumors, invalidating the results of the study (177, 178). Subsequently, H. hepaticus infection was found to be widespread among commercially available mice in the USA (148). Helicobacter bi/is has also been associated with hepatitis in aged, inbred strains of mice (48) and, like H. hepaticus, was isolated from commercially available mice (148). The other Helicobacter spp. which have been shown to colonize the mouse intestinal tract are Helicobacter muridarum (94) and Helicobacter trogontum (104). The closely related "Flexispira rappini" has also been isolated from the mouse intestinal tract (145).

Prior to this study, no investigation had been made of the presence or prevalence of these murine helicobacters in Australian mouse colonies. This study aimed to investigate the types of Helicobacter species naturally colonizing either conventional or SPF laboratory mice. In a broader context, the question being asked was if there exists a "conventional" mouse, and are the mucosa-associated intestinal helicobacters "autochthonous microbiota". That is, are the same types of spiral microorganisms present in all mice, indicating a long evolutionary relationship between bacterium and host. Chapter4 56

4. 2 Experimental design Conventional or Specific Pathogen Free (SPF), Quackenbush/Swiss (Q/S) mice (n=23) were obtained from four separate animal facilities in Sydney. The origin of all mice was the Gunn Building animal facility, Sydney University. Mice were then rederived by cesarean derivation at the Combined Universities Laboratory Animal Services (CULAS) facility with controlled out-breeding and maintained at SPF category 4. From here mice were taken to the facilities from which they were obtained for this study and maintained either under conventional or SPF category 1 conditions. These facilities were; The UNSW Animal Breeding and Holding Unit (ABHU, n=6, conventional), The Children's Medical Research Institute (CMRI, n=6, SPF), The Heart Research Institute (HRI, n=8, conventional), and The UNSW Microbiology Animal Facility, (UNSW, n=3, conventional). All mice obtained were female ex-breeding stock due to be euthanased.

Mice were sacrificed and the liver and gastrointestinal tract (GIT) removed aseptically for culture and histological processing according to the methods described (Chapter 2). The liver removed first and divided into two, half for culture and half for fixation. Four regions of the GIT, stomach, caecum, small bowel (8 cm immediately above the ileal­ caecal junction) and large bowel (colon, 6 cm immediately below the ileal-caecal junction), were located and samples taken. Figure 4.1 illustrates the isolation protocol in detail. In the case of SPF mice from the CMRI facility, only caecum and liver samples were taken for culture while all samples were fixed for histological processing.

After several passages, and when cultures appeared to be pure based on examination of plates and observation of wet mounts using phase contrast microscopy, DNA was isolated from cultures using one of the methods described (Chapter 2). Cultures were also preserved in BHIG in liquid nitrogen. Morphology and growth characteristics, in conjunction with a battery of PCRs, were used to classify isolates into 10 groups, designated A-J, as described in the following sections. VI

-...J

~

n

f

processing

HBA

filtration

l

histological

for

fix

selective

CSA

"

specimen

l

tissue

rnicroaerophilic

'--..

HBA

+

inoculation

direct

/

-CJ1

sample

CSA

or

liver

I

+

culture

scraping

HBA

mucus homogesised

filtration

l

selective

CSA

+

protocol

anaerobic

HBA

Isolation

+

4.1

direct inoculation

CSA Figure Chapter4 58

4.3 Results A total of 110 separate cultures of motile, spiral or curved bacteria were obtained (ABHU=48, CMRI=9, HRI=36, UNSW=17). The nomenclature devised here to identify isolates indicated the animal facility, mouse number, tissue type and in some cases a distinguishing feature of the isolates morphology. Three general morphological types were observed. These were; morphology type 1, a quite thin, spiral shape, designated "sp" (for spirillum), morphology type 2, a curved to straight, fusiform rod designated "fr" (similar to H. bi/is, H. trogontum and "F. rappini''), and morphology type 3, a thick, rod shaped organism which ranged from being straight but flexible or slightly curved to being a loose spring like form designated "fat S" (Figure 4.2). A degree of morphological variation existed within the group of isolates classified as having morphology type 1. These variations were difficult to detect without simultaneous direct comparison with all other variants. This group therefore, was not further divided according to morphological observation. Numbers of isolates which possessed each morphology type are presented (Table 4.1).

A rapid urease test was performed to assist in the separation of isolates. All isolates with morphology type 2 were urease positive, while all type 3 isolates were negative. Isolates with morphology type 1 had variable urease activity.

DNA isolated from the 110 bacterial cultures was firstly subjected to the general bacterial 16S rDNA PCR using primers F27(UFP) and R1492(URP) (Table 2.1) to ensure the suitability of DNA samples in amplification reactions. All templates gave a positive result and therefore were able to be used further in specific PCRs to determine the presence and type of Helicobacter species in samples. Helicobacter genus specific PCR was carried out to determine the number of isolates which were members of this genus. Non­ helicobacters were considered to be those whose DNA did not amplify in either generic Helicobacter PCR, with primers F27(UFP) and R274, or with primers H276f and H676r (Table 2.1). There was 100% agreement between results obtained for both reactions. The number of Helicobacter PCR positive DNA samples, for each morphology type, is presented (Table 4.2). In summary, all isolates with morphology type 2 were helicobacters and all isolates with morphology type 3 were non-helicobacters. Approximately half of the morphology type 1 isolates were helicobacters. However, all morphology type 1 isolates from CMRI were helicobacters and all morphology type 1 isolates from HRI were non-helicobacters. Figure 4.2 Phase contrast photomicrographs showing the three characteristic morphology types for organisms isolated in this study. These morphology types were describes as being; type 1, "spirillum-like"; type 2, "Flexispira-like"; type 3, fat S-shaped. (A) morphology type 1, bacterial group C. (B) morphology type 1, bacterial group D (C) morphology type 1, bacterial group I (D) morphology type 2, bacterial group A (E) morphology type 3, bacterial group J I ,I ,, ) • -I-. I A • • .,.,. o, .... - '~,; - •.. .,., / \1 \ . . •, .. 4 • ~ .. • • I • . ' ,; • • ' ' . \ . , .\ / , /' --., r I • , \ ~)' I I , ~ ..,._ , :i , .I , • • I ... ' - -· 't , ' ., ...... I' \ ,.·. -.! ' ·- \ . B .. ..• <. . ., ,· 1, . . , • l • , ~· 1. t/ . / ·/ • ? ;_., ...... _, - ~' \ \ ., .._ ; • ...... ·- . , .... ; ... , .. • • \ . . 11 -\./ r ,,__:::-. .,. ,, ..'. .. • -,,-- J ,....__ E ~~ .. "' ~ ....._ , t • . • ...... ~ 'l. I ( ,. / • , . \ .• . . . . - I/ -'· . ., .. .. ' ... ' / . ,·, I .... - ' ., . • -, • .. ·. ,c /\;_I .. '' )- .. . -/ , • I .. ~ • • - ' 'y.,. , ... • . I ., V- - \ \ I • • . ' " "J. \ \ y i .. Chapter4 60

Table 4.1 Number (and percentage) of isolates, with each of 3 morphology types, cultured from mice originating from different animal facilities.

Morphology Type

Animal Facility 1 2 3

ABHU 23 15 10 n=48 (48%) (31%) (21%)

CMRI 9 0 0 n=9 (100%) (0%) (0%)

HRI 21 15 0 n=36 (58%) (42%) (0%)

UNSW 9 6 2 n=17 (53%) (35%) (12%)

Total 62 36 12 n=llO (56%) (33%) (11%) Chapter4 61

The non-helicobacter samples were therefore, either morphology type 1 or 3 These isolates were designated as group I (morphology type 1) or group J (morphology type 3), and will be referred to by these group classifications for the remainder of this thesis. Further characterization of these isolates will be presented in Chapters 7 and 8.

A number of the Helicobacter PCR positive samples produced enlarged products when amplified in reactions with both the universal bacterial primer set, F27(UFP) and Rl492(URP), and the Helicobacter generic primer set, F27(UFP) and R274. This was believed to be due to the presence of an intervening sequence (IVS) inserted into the l 6S rRNA gene 5' of nucleotide position 274 (E.coli numbering). Additionally, some Helicobacter positive samples when amplified in either of the above reactions gave product bands of two different sizes, one of the expected size and one the same size as the enlarged products, indicating that these isolates possibly possessed 16S rRNA operons which were polymorphic for the presence of an inserted IVS.

Based on the size of the amplification product(s) obtained from PCR utilizing primers F27(UFP) and R274, Helicobacter PCR positive samples were divided into those possessing 16S rDNA without an inserted IVS, those with an IVS, and those polymorphic for the presence of an IVS (Table 4.4). All but one of the polymorphic IVS samples came from isolates with morphology type 2 (15/15 for ABHU and 6/6 for UNSW). None of the HRI samples with morphology type 2 however, were IVS polymorphic, all producing a single enlarged band when amplified in the generic Helicobacter PCR utilizing primers F27(UFP) and R274.

The helicobacter positive samples were then subjected to a range of species specific PCRs. The logic of the PCR classification system which was adopted is represented in the flow diagram (Figure 4.4) and described below. As there was no conclusive evidence that IVS polymorphic l 6S rRNA operons did in fact exist in the isolates so classified, the possibility that these DNA samples simply originated from mixed cultures of IVS positive and negative species was considered. Also, several of the species being tested for were IVS negative, so it was possible that mixed cultures of IVS negative species also existed. Where there was any possibility of a mixed culture, all relevant PCRs were performed on the DNA sample. For example, a sample containing 16S rDNA of normal size, without an inserted IVS, was subjected to H. muridarum and H. hepaticus species specific reactions, and samples containing 16S rDNA polymorphic for an IVS were subjected to H. muridarum, H. hepaticus and H. bilis PCRs. Chapter4 62

Table 4.2 Number (and percentage) of Helicobacter PCR positive isolates with each of the three morphology types.

Morphology Type (% helicobacters) Animal Facility 1 2 3

ABHU 18/23 15/15 0/10 (78%) (100%)(0%)

CMRI 9/9 NA NA (100%)

HRI 0/21 15/15 NA (0%) (100%)

UNSW 5/9 6/6 0/2 (56%) (100%)(0%)

Total 32/62 36/36 0/12 (52%) (100%)(0%) Chapter 4 63

M 1 2 3 4 5 6 7 8 9

Figure 4.3 PCR products from the Helicobacter genus specific reaction using primers F27 (UFP) and R27 4 (Table 2.1) showing examples of normal size product (smaller band, -300 bp) and possible polymorphic rDNA for the presence of an inserted IVS (double bands, -500 bp and -300bp). All DNA templates were extracted from bacterial isolates from mice housed at the UNSW facility. DNA separated in 2% agarose/TAE.

lane M Xl 74/Hae III (200 ng) 1 UNSWlcaest 2 UNSWlcaefr 3 UNSWlLBst 4 UNSW2LBfr 5 UNSW3caefr 6 UNSWlcaesp 7 UNSWlcaefatS 8 Helicobacter muridarum positive control 9 "no DNA" control Chapter4 64

Table 4.4 Intervening sequence (IVS) status of Helicobacter PCR positive isolates.

Morphology Type Animal Facility 1 2

ABHU IVS negative 9/18 0/15 n=33 IVS positive 8/18 0/15 IVS polymorphic 1/18 15/15

CMRI IVS negative 9/9 NA n=9 IVS positive 0/9 NA IVS polymorphic 0/9 NA

HRI IVS negative NA 0/15 n=15 IVS positive NA 15/15 IVS polymorphic NA 0/15

UNSW IVS negative 4/5 0/6 n=ll IVS positive 1/5 0/6 IVS polymorphic 0/5 616

Total IVS negative 22/32 0/36 n=68 IVS positive 9/32 15/36 mixed IVS 1/32 21/36 Chapter 4 65

Helicobacter genus specific PCR F27(UFP)/R274 and H276f/H676r I t + r negative positive no further PCR (Groups I and J) IVS positive IVS polymorphic I t t morphology type 2 morphology type 1

H. bilis PCR H. bilis PCR C62/C12 C62/C12 'I 'I t t f t positive negative positive negative l Group' B Group' D Group A Group C IVS negative H. bilis

H. hepaticus PCR B38/B39 H. muridarum PCR H276f/HmurR I I + + + t negative positive negative positive I H. trogontum PCR B72/B39 Group E H. muridarum'

negative positive Group H

Figure 4.4 Group F Group G PCR classification of bacterial groups A-J H. hepaticus' H. tro!(ontum' Chapter4 66

Based on the PCR results, the helicobacter positive samples were divided into 8 groups designated A-H with the following profiles. Groups A-D were those 16S rDNA species which were IVS positive (or IVS polymorphic), and were from cultures with either morphology type 2 (groups A and B) or morphology type 1 (groups C and D). These four groups were either positive (groups A and C) or negative (groups B and D) in the H. bilis specific PCR using primers C62 and C12 (Table 2.1). Group A therefore, contained H. bilis PCR positive isolates which were IVS positive or IVS polymorphic, had morphology type 2 (fusiform) and were urease positive. These were tentatively classified as H. bilis, although it was noted that some group A isolates were IVS polymorphic which has not been reported previously in H. bilis. Group B isolates were considered to be "F. rappini"-like. This organism has a similar morphology to H. bilis as well as being urease positive, and some isolates possess 16S rDNA including an inserted IVS (48). This description fitted with the profile of group B isolates. However, all group B isolates were IVS polymorphic which has not been previously reported for "F. rappini. Isolates in groups C and D (morphology type 1) were considered to be previously unrecognized members of the genus Helicobacter. This conclusion was made based on their being morphologically very different from bacterial groups A and B, which are similar to H. bilis and "F. rappini'', and these are the only murine helicobacters reported to possess IVSs in their 16S rRNA genes. In summary, all of the morphology type 2 isolates and all polymorphic IVS samples (both morphology type 1 and type 2) were therefore able to be classified into bacterial groups A, Band D. No bacterial group C isolates were IVS polymorphic. A single sample arising from a mixed culture was detected in one of the polymorphic IVS samples which was amplified in the H. muridarum specific PCR. This organism was classified as B/E to indicate the presence of both of these bacterial groups.

The remaining groups of helicobacters (E-H) consisted of those isolates having 16S rDNA without an inserted IVS. Isolates of H. muridarum (group E), H. hepaticus (group F) and H. trogontum (group G) fell into this category. Group H were 16S rDNA IVS negative samples which could not be classified by PCR into any of these three species groups. All isolates without an IVS were amplified in PCRs utilizing both primer set H276f and HmurR, and primer set B38 and B39 to ensure the presence of a mixed DNA sample originating from known murine Helicobacter species was not overlooked.

Results obtained from these reactions indicated that there were no samples containing either H. hepaticus or H. trogontum DNA. As there was no positive PCR for the identity of isolates categorized in group H the possibility of DNA from such organisms being present in samples classified as H. muridarum (group E) could not be discounted at this point. For the purposes of this chapter however, all DNA samples which gave a single Chapter4 67

300 bp band in the Helicobacter specific PCR, using primers F27(UFP) and R274, were classified as either H. muridarum (group E, n=6) or Helicobacter sp. (group H, n=l 7).

All isolates were therefore classified into 10 groups (A-J) and expressed as a percentage of the total number of isolates obtained from each of the 4 facilities (Table 4.5). The total number of 16S rDNA species reported here (n=l 11) is greater than the original number of DNA samples (n=l 10) since both bacterial groups (Band E) detected in the single mixed culture have been included. Although this sample was detected as being a mixed culture, it was also considered as having 16S rRNA operons polymorphic for the presence of an IVS since all other bacterial group B isolates were also polymorphic samples. The proportion of isolates classified into bacterial groups A-D, which were also polymorphic in their 16S rDNA size profiles, is presented (Table 4.6). All members of bacterial groups A, B and D from facilities ABHU and UNSW were polymorphic (n=22) and bacterial group C isolates from these same facilities were not IVS polymorphic (n=9). Bacterial group A was the only IVS positive group detected in HRI isolates all of which were not polymorphic (n=15)

The percentage of mice from which bacterial groups (A-J) were isolated is presented (Figure 4.5). These values were also divided according to animal facilities (Figure 4.6). Isolates originating from mice housed at animal facilities ABHU and UNSW showed a high level of species diversity, while isolates from CMRI and HRI animals had limited bacterial diversity. A single group of helicobacters was detected in mice from both of these facilities and one non helicobacter group (group I) was also detected in HRI mice.

The number (and percentage) mouse tissue samples from which the different bacterial groups were isolated was also calculated (Table 4.7 and Figure 4.7). The CMRI facility was omitted from this calculation since isolations were only made from liver and caecal samples in these mice. Chapter4 68

Table 4.5 Number (and percentage) of isolates classified in groups A-J from each animal facility.

Animal Facility Isolate Group ABHU CMRI HRI UNSW Total n=48 n=9 n=36 n=18 n=lll

A (H. bilis) 3 (6%) 0 15 (42%) 1 (6%) 19 (17%)

B 12 (25%) 0 0 5 (28%) 17 (15%)

C 8 (17%) 0 0 1 (6%) 9 (8%)

D 1 (2%) 0 0 0 1 (1%)

E (H. muridarum) 3 (6%) 0 0 3 (17%) 6 (5%)

F (H. hepaticus) 0 0 0 0 0

G (H. trogontum) 0 0 0 0 0

H 6 (13%) 9 (100%) 0 2(11%) 17 (15%)

I 5(10%) 0 21 (58%) 4 (22%) 30 (27%)

J 10 (21 %) 0 0 2 (11%) 12(11%)

Table 4.6 Number (and percentage) of isolates cultured from mice originating from different animal facilities, classified in bacterial groups A-D, which were polymorphic for an IVS.

Animal Facility

Bacterial Group ABHU HRI UNSW

A 3/3 0/15 1/1

B 12/12 NA 5/5

C 0/8 NA 0/1

D 1/1 NA NA Chapter 4 69

70

60

so

Q) C') 40 ,._(Cl C: Q) u L. Q) 30 Q,

20

10

0 A B C D E F G H J bacterial group

Figure 4.5 Percentage of mice from which bacterial groups (A-J) were cultured. Chapter 4 70

ABHU CMRI

100 100

90 f--- 90

80 f--- 80

70 f--- 70 ~ en en~ 0:, 60 60 ...... 0:, C C ~ so ~ so ..~ ..~ ~ 40 ~ 40 Cl. Cl. 30 30 20 20 1 0 -- 10 0 0 A B C D E F G H I J A B C D E F G H I J bacterial group bacterial group

HRI UNSW

100 100 90 90 ,- 80 80 - 70 - 70 - ~ ~ en - en 60 - ...0:, 60 ...0:, C - C ~ so ~ so -- ~ ~ ~ - .. .. 40 ~ 40 Cl. Cl. 30 - 30 - t-

20 - 20 t-

10 ,_ 1 0 - 0 I I 0 I ABCDEFG H IJ A B C D E F G H I J bacterial group bacterial group

Figure 4.6 Percentage of mice from different animal facilities from which bacterial groups (A-J) were isolated. n::r Cl "d ~ '"I ~ 80 • A 70 DB •c 60 - • D I b acten'al group Q) er) so ...(Cl ;; 40 c.:, L. ci:, 30 c. 20

10

0 liver stomach small caecum large bowel bowel tissue type

Figure 4.7 Percentage of different mouse tissue types from which bacterial groups (A-J) were isolated.

----..} Chapter4 72

4.4 Discussion and Summary A wide range of spiral bacteria was cultured from the mice used in this study, a large percentage (62%) of which were identified as being members of the Helicobacter genus. The non-helicobacter isolates were divided morphologically into 2 groups, I and J. One of these groups (I) was indistinguishable in growth characteristics and morphology from many of the Helicobacter isolates cultured in this study from mouse intestinal tissue.

Two of the previously described murine helicobacters, H. hepaticus and H. trogontum, were not isolated from any of the animals used in this study. This finding was unexpected, particularly as H. hepaticus has been found to be widespread in colonies of laboratory mice in the USA (137, 148). Until now, no study had been made to determine the presence or prevalence of these organisms in Australia. H. bilis (group A) and H. muridarum (group E) positive samples made up 28% and 9% respectively of the total number of helicobacter isolates leaving 63% of isolates without a definitive species classification. Although 25% of the unclassified helicobacters were considered to be "F. rappini'' -like, the remaining 38% were without obvious taxonomic affiliation within this genus and it is possible they represent up to three previously unreported species (groups C, D and H). Elucidation of such relationships requires more detailed examination, both genetic and biochemical. Relationships between these groups, based on 16S rDNA sequence analysis, is presented in the following chapters (Chapters 5 and 6).

A number of DNA samples appeared to possess operons of the 16S rRNA which were polymorphic for the presence of an intervening sequence. The evidence for this was the presence of two different sized PCR products after amplification with the generic Helicobacter primer set F27(UFP) and R274. In 21/22 of the IVS polymorphic samples the organism observed in cultures by phase contrast microscopy had a type 2 morphology. However, the presence of IVS polymorphic 16S rDNA was not constant for all bacterial group divisions among animal facilities. The 16S rDNA from all bacterial group A isolates from ABHU and UNSW were polymorphic for an IVS, whereas all group A isolates from HRI had only IVS positive 16S rDNA. All group B isolates were polymorphic, as was the single group D isolate with morphology type 1. However, organisms cultured on different occasions, by the author and other members of the same research group, have been classified into bacterial group D using the same criteria as developed for this study, and these do not all possess polymorphic 16S rDNA (unpublished observation).

There is a strong precedence for different rRNA operons within a single bacterium to be polymorphic for the presence of an IVS (68-70, 98). That is, different operons of the rRNA gene of a single organism differ in terms of whether they possess an inserted NS or not. The 16S rRNA gene in Campylobacter helveticus has been shown to contain an IVS in some strains and to be polymorphic for the presence of an IVS other strains (98), Chapter4 73

so not all members of a single species have the same IVS profile. This polymorphic IVS status has not been previously reported in the 16S rDNA of Helicobacter species, but 23S rRNA genes in helicobacters have been shown to be polymorphic in a number of isolates (68, 70). There appears to be no reason, therefore, why the 16S rRNA operons of Helicobacter species may not also be able to be polymorphic for the presence of an IVS. The results described in this chapter present such a possibility, although this line of investigation was not pursued in further experiments.

The diversity of bacterial groups cultured varied significantly among the different animal facilities. Mice originating from animal facilities ABHU and UNSW were colonized by a wide range of different helicobacter isolate groups as well as both morphology types of non-helicobacters. All mice from both of these facilities were colonized by group B helicobacters with other groups isolated from less than 100% of animals. This situation was in contrast with the isolation profile for animals originating from animal facilities CMRI and HRI. In mice from both of these facilities only one helicobacter group was isolated (Hand A respectively). Furthermore, only non-helicobacter group I was isolated from HRI derived mice and neither non-helicobacter group was isolates from CMRI mice. To summarize, the CMRI and HRI mice were much more limited in the range of bacterial groups detected than were ABHU and UNSW mice. This limited bacterial diversity was expected in the case of the SPF mice from CMRI. In such cases a single dominant bacterial species can thrive without competition from other species which inhabit the same ecological niche. The case of the conventional mice from HRI containing only one group of helicobacter (group A, H. bilis) and only one of the non-helicobacter groups (group I) was, however, unexpected.

When isolates were separated according to the location in the mouse from which they were cultured, as expected, very few helicobacters or non-helicobacters were isolated from liver or stomach samples. In fact, no helicobacters were isolated from livers of the conventional mice, and only a single group I isolate was cultured from the liver of one HRI mouse. Although H. bilis (bacterial group A) was isolated from 7/8 HRI, 3/6 ABHU and 1/3 UNSW mice it was not isolated from any liver samples. This may be due to the fact that the animals used in this study were under 12 months old. Helicobacters were also cultured from very small numbers of stomach samples ( 1/8 for HRI and 2/6 for ABHU) and non-helicobacter group J was only isolated from 3/6 ABHU stomach samples. By far the majority of cultures originated from caecal, small bowel and large bowel samples. Slightly higher numbers of isolates and were obtained from caecal and large bowel samples compared to small bowel samples, as was the prevalence of most bacterial groups. However, the numbers of mice investigated was too small to attach any real significance to these values. The difficulty and time involved in culturing many Chapter4 74

Helicobacter species, and the small numbers of animals used in this study, requires a more sensitive and reliable technique for detecting these organisms without the need to culture.

The results presented in this chapter, although involving small numbers of animals, highlight the vast differences which can occur in the normal microbiota of laboratory mice. An awareness of these possible differences is essential in helicobacter research. In particular, the differences in antigen exposure among animals could impact significantly on helicobacter vaccine studies. A further important observation, highlighted by these results, is in the definition of conventional and SPF mice. SPF animals are defined as animals that are free of specific microorganisms and parasites, but not necessarily free of others not specified (167). One of the major problems with the term SPF is that it sounds like a well defined category. Earlier studies have documented wide variation between the numbers and species of facultatively anaerobes in SPF units in Australia (114). It is widely assumed that SPF animals are Helicobacter-free, and this is clearly not the case. Concern over the presence of lower bowel helicobacters has resulted in some major animal facilities now screening for the presence of these organisms, particularly H. hepaticus which is known to be associated with liver disease in mice (46).

The following chapters (Chapters 5-8) examine more closely the identity of some of the bacterial groups described here, followed by a reexamination using PCR of the samples taken here and fixed for histological examination (Chapter 9). Chapter 5 75

Chapter 5

Ribosomal RNA Gene Sequences and Inferred Phylogenetic Relationships Among Helicobacter Species

5. 1 Background Gene sequences of the 16S ribosomal RNA have been used extensively to measure the genetic relatedness of Helicobacter species and to assist in classification and speciation (13, 21, 26, 27, 42, 48, 49, 57, 94, 104, 126, 149, 153, 156, 157). While not by themselves sufficient for the definition of new species or classification of isolates into existing taxa (taxonomy), phylogenies reconstructed from 16S rDNA sequences are widely used to illustrate proposed evolutionary relationships by the measurement of genetic distances between selected organisms. Phylogenetic studies involving 16S rRNA gene sequences have also proved useful at the diagnostic level enabling the development of probes which can be used for the recognition of unidentified strains. The use of rRNA gene sequence data in phylogenetic studies is considered an essential piece of systematic information used for revealing natural relationships among organisms which ideally determines taxonomic placements.

The aim of this study was to determine the 16S rRNA gene sequence of isolates representing the different groups of helicobacters defined in Chapter 4 and to include these sequences in a phylogenetic tree reconstructed from 16S rRNA gene sequences of Helicobacter and closely related species.

5. 2 Experimental design Isolates selected for sequence determination represented helicobacter positive bacterial groups A, C, D and H described in Chapter 4. Four additional isolates which were cultured from conventional Q/S mice housed at the UNSW animal facility were included in this study. Three of these isolates fitted the morphological and PCR profile of bacterial group Das defined in Chapter 4. These isolates were designated UNSW1.7col, UNSW1.7sp (isolated by Jani O'Rourke) and UNSWMCSpl (isolated by the author). The fourth additional isolate fitted the profile of bacterial group C. This isolate was designated UNSW3. l lcae, again isolated by Jani O'Rourke. 16S rDNA sequences from Helicobacter muridarum isolates UNSW1.6cae and UNSW1.7st, representing group E, which had been determined previously (Chapter 3) were also included here in the phylogenetic analysis. No organisms representing group B were included. This was Chapter 5 76

primarily due to time constraints and the slightly more technically difficult task of sequencing polymorphic operons. It was felt that this group of organisms warranted a separate, more detailed study which was not performed as part of the research presented in this thesis.

The 16S rDNA was amplified by PCR using the modified consensus primers F27(UFP) and R1492(URP) (Table 2.1) complementary to the conserved 5' and 3' ends of the 16S rRNA gene. DNA amplification reactions were prepared as described (Chapter 2) with a thermocycling profile as follows. An initial denaturation step at 94°C for 3 minutes, followed by 5 cycles of 94°C for 45 seconds, 53°C for 45 seconds and 72°C for 1 minute, then 28 cycles of 94°C for 45 seconds, 48°C for 45 seconds and 72°C for 1 minute. This was followed by a hold a 72°C for 10 minutes (FfS 320, Corbett Research). The product from several amplification reactions was combined and purified then used as the template DNA in sequencing reactions. 16S rDNA sequences were determined using the methods described (Chapter 2) with thirteen oligonucleotide primers enabling sequencing of both strands of the 16S rDNA with contiguous overlaps (Table 2.2). Sequences were aligned and a phylogeny of the genus Helicobacter, together with representative Campylobacter and Arcobacter species, inferred based on a pairwise distance comparison of all sequences included in the analysis.

5.3 Results Near complete 16S rDNA sequences for 16 isolates (group A=3, group C=4, group D=3, group E=2 and group H=4) were determined and have been submitted to the GenBank data base (Table 5.1).

Sequences from the group A isolates were identical to one another and had 99.4% sequence identity to Helicobacter bilis. Group C isolates had between 99.6-100% sequence identity with one another and 99.2-99.4% sequence identity to H. bilis. Group C isolates had slightly lower sequence identities (98.6-98.8%) with the consensus sequence from group A isolates in this study. Sequences from group D isolates had 98.6- 99 .3% sequence identity with one another and 97.6-98.3% identity with H. bilis. Again, these isolates had very high percentages of sequence identity with group A isolates (96.9- 97. 7%) and group C isolates (97.9-98.9%). The sequences from all of these isolates (groups A, C, and D) possessed the same 200 bp IVS also present in H. bilis. Chapter 5 77

Table 5.1 GenBank accession numbers for the 16S rRNA gene sequences of Helicobacter isolates determined during this study.

Group Isolate Accession Number

Group A HRI3caefr AF054570 HRI6caefr AF054571 HRI7stomfr AF054572

Group C UNSW3.l lcae AF054573 ABHU3cae AF054574 ABHU4stomsp AF054575 UNSW3SBsp AF054576

GroupD UNSW1.7col AF054577 UNSW1.7sp AF054578 UNSWMCSpl AF054579

GroupE UNSW1.6cae AF013464 UNSW1.7st AF010140

Group H ABHU4caesp AF000223 CMRI2cae AF000221 CMRI2liv AF000222 UNSWlcaesp AF000224 Chapter 5 78

As presented in Chapter 3, two suspected H. muridarum isolates (group E) had 99.6% sequence identity with each other and 99.6% and 99.2% identity with H. muridarum. Finally, the sequences from the 4 group H isolates were identical and had 98.2% identity to Helicobacter rodentium, and 96.2% identity to Helicobacter pullorum. A closer examination of this group of organisms will be presented in Chapter 6.

A multiple sequence alignment of the determined 16S rRNA gene sequences of these isolates is presented in Appendix 1. The multiple sequence alignment of these sequences plus representative 16S rDNA sequences from other Helicobacter and closely related species formed the basis for the phylogenetic analysis. The 16S rRNA sequence of Escherichia coli was used as an outgroup in the tree reconstruction. The GenBank accession numbers for sequences included in the phylogenetic tree are found in Table 5.2. Pairwise comparisons of the 16S rRNA sequences are presented in a similarity matrix (Table 5.3). The uncorrected percentage sequence similarities (upper triangular half) were calculated from the aligned sequences with a total length of 1744 bases (including IVS) prior to ambiguous positions (insertions and unidentified bases) being removed. Internal gaps (independent of length) in the sequence alignment were included, but end gaps were excluded, and percentage similarities calculated based on the number of positions compared between individual sequences. Corrected genetic distance measurements used in the phylogenetic reconstruction were calculated after all ambiguous positions were removed leaving an alignment of 1332 bases (lower triangular half, genetic distance XlOO). Figure 5.1 is the phylogenetic tree reconstructed from the calculated genetic distances corrected for multiple base changes by the method of Jukes and Cantor (73) using the neighbor-joining method for tree reconstruction (140). Bootstrap values indicate the number of times the group of species to the right of a node occurred among different trees ( out of 100 resamplings of the data) (31 ). Only values which were greater than 50% are reported.

Transmission electron micrographs showing examples of bacterial groups A, C, D and H are presented (Figures 5.2-5.4). An electron micrograph of bacterial group His presented in the following chapter (Chapter-6). Chapter 5 79

Table 5.2 GenBank accession numbers for the 16S rRNA gene sequences of organisms used in phylogenetic analysis.

Organism Accession Referenceh Number•

Arcobacter butzleri L14626 (157) Arcobacter cryaerophilus L14624 (157) Campylobacter coli L04312 (185) Campylobacter jejuni L04315 (185) Escherichia coli 101695 (14) "Flexi.spira rappini" M88137 (26) "Gastrospirillum hominis" I L10079 (153) "Gastrospirillum hominis" 2 L10080 (153) Helicobacter acinonyx M88148 (26) Helicobacter bilis U18766 (48) Helicobacter bizzazeronii Y09404 (72) Helicobacter canis Ll3464 (157) Helicobacter cholecystus U46129 (49) Helicobacter cinaedi M88150 (157) Helicobacter felis M37642 (126) Helicobacter fennelliae M88154 (157) Helicobacter hepaticus L39122 (8) Helicobacter muridarum M80205 (94) Helicobacter mustelae M35048 (126) H elicobacter nemestrinae X67854 (152) Helicobacter pametensis M88147 (21) Helicobacter pullorum L36141 (156) Helicobacter pylori M88157 (157) Helicobacter rodentium U96297 (149) H elicobacter salomonis U89351 (72) Helicobacter sp. Bird-B M88139 (21) Helicobacter sp. Bird-C M88144 (21) Helicobacter sp. CLO 3 M88151 (157) Helicobacter suncus AB006147 Unpublished Helicobacter trogontum U65103 (104) Helicobacter westmeadii U44756 (168) Woline/la succinogenes M88159 (185)

a Sequences are available directly through the GenBank accession numbers. h References for those sequences which have been published. Chapter 5 80

Table 5.3 Similarity matrix for species used in phylogenetic reconstruction. Upper triangle values are percentage sequence identities. Lower triangle values are genetic distance measurements corrected for multiple base changes by the method of Jukes and Cantor for sequence positions which were unambiguous for all sequences in the analysis. Isolates whose 16S rDNA sequence was determined during this study are highlighted in bold text.

Sequences are in the order;

1 Arcobacter butzleri 25 Helicobacter hepaticus 2 Arcobacter cryaerophilus 26 Helicobacter trogontum 3 Campylobacter coli 27 H elicobacter cholecystus 4 Campylobacter jejuni 28 Helicobacter pametensis 5 Helicobacter sp. CLO 3 29 Helicobacter suncus 6 Helicobacter fennelliae 30 Helicobacter mustelae 7 HRI3caefr (group A) 31 Helicobacter sp. Bird-B 8 HRI6caefr (group A) 32 Helicobacter sp. Bird-C 9 HRl7stomfr (group A) 33 Helicobacter pullorum 10 ABHU3cae (group C) 34 CMRI2cae (group H) 11 UNSW3.llcae (group C) 35 UNSWlcaesp (group H) 12 UNSW3SBsp (group C) 36 CMRI2liv (group H) 13 ABHU4stomsp (group C) 37 ABHU4caesp (group H) 14 UNSWMCSpl (group D) 38 Helicobacter rodentium 15 UNSW1.7col (group D) 39 Helicobacter acinonyx 16 UNSW1.7sp (group D) 40 Helicobacter pylori 17 Helicobacter bi/is 41 Helicobacter nemestrinae 18 Helicobacter canis 42 Helicobacter bizzozeronii 19 "Flexispira rappini" 43 H elicobacter salomonis 20 Helicobacter cinaedi 44 "Gastrospirillum hominis" 2 21 Helicobacter westmeadii 45 Helicobacter felis 22 UNSWl. 7st (group E) 46 "Gastrospirillum hominis" 1 23 UNSW1.6cae (group E) 47 Wolinella succinogenes 24 Helicobacter muridarum 48 Escherichia coli Chapter 5 81

2 3 4 5 6 7 8 9 1 0 11 1 2

I. Arcobacter butzleri 97.3 86.3 86.8 86.8 86.1 84.9' 84.8 84.9 84.6 84.5 84.6 2. Arcobacter cryaerophilus 2.44 86.7 87.2 86.0 85.0 84.0 84.0 84.0 83.9 83.7 83.8 3. Campylobacter coli 14.53 14.08 98.6 86.7 87.5 86.1 86.1 86. I 85.7 85.4 85.6 4. Campylobacter jejuni 13.99 13.45 1.44 86.6 87.2 85.8 85.8 85.8 85.4 85.1 85.3 5. Helicobacter sp. CLO 3 13.81 14.81 14.35 14.44 95.0 95.4 95.4 95.4 94.9 94.7 94.7 6. Helicobacter fennelliae 14.99 16.38 13.45 13.81 4.73 96.0 96.0 96.0 95.2 95.1 95.0 7. HRI3caefr 14.17 15. 18 13.00 13.36 4.17 3.46 100.0 100.0 98.8 98.6 98.6 8. HRI6caefr 14.17 15. 18 13.00 13.36 4.17 3.46 0.00 100.0 98.8 98.6 98.6 9. HRI7stomfr 14.17 15.18 13.00 13.36 4.17 3.46 0.00 0.00 98.8 98.6 98.6 10. ABHU3cae 14.35 14.99 13.27 13.63 4.65 4.01 I. 13 1.13 1.13 99.9 99.7 11. UNSW3.llcae 14.35 14.99 13.27 13.63 4.65 4.01 1.13 I. 13 I. 13 0.00 99.6 12. UNSW3SBsp 14.35 15.08 13.27 13.54 4.73 4.09 1.21 1.21 1.21 0.08 0.08 13. ABHU4stomsp 14.35 14.99 13.27 13.63 4.65 4.01 1.13 I. 13 1.13 0.00 0.00 0.08 14. UNSWMCSpl 14.81 15.45 13.54 13.99 4.97 4.81 2.21 2.21 2.21 1.06 1.06 I. 13 15. UNSW1.7col 14.90 15.54 13.63 14.08 5.05 4.89 2.29 2.29 2.29 I. 13 1.13 1.21 16. UNSW1.7sp 14.63 15.27 13.36 13.81 4.81 4.65 2.05 2.05 2.05 0.91 0.91 0.98 17. Helicobacter bi/is 14.35 15.08 13.00 13.36 4.25 3.69 0.68 0.68 0.68 0.45 0.45 0.53 18. Helicobacter canis 14.17 14.99 13.09 13.45 3.85 4.01 1.06 1.06 1.06 1.21 1.21 1.29 19. "Flexispira rappint' 14.63 15. 18 12.47 12.91 4.17 4.17 1.44 1.44 1.44 1.36 1.36 1.44 20. Helicobacter cinaedi 14.63 15.18 13.27 13.72 4.41 3.77 1.44 1.44 1.44 1.82 1.82 1.90 21. Helicobacter westmeadii 14.81 15.27 13.45 13.90 4.8 I 3.61 1.90 1.90 1.90 2.13 2.13 2.21 22. UNSW1.7st 14.08 15.18 13.99 13.99 4.97 4.17 3.22 3.22 3.22 2.91 2.91 2.83 23. UNSW1.6cae 14.26 15.36 14.17 14.17 5.13 4.33 3.38 3.38 3.38 3.06 3.06 2.99 24. Helicobacter muridarum 14.17 15.27 14.08 14.08 5.21 4.09 3.14 3.14 3.14 2.83 2.83 2.75 25. Helicobacter hepaticus 14.35 15.45 13.72 13.99 4.57 4.01 2.29 2.29 2.29 1.75 1.75 1.82 26. Helicobacter trogontum 13.99 15.08 14.17 14.53 4.25 3.77 2.99 2.99 2.99 2.91 2.91 2.99 27. Helicobacter cholecystus 14.44 15.08 13.09 13.82 5.37 4.17 3.22 3.22 3.22 3.06 3.06 3.14 28. Helicobacter pametensis 14.17 14.72 12.82 12.73 4.57 4.09 3.30 3.30 3.30 3.14 3.14 3.22 29. Helicobacter suncus 13.45 14.26 13.72 13.45 4.49 5.21 3.14 3.14 3.14 3.06 3.06 3.14 30. Helicobacter mustelae 13.72 14.35 13.54 13.27 4.33 5.05 3.61 3.61 3.61 3.46 3.46 3.54 3 I. Helicobacter sp. Bird-B 13.45 14.44 I 3.45 13.18 4.49 4.97 3.38 3.38 3.38 3.30 3.30 3.38 32. Helicobacter sp. Bird-C 14.26 15.18 13. 18 12.73 4.01 4.65 2.83 2.83 2.83 2.83 2.83 2.75 33. Helicobacter pullorum 13.36 14.26 13. 18 13.09 3.54 3.61 3.06 3.06 3.06 3.77 3.77 3.85 34. CMRI2cae 14.72 15.36 I 3. 18 13.27 4.41 4.17 4.81 4.81 4.81 4.97 4.97 5.05 35. UNSWlcaesp 14.72 15.36 13. 18 13.27 4.41 4.17 4.81 4.81 4.81 4.97 4.97 5.05 36. CMRI2liv 14.72 15.36 I 3.18 13.27 4.41 4.17 4.81 4.81 4.81 4.97 4.97 5.05 37. ABHU4caesp 14.72 15.36 13.18 13.27 4.41 4.17 4.81 4.81 4.81 4.97 4.97 5.05 38. Helicobacter rodentium 13.54 14.53 12.82 12.82 4.81 4.81 4.09 4.09 4.09 4.25 4.25 4.33 39. Helicobacter acinonyx 17.90 16.10 15.08 14.81 5.93 6.59 5.85 5.85 5.85 6.5 I 6.51 6.59 40. Helicobacter pylori 14.63 15.73 14.90 14.63 5.29 6.10 5.53 5.53 5.53 6.34 6.34 6.42 41. Helicobacter nemestrinae 15.18 16.10 15.36 15.27 7.08 7.58 6.75 6.75 6.75 7.41 7.41 7.49 42. Helicobacter bizwzeronii 16.19 16.85 15.45 15.08 6.10 6.18 6.26 6.26 6.26 6.75 6.75 6.83 43. Helicobacter salomonis 16.29 17.04 15.73 26.36 6.42 6.26 6.34 6.34 6.34 6.83 6.83 6.92 44. "Gastrospirillum hominis" 2 16.10 16.94 15.45 14.99 6.10 6.02 6.26 6.26 6.26 6.59 6.59 6.67 45. Helicobacter felis 16.10 16.74 15.64 15.18 6.02 6.10 6.18 6. 18 6.18 6.67 6.67 6.75 46. "Gastrospirillum hominis" I 16.57 17.04 15.73 15.64 6.18 6.42 6.67 6.67 6.67 7.33 7.33 7.41 47. Woline/la succinogenes 14.72 15.18 14.08 13.99 7.33 7.33 6.26 6.26 6.26 6.34 6.34 6.26 48. Escherichia coli 24.56 24.77 24.40 25.09 24.04 25.09 24.67 24.67 24.67 24.04 24.04 24.15 Chapter 5 82

13 14 15 1 6 17 18 19 20 21 22 23 24

I. Arcobacter butzleri 84.6 84.3 83.7 84.4 85.0 85.2 85.1 85.0 84.6 85.1 84.7 85.4 2. Arcobacter cryaerophilus 83.9 83.5 83.0 83.7 84.4 84.7 84.6 84.4 84.1 84.2 83.8 84.5 3. Campylobacter coli 85.7 85.2 84.7 85.6 86.3 86.6 87. I 86.4 86.1 85.5 85.1 85.8 4. Campylobacter jejuni 85.4 84.9 84.3 85.2 86.0 86.3 86.8 86.0 85.7 85.5 85. I 85.8 5. Helicobacter sp. CLO 3 94.9 94.3 93.9 94.8 95.4 95.9 95.5 95.3 94.9 94.4 93.9 94.4 6. Helicobacter Jennelliae 95.2 94.2 93.8 94.7 95.8 95.5 95.4 95.8 95.8 95.1 94.6 95.3 7. HRI3caefr 98.8 97.1 96.9 97.7 99.4 98.6 98.0 98.0 97.8 96.1 95.7 96.2 8. HRI6caefr 98.8 97.1 96.9 97.7 99.4 98.6 98.0 98.0 97.8 96.1 95.7 96.2 9. HRI7stomfr 98.8 97.1 96.9 97.7 99.4 98.6 98.0 98.0 97.8 96.1 95.7 96.2 10. ABHU3cae 100.0 98.4 98.1 98.9 99.4 98.2 98.0 97.7 97.4 96.3 95.9 96.5 11. UNSW3.llcae 99.9 98.3 98.0 98.9 99.2 98.1 97.9 97.6 97.3 96.2 95.8 96.4 12. UNSW3SBsp 99.7 98.1 97.9 98.7 99.2 98.0 97.8 97.5 97.2 96.2 95.8 96.3 13. ABHU4stomsp 98.4 98.1 98.9 99.4 98.2 98.0 97.7 97.4 96.3 95.9 96.5 14. UNSWMCSpl 1.06 98.6 99.3 97.8 96.7 97.7 97.1 96.9 95.4 94.9 95.6 15. UNSW1.7col 1.13 0.23 99.1 97.6 96.4 97.3 96.7 96.5 95.0 94.7 95.2 16. UNSW1.7sp 0.91 0.15 0.23 98.3 97.2 98.2 97.6 97.5 95.9 95.5 96.1 I 7. Helicobacter bilis 0.45 1.52 1.59 1.36 98.7 98.6 98.1 97.8 96.2 95.8 96.3 18. Helicobacter canis 1.2 I 2. I 3 2.21 1.98 0.98 97.9 97.5 97.4 96.3 95.8 96.4 19. "Flexispira rappini"' 1.36 1.29 1.36 1.13 0.91 1.59 98.7 98.2 96.2 95.8 96.3 20. Helicobacter cinaedi 1.82 1.90 1.98 I. 75 1.36 1.90 1.06 98.9 95.7 95.3 96.1 21. Helicobacter westmeadii 2. I 3 2.21 2.29 2.05 1.82 2.05 1.52 0.60 95.4 94.9 95.4 22. UNSW1.7st 2.91 3.30 3.38 3. 14 3.14 3.22 3.14 3.69 4.09 99.6 99.6 23. UNSW1.6cae 3.06 3.46 3.38 3.30 3.30 3.38 3.30 3.85 4.25 0. 15 99.2 24. Helicobacter inuridarum 2.83 3.22 3.30 3.06 3.06 3.14 3.22 3.61 4.01 0.38 0.53 25. Helicobacter hepaticus 1.75 2.36 2.44 2.21 2.21 2.44 2.21 2.9 I 3.22 1.90 2.05 1.98 26. Helicobacter trogontum 2.9 I 3.77 3.85 3.61 3.38 3.46 4.09 3.38 3.46 3.54 3.69 3.46 27. Helicobacter cholecystus 3.06 3.69 3.77 3.54 2.75 2.99 3.06 3.77 3.93 3.85 4.01 3.46 28. Helicobacter pametensis 3. I 4 3.93 4.01 3.77 2.83 3.22 2.99 4.01 4.09 3.93 4.09 3.85 29. Helicobacter suncus 3.06 3.93 4.01 3.77 2.91 2.75 3.30 3.85 4.09 3.85 4.01 3.77 30. Helicobacter mustelae 3.46 4. I 7 4.25 4.01 3.30 3.22 3.69 4.41 4.49 3.93 4.09 3.85 31. Helicobacter sp. Bird-B 3.30 4.09 4.17 3.93 3.14 2.83 3.54 4.01 4.09 3.85 4.01 3.6 I 32. Helicobacter sp. Bird-C 2.83 3.77 3.85 3.61 2.52 2.52 2.83 3.69 4.01 3.30 3.46 3.22 33. Helicobacter pullorum 3.77 4.4 I 4.49 4.25 3.46 3.46 3.61 4.01 4.17 4.33 4.49 4.41 34. CMRl2cae 4.97 5.53 5.61 5.37 4.57 5.13 4.73 5.05 5.37 5.29 5.45 5.45 35. UNSWlcaesp 4.97 5.53 5.61 5.37 4.57 5. I 3 4.73 5.05 5.37 5.29 5.45 5.45 36. · CMRI2liv 4.97 5.53 5.6 I 5:31 4.57 5.13 4.73 5.05 5.37 5.29 5.45 5.45 37. ABHU4caesp 4.97 5.53 5.61 5.37 4.57 5. I 3 4.73 5.05 5.37 5.29 5.45 5.45 38. Helicobacter rodentium 4.25 5. I 3 5.2 I 4.97 3.85 4.41 4.33 4.33 4.65 5.21 5.37 5.05 39. Helicobacter acinonyx 6.51 7.08 7.16 6.92 6.26 6.10 6.59 6.83 7.16 6.75 6.92 6.83 40. Helicobacter pylori 6.34 7.16 7.25 7.00 5.93 5.93 6.18 6.59 7.08 6.10 6.26 6.34 41. Helicobacter nemestrinae 7.41 8.24 8.24 8.08 7.08 7.25 7 .41 7.58 7 .9 I 7.33 7.41 7.49 42. Helicobacter bizzozeronii 6.75 7.25 7.33 7.08 6.34 6.34 6.42 6.5 I 6.92 6.67 6.83 6.59 43. Helicobacter salomonis 6.83 7.33 7.4 I 7 .16 6.59 6.34 6.67 6.75 6.92 7.00 7. 16 6.92 44. "Gastrospirillum hominis" 2 6.59 6.75 6.83 6.59 6.34 6.26 6.42 6.5 I 6.67 6.75 6.92 6.5 I 45. Helicobacter Jelis 6.67 7. 16 7.25 7.00 6.42 6.34 6.51 6.59 6.92 6.83 7.00 6.75 46. "Gastrospirillum hominis" I 7.33 7.66 7.74 7.49 6.92 7.00 6.75 7.00 7.4 I 7.00 7. 16 7.08 47. Wolinella succinogenes 6.64 6.75 6.83 6.59 6.34 6.26 6.67 6.75 6.92 6.67 6.67 6.42 48. Escherichia coli 24.04 23.73 23.84 23.53 24.35 23.94 24.04 24.46 24.56 24.15 24.25 24.25 Chapter 5 83

25 26 27 28 29 30 31 32 33 34 35 36

1. Arcobacter butzleri 85.1 85.5 85.3 85.6 85.8 86.1 86.1 85.5 86.2 85.0 85.0 85.0 2. Arcobacter cryaerophilus 84.3 84.7 84.8 85.2 85.4 85.8 85.3 84.8 85.4 84.4 84.4 84.5 3. Campylobacter coli 86.2 85.6 86.5 86.9 86.0 86.5 86.4 86.6 86.6 86.4 86.4 86.4 4. Campylobacter jejuni 86.0 85.3 86.8 87.0 86.3 86.8 86.7 87.0 86.7 86.4 86.4 86.4 5. Helicobacter sp. CLO 3 95.1 95.6 94.4 95.0 94.8 95.3 95.2 95.4 96.2 95.4 95.4 95.4 6. Helicobacter fennelliae 95.5 96.0 95.5 95.4 94.0 94.5 94.6 94.7 95.9 95.5 95.5 95.5 7. HRI3caefr 97.2 96.5 96.4 96.1 95.9 95.5 95.9 96.1 96.5 94.7 94.7 94.7 8. HRI6caefr 97.2 96.5 96.4 96.1 95.9 95.5 95.9 96.1 96.5 94.7 94.7 94.7 9. HRl7stomfr 97.2 96.5 96.4 96.1 95.9 95.5 95.9 96.1 96.5 94.7 94.7 94.7 10. ABHU3cae 97.5 96.3 96.3 96.0 95.8 95.5 95.8 96.1 95.6 94.4 94.4 94.4 11. UNSW3.llcae 97.4 96.1 96.1 95.9 95.7 95.4 95.7 96.0 95.5 94.3 94.3 94.3 12. UNSW3SBsp 97.3 96.1 96.0 95.8 95.7 95.4 95.7 96.0 95.4 94.2 94.2 94.2 13. ABHU4s tomsp 97.5 96.3 96.3 96.0 95.8 95.5 95.8 96.1 95.6 94.4 94.4 94.4 14. UNSWMCSpl 96.4 95.0 95.1 94.8 94.5 94.5 94.6 94.9 94.4 93.5 93.5 93.5 15. UNSW1.7col 96.0 94.6 94.7 94.3 94.2 94.1 94.2 94.5 94.1 93.1 93.1 93.1 16. UNSW1.7sp 97.0 95.6 95.6 95.3 95.1 95.0 95.1 95.4 95.0 94.0 94.0 94.0 17. Helicobacter bi/is 97.3 96.1 96.8 96.5 96.1 95.9 96.2 96.5 96.2 94.9 94.9 94.9 18. Helicobacter canis 97.2 96.1 96.6 96.2 96.6 96.2 96.7 96.8 96.3 94.5 94.5 94.5 19. "Flexispira rappint' 97.4 95.6 96.5 96.3 95.7 95.7 95.8 96.3 96.0 94.9 94.9 94.9 20. Helicobacter cinaedi 96.7 96.1 95.7 95.4 95.1 95.1 95.4 95.6 95.5 94.4 94.4 94.4 21. Helicobacter westmeadii 96.4 96.1 95.6 95.3 95.0 94.8 95.2 95.2 95.4 94.3 94.3 94.3 22. UNSW1.7st 97.8 96.1 95.8 95.4 95.4 95.4 95.6 96.0 95.2 94.2 94.2 94.2 23. UNSW1.6cae 97.3 95.7 95.3 95.0 95.0 95.0 95.1 95.6 94.8 93.7 93.7 93.7 24. Helicobacter muridarum 97.7 96.2 96.1 95.6 95.4 95.7 95.8 96.1 95.2 94.0 94.0 94.0 25. Helicobacter hepaticus 97.0 96.3 96.1 96.3 96.2 96.3 96.3 95.9 94.5 94.5 94.5 26. Helicobacter trogontum 2.67 95.6 95.9 96.2 96.2 96.8 96.0 96.0 95.1 95.1. 95.1 27. Helicobacter cholecystus 3.30 4.25 98.5 96.8 96.8 97.6 97.6 96.8 95.1 95.1 95.1 28. Helicobacter pametensis 3.22 3.85 1.36 96.7 96.8 97.8 97.9 97.2 95.8 95.8 95.8 29. Helicobacter suncus 3.06 3.22 2.83 2.67 98.5 98.5 98.1 95.4 93.9 93.9 93.9 30. Helicobacter mustelae 3.22 3.38 2.99 2.67 1.06 98.5 97.8 95.4 94.0 94.0 94.0 31. Helicobacter sp. Bird-B 3.06 2.83 2.21 2.05 1.06 2.21 98.3 95.9 94.6 94.6 94.6 32. Helicobacter sp. Bird-C 3.06 3.61 1.98 1.67 1.67 1.90 1.59 96.1 94.8 94.8 94.8 33. Helicobacter pullorum 3.77 3.61 3.14 2.52 4.17 4.17 3.85 3.46 96.2 96.2 96.2 34. CMRI2cae 5.21 4.97 4.73 3.93 5.69 5.69 5.29 4.89 3.46 100.0 100.0 35. UNSWlcaesp 5.21 4.97 4.73 3.93 5.69 5.69 5.29 4.89 3.46 0.00 100.0 36. CMRI21iv 5.21 4.97 4.73 3.93 5.69 5.69 5.29 4.89 3.46 0.00 0.00 37. ABHU4caesp 5.21 4.97 4.73 3.93 5.69 5.69 5.29 4.89 3.46 0.00 0.00 0.00 38. Helicobacter rodentium 4.8 I 4.73 4.25 4.01 4.73 5.05 4.49 4.57 3.69 1.75 1. 75 1.75 39. Helicobacter acinonyx 6.26 7.00 5.53 5.29 6.02 5.69 5.93 5.61 4.97 6.34 6.34 6.34 40. Helicobacter pylori 6.10 6.67 5.45 5.05 6.02 5.53 5.85 5.21 4.65 5.53 5.53 5.53 41. Helicobacter nemestrinae 7.16 7.74 6.34 6.26 6.92 6.42 6.75 6.10 6.51 7.16 7.16 7.16 42. Helicobacter bizzozeronii 6.26 6.75 5.21 5.45 6.59 5.93 6.18 5.93 5.13 6.10 6.10 6.10 43. Helicobacter salomonis 6.34 6.83 5.21 5.45 6.51 5.93 6.02 6.10 5.05 6.59 6.59 6.59 44. "Gastrospirillum hominis" 2 6.26 6.59 4.89 5.29 6.51 5.77 6.02 6.10 4.89 6.26 6.26 6.26 45. Helicobacter felis 6.02 6.67 5.29 5.53 6.42 5.85 6.10 6.10 4.97 6.34 6.34 6.34 46. "Gastrospirillum hominis" 1 6.92 7.41 6.83 6.59 7.41 6.92 6.92 6.83 5.93 6.02 6.02 6.02 41. Wolinella succinogenes 7.00 6.34 5.85 5.77 6.02 5.77 5.77 5.45 6.83 7.99 7.99 7.99 48. Escherichia coli 24.15 24.04 24.46 24.35 24.35 24.46 24.46 24.46 24.25 25.51 25.51 25.51 Chapter 5 84

37 38 39 40 41 42 43 44 45 46 47 48

I. Arcobacter butzleri 85.0 85.8 84.8 84.7 84.1 82.9 83.1 83.3 83.9 82.6 85.2 76.6 2. Arcobacter cryaerophilus 84.4 85.1 84.0 84.0 83.4 82.6 82.6 82.9 83.3 82.1 84.8 76.1 J. Campylobacter coli 86.4 86.8 84.8 84.9 84.4 83.5 83.6 83.8 84.3 83.3 85.8 75.5 4. Campylobacter jejuni 86.4 86.9 85.2 85.1 84.5 83.9 83.9 84.2 84.7 83.4 85.9 75.8 5. Helicobacter sp. CLO 3 95.4 95.0 93.9 93.9 92.3 92.8 92.8 93.2 93.4 92.7 92.3 78.0 6. Helicobacter fennelliae 95.5 94.8 92.9 93.0 91.6 92.5 92.7 93.1 93.4 92.3 92.6 77.1 7. HRI3caefr 94.7 95.5 93.0 93.1 92.2 91.9 92.1 92.1 92.7 91.4 92.9 75.8 8. HRI6caefr 94.7 95.5 93.0 93.1 92.2 91.9 92.1 92.1 92.7 91.4 92.9 75.8 9. HRl7stomfr 94.7 95.5 93.0 93.1 92.2 91.9 92.1 92.1 92.7 91.4 92.9 75.8 10. ABHU3cae 94.4 95.2 92.3 92.4 91.4 91.4 91.5 91.6 92.1 90.8 92.8 76.0 11. UNSW3.llcae 94.3 95. l 92.1 92.2 91.2 91.2 91.3 91.4 91.9 90.6 92.6 75.8 12. UNSW3SBsp 94.2 95.1 92.1 92.1 91.2 91.2 91.3 91.4 91.9 90.6 92.7 75.9 13. ABHU4stomsp 94.4 95.2 92.3 92.4 91.4 91.4 91.5 91.6 92.1 90.8 92.8 76.0 14. UNSWMCSpl 93.5 94.0 91.5 91.3 90.3 90.4 90.6 91.1 91.2 90.0 92.2 76.1 15. UNSW1.7col 93.1 93.6 91.2 90.8 89.9 90.0 90.2 90.8 90.9 89.7 91.7 75.6 16. UNSW1.7sp 94.0 94.5 92.0 91.6 90.7 90.9 91.0 91.6 91.7 90.5 92.5 76.4 17. Helicobacter bilis 94.9 95.7 92.8 92.9 92.0 91.8 91.9 92.1 92.6 91.2 92.9 76.3 18. Helicobacter canis 94.5 95.3 93.5 93.4 92.2 92.2 92.6 92.5 93.0 91.6 93.0 77.0 19. "Flexispira rappint' 94.9 95.4 92.7 92.8 91.8 91.7 91.8 92.0 92.5 91.4 92.8 76.6 20. Helicobacter cinaedi 94.4 95.2 92.7 92.5 91.5 91.6 91. 7 92.0 92.5 91.1 92.7 76.2 21. Helicobacter westmeadii 94.3 95.0 92.J 91.8 91.2 91.2 91.5 91.9 91.9 90.7 92.4 75.9 22. UNSW1.7st 94.2 94.4 92.4 92.8 91.6 91.9 91.8 92.0 92.2 91.3 92.8 76.4 23. UNSW1.6cae 93.7 93.9 92.0 92.4 91.2 91.5 91.4 91.6 91.9 90.9 92.5 76.1 24. Helicobacter inuridarum 94.0 94.5 92.6 92.9 91.7 92.0 91.9 92.3 92.6 91.2 93.1 76.8 25. Helicobacter hepaticus 94.5 95.0 93.3 93.0 92.0 92.3 92.5 92.5 93.1 91.5 92.6 77.0 26. Helicobacter trogontum 95.1 95.2 92.4 92.4 91.3 91.8 92.1 92.4 92.6 9 I. I 93.2 76.7 27. Helicobacter cholecystus 95.1 95.7 93.8 93.4 92.6 93.1 93.4 93.6 93.7 91.4 93.8 76.5 28. Helicobacter pametensis 95.8 95.8 93.9 93.9 92.7 92.9 93.2 93.2 93.6 91.7 93.8 76.7 29. Helicobacter suncus 93.9 95.0 93.3 92.9 92.0 92.0 92.3 92.4 92.7 91.1 93.4 76.6 30. Helicobacter mustelae 94.0 94.8 93.8 93.5 92.6 92.6 92.9 93.0 93.4 91.4 93.3 76.6 31. Helicobacter sp. Bird-B 94.6 95.3 93.6 93.2 92.4 92.3 92.8 92.7 93.1 91.5 93.8 76.8 32. Helicobacter sp. Bird-C 94.8 95.2 93.6 93.6 92.7 92.4 92.5 92.5 92.9 91.5 94.2 76.6 33. Helicobacter pullorum 96.2 96.0 94.4 94.2 92.6 93.2 93.6 93.7 94.1 92.3 92.7 76.7 34. CMRI2cae 100.0 98.2 92.8 93.3 91.6 92.3 92.2 92.7 92.7 92.4 91.8 75.9 35. UNSWlcaesp 100.0 98.2 92.8 93.3 91.6 92.3 92.2 92.7 92.7 92.4 91.8 75.9 36. CMRl2liv 100.0 98.2 92.8 93.3 91.6 92.3 92.2 92.7 92.7 92.4 91.8 75.9 37. ABHU4caesp 98.2 92.8 93.3 91.6 92.3 92.2 92.7 92.7 92.4 91.8 75.9 38. Helicobacter rodentium 1.90 93.1 93.2 91.8 91.7 91.8 92.3 92.3 91.3 93.0 76.2 39. Helicobacter acinonyx 6.42 6.34 97.7 95.7 95.9 96.1 96.3 96.3 94.6 90.3 75.5 40. Helicobacter pylori 5.61 5.77 2.13 96.8 94.8 94.9 95.0 95.3 94.2 90.4 74.8 41. Helicobacter nemestrinae 7.08 7.25 4.09 2.91 93.9 94.2 93.7 94.3 93.1 89.5 75.6 42. Helicobacter bizzozeronii 6.10 6.83 3.77 4.49 5.37 99.1 98.7 98.7 96.1 89.3 75.3 43. Helicobacter salomonis 6.59 7.08 3.93 4.49 5.29 0.91 98.9 99.0 96.2 89.3 76.0 44. "Gastrospirillum hominis" 2 6.26 6.75 3.93 4.49 5.69 1.21 1.06 98.7 96.2 89.7 75.3 45. Helicobacter felis 6.34 6.83 3.77 4.25 5.21 0.83 0.68 0.98 95.9 90.1 76.1 46. "Gastrospirillum hominis" I 6.02 7.08 4.65 5.13 6.02 3.38 3.46 3.46 3.38 88.6 75.0 47. Woline/la succinogenes 7.99 6.67 8.75 8.58 9.60 9.09 9.34 9.00 9.09 9.68 78.1 48. Escherichia coli 25.5 I 25.19 25.82 26.46 25.82 25.82 25.51 26.35 25.82 26.35 22.61 Figure 5.1

Phylogenetic tree for organisms belonging to the genus Helicobacter and other members of the epsilon subdivision of the proteobacteria derived from near complete 16S rRNA gene sequences. Genetic distances were corrected for multiple base changes by the method of Jukes and Cantor (73) and the tree reconstructed from a pairwise distance matrix by using the neighbor-joining method of Saitou and Nei (140). Scale bar represents a genetic distance between nucleotide sequences of 5%. Bootstrap values (for branches present in more than 50% of 100 resamplings of the data) are indicated at the nodes. Sequences for organisms in coloured, boldface type were determined during this study and represent bacterial groups A, C, D, E and Has described in the text. 85 Chapter 5

Helicobacter salomonis Helicobacter felis Helicobacter bizzazeronii 11 11 Gastrospirillum hominis 2 11 11 hominis 1 100 ...______Gastrospirillum Helicobacter pylori ------Helicobacter nemestrinae ..._____ Helicobacter acinonyx ._____ Helicobacter sp. CLO 3 Helicobacter pullorum 55 ABHU4caesp 100 UNSWlcaesp ] Group H 100 CMRI2cae CMRI2liv Helicobacter rodentium Helicobacter fennelliae Helicobacter trogontum Helicobacter hepaticus 98 UNSW1.6cae ] ._____, UNSW1.7st Group E 100 Helicobacter muridarum Helicobacter canis 62 6 HRI3caefr HRI6caefr JGroup A HR17stomfr Helicobacter bilis UNSWJSBsp UNSWJ.llcae C 100 ABHU4stomsp ] Group ABHU3cae

79 UNSWl. 7col ] 10 UNSWMCSpl Group D UNSW1.7sp 11 11 Flexispira rappini 74 Helicobacter westmeadii 100 1oo Helicobacter cinaedi Helicobacter pametensis Helicobacter cholecystus Helicobacter sp. Bird-B 6 Helicobacter suncus Helicobacter mustelae Helicobacter sp. Bird-C .______Woline/la succinogenes 100 Campylobacter jejuni Campylobacter coli Arcobacter cryaerophilus ______1_ 00_;---- Arcobacter butzleri Escherichia coli

5% Figure 5.2 Negatively stained preparations of a bacterial group A isolate, HI6caefr, showing; (A) its rod-shaped morphology and multiple polar flagella, magnification 20 000. (B) higher magnification showing detail of periplasmic fibrils entwining the organism, magnification 80 000. A B Figure 5.3 Negatively stained preparations of two morphologically similar bacteria belonging to different bacterial groups. (A) Bacterial group C isolate, ABHU4stomsp, magnification 32 000 (B) Bacterial group D isolate, MCSpl, magnification 28 000 A B Figure 5.4 Negatively stained preparation of an isolate from bacterial group E, UNSW1.7st, which is indistinguishable from Helicobacter muridarum. (A) These bacteria have a spiral morphology, periplasmic fibrils and multiple, bipolar, sheathed flagella, magnification 30 000. (B) Higher magnification showing the flagella and periplasmic fibres in more detail, magnification 120 000. A Chapter 5 89

5. 4 Discussion and Summary The 16S rRNA gene sequences from 16 helicobacters cultured from mouse tissue samples was determined and a phylogeny for the genus Helicobacter reconstructed. Some of these sequences were concluded to be from recognized species (H. niuridarum and H. bilis) while others were from previously undescribed species. In the phylogenetic analysis, sequences from all isolates grouped, as expected, with the lower bowel helicobacters. The four sequences from group H isolates were identical to one another and had highest sequence similarity with the most recently described helicobacter, H. rodentium (149). This group of isolates was examined in more detail and will be discussed in Chapter 6. The sequences from group E, H. muridarum, isolates were positioned, as expected, on a branch with H. muridarum.

The remaining 10 sequences determined in this study ( originating from groups A, C and D) were found to cluster in the group of organisms containing H. bilis and "Flexispira rappini"' as well as Helicobacter canis, Helicobacter westmeadii and Helicobacter cinaedi. Although there was very high sequence homology between all of these isolates, each of the three groups formed distinct clusters in the tree reconstruction, supported by high bootstrap values. Interestingly, corrected genetic distance measurements, on which the phylogenetic reconstruction was based, placed the group C isolates (with morphology type 1, or spirillum like) closer to H. bilis than they did the group A isolates to H. bilis. This differed from the tentative classification of group A as H. bilis isolates based not only on sequence similarity, but on shared morphological and growth characteristics as well as urease activity. Although morphology can be a very unreliable as a method for distinguishing between isolates, there were marked difference between the fusiform rod shape of H. bilis, group A and group B isolates (morphology type 2), and the thin spirillum like morphology of group C and D isolates (morphology type 1) which was considered significant in terms of taxonomy. This complex of sequences with very high percentage identities represents a clear case where the sensitivity of the 16S rDNA is insufficient for the elucidation of relationships between closely related bacteria . For these organisms to be assigned to existing taxa or for new species to be recognized, more sensitive analyses need to be performed encompassing a broad range of systematic characteristics. Such analyses should include extensive analysis of proteins and biochemical systems present. Further genetic characterization, ideally, should involve whole genome studies (for example, total DNA-DNA hybridization techniques) applied to these isolates and closely related helicobacters. Such investigations would allow for the elucidation of relationships based on the entire genetic composition of organisms and expression of proteins under standardized conditions rather than on the sequence of a single, albeit evolutionarily significant, gene. Chapter 5 90

The criteria for the designation of new Helicobacter species are not clear. Along with Campylobacter and Arcobacter spp. one of the major difficulties arising in terms of taxonomy and systematics is the general biochemical unreactivity of these bacteria (119, 174). Minimum standards have been proposed for the description of new species in the family Campylobacteraceae (which includes only Campylobacter and Arcobacter genera) ( 170). These include descriptions of; cell morphology, staining behaviour, motility, colony morphology, growth conditions (including culture medium, temperature range and gaseous requirements), biochemical properties, antimicrobial sensitivities, molecular data and a description of the natural habitats of the proposed species. Many, if not all, of these criteria would also be appropriate for Helicobacter species, although such a system for species designation has not been proposed.

In terms of molecular data, analysis of 16S rRNA gene sequences is the most powerful at a level above that of species. Total DNA-DNA hybridization, cellular fatty acid profiles and protein profiling are all measures, either directly or indirectly, of the genetic content and organization of a cell ( 119). These methods are also more useful for elucidating relationships between closely related species than 16S rRNA gene sequence analysis. There is general agreement that bacterial species are phenotypically distinct groups of strains with DNA-DNA hybridization values of 70% or more (180). Strains with this level of DNA relatedness in hybridization studies will usually have 16S rRNA gene sequences with greater than 97% identity (154), although organisms having almost 100% 16S rDNA sequence identity have been reported as having DNA-DNA reassociation values significantly less than 70% (39).

16S rRNA gene sequences accumulate changes at a low evolutionary rate and in a neutral or clocklike manner, which do not confer any selective advantage on the cell (74, 123). Analysis of these sequences is useful for distinguishing moderately divergent populations. Sequence differences however, would have little or no bearing on ecological differences between populations. There are numerous examples of closely related, but ecologically distinct bacterial populations, which cannot be distinguished by 16S rRNA gene sequences (123). Furthermore, any particular DNA-DNA hybridization cut off value is increasingly being seen as arbitrary and lacking in true significance in terms of defining bacterial species as distinct ecological units (174). Housekeeping protein-coding genes have been put forward as more suitable candidates for distinguishing closely related, but ecologically distinct populations. Sequence analyses of protein-coding genes have shown that ecologically distinct populations, which cannot be distinguished on the basis of 16S rRNA gene sequences, almost always fall into separate DNA sequence Chapter 5 91

clusters, and they are able to reveal ecologically distinct populations not distinguished by other molecular techniques (123).

It has been recommended that the molecular criteria for species determination should include 16S rRNA gene sequence, DNA-DNA hybridization and protein-coding gene sequences (based on diversity at least two unlinked loci) (123). In a truly polyphasic taxonomy, these molecular studies would be combined with cellular fatty acid and whole cell protein analysis (174). Chapter 6 92

Chapter 6

Description of a new Helicobacter species

6. 1 Background The 16S rDNA sequences obtained from the 4 isolates representing group H (Chapter 5) were identical to one another, yet significantly different from those of other Helicobacter species contained in the GenBank database. The presented study was performed to determine if these isolates represented a new species of Helicobacter.

6. 2 Experimental design The isolates examined as possible members of a new Helicobacter species included the 4 isolates whose 16S rRNA gene sequence was determined and analyzed previously, ABHU4caesp, CMRI2cae, CMRI2liv and UNSWlcaesp (Chapter 5) plus 2 additional isolates which were also placed in group H (Chapter 4), designated ABHU6SBsp and UNSW2caesp.

Phenotypic tests commonly used for typing of helicobacters and campylobacters were performed. These included urease, catalase and oxidase tests as well as those tests contained in the API Campy (bioMerieux) system (Chapter 2). Tolerance to 1.0% (w/v) glycine and 1.5% (w/v) NaCl was determined by culturing bacteria on HBA supplemented with each of these compounds. Growth at 25°C and 42°C was tested by culturing on HBA at these incubation temperatures. Susceptibility to nalidixic acid, cephalothin and metronidazole was also determined. The ultrastructure of isolates was investigated by transmission electron microscopy on negatively stained preparations.

6.3 Results

6.3.1 Isolation and Growth Characteristics After 3 to 5 days of incubation at 37°C, in an anaerobic jar with an anaerobic gas generating kit (BR 38, Oxoid), on HBA, growth appeared as a thin, transparent, spreading film. Single colonies were isolated when the initial inoculation of mouse intestinal mucus scraping was made onto 0.65 µm filters as described (Chapter 2). Single colonies had irregular edges, consistent with a spreading, motile organism. Examination using phase contrast microscopy revealed motile, spiral shaped bacteria. Chapter 6 93

The isolates grew on HBA at 37°C under anaerobic conditions. They did not grow on HBA under microaerophilic conditions (BR 56, Oxoid) or on CSA under anaerobic conditions. They grew at 25°C but not at 42°C. These bacteria were Gram negative.

6.3.2 Ultrastructure Negatively stained preparations examined by transmission electron microscopy showed isolates to be spiral or curved rods approximately 0.3 µm wide and 1.5-4.5 µm long. They possessed single, bipolar, unsheathed flagella and no periplasmic fibrils (Figure 6.1).

6.3.3 Biochemical and Physiological Characteristics The six strains examined did not grow in the presence of 1% glycine or 1.5% NaCl. They were resistant to nalidixic acid and cephalothin but sensitive to metronidazole. They were urease, catalase and oxidase negative. One strain (ABHU6SBsp) reduced nitrate to nitrite. They did not hydrolyze hippurate, alkaline phosphatase or indoxyl acetate and gamma-'2-glutamyl transpeptidase was not present. A comparison of some of these characteristics with other Helicobacter species is given in Table 6.1.

In addition, all of the isolates which had been classified into group H (ABHU=6, CMRl=9, HRl=0, UNSW=2) were urease negative and initially cultured under anaerobic conditions.

6.3.4 Phylogenetic Analysis The 16S rDNA sequences determined for four isolates were identical (Chapter 5) with 96.2% sequence identity to Helicobacter pullorum. These sequences have been submitted to GenBank under the accession numbers AF000221-AF000224 (Chapter 5). Figure 6.1 Negatively stained preparation for group H isolate, CMRI2cae showing spiral morphology and single bipolar flagella, magnification 13 000. Chapter 6 94 \0 VI

~

::r' ..,

(") °'

.§

on

cine

+ + +

+ +

1%

ND

ND

ND

I

-(0/6)

Growth

-

-

-

+ + +

+

+ + + + + + + + + + + + +

42°C

-(0/6)

at

Growth

-

-

- - - -

-

+ + + + +

+ +

ND

ND

acatate

-(0/6)

lndoxyl

hydrolysis

- -

- - - -

-

+ +

+

+ + + +

+

ND

ND

ND

ND

ND

Glutamyl

Gamma-2-

transpeptidase

speciesa

-

- -

+ +

+ +

+

+ +

+ + +

+ +

ND

ND

-(1/6)

-(0/6) -(0/6)

Alkaline

hydrolysis

phosphatase

Helicobacter

-

-

- - - -

+

+

+ + + + + + +

+

other

Nitrate

-(1/6)

reduction

from

- - --

--

- -

-

+ + +

+

+

+ +

isolates

Urease

-(0/6)

H

production

group

-

+

+ + + + + + + + + + + + + + + + + + + + +

+ +

(14/16)

-(0/6)b

Catalase

+

production

3

Bird-B

Bird-C

CLO

sp.

which differentiate

sp. sp.

sp.

cholecystus

trogontum

acinonyx

bilis

pullorum pametensis

hepaticus muridarum

canis

mustelae

nemistrinae pylori

cinaedi

felis

fennelliae

isolates)

rappini"

H

Taxon

6.1

"Flexispira

Table Characteristics

Helicobacter (group

Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter

Helicobacter

Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter Helicobacter n Sensitivitl to ::r .g Nalidixic G+C ..... 0 Taxon acid Cephalothin Periplasmic No. of Distribution Sheathed content ""'I (30 µg disc) (30 µg disc) fibers flagella/cell of flagella (mol%) O'I

Helicobacter sp. R(6/6) R(6/6) . 2 Bipolar . ND (group H isolates)

Helicobacter pullorum s R - 1 Monopolar - 34-35 Helicobacter sp. CLO 3 I R - ND ND ND 45 Helicobacter fennelliae s s - 2 Bipolar + 35 Helicobacter trogontum R R + 5-7 Bipolar + ND Helicobacter hepaticus R R - 2 Bipolar + ND Helicobacter muridarum R R + 10-14 Bipolar + 34 Helicobacter canis s I - 2 Bipolar + 48 Helicobacter bilis R R + 3-14 Bipolar + ND "Flexispira rappini" R R + 10-20 Bipolar + 34 Helicobacter cinaedi s I - 1-2 Bipolar + 37-38 Helicobacter pametensis s s - 2 Bipolar + 38 Helicobacter cholecystus I R - 1 Monopolar + ND Helicobacter sp. Bird-B s R - 2 Bipolar + 31 Helicobacter sp. Bird-C s R - 2 Bipolar + 30 Helicobacter mustelae s R - 4-8 Lateral + 36 Helicobacter pylori R s - 4-8 Bipolar + 35-37 Helicobacter nemistrinae R s - 4-8 Bipolar + 24 Helicobacter acinonyx R s - 2-5 Bipolar + 30 Helicobacter felis R s + 14-20 Bipolar + 42

a Data were obtained from references (48, 49, 104, 156) and this study. +, positive reaction; -, negative reaction; S, susceptible; R, resistant; ND, not determined.

b The numbers in parentheses are, numbers of strains positive/number of strains tested. "°O'I Chapter 6 97

6. 4 Discussion and Summary The six isolates examined here could only be cultured on HBA in the gaseous conditions produced by the anaerobic gas generating kit (BR 38, Oxoid). While many helicobacters may be cultured anaerobically they are all also culturable under microaerophilic conditions. Some helicobacters show improved growth in an atmosphere produced by

the anaerobic gas generating kit without the addition of the catalyst, which sequesters H2 molecules. The gaseous mixture produced by this combination is still microaerophilic but has a different oxygen and hydrogen tension to that produced by the microaerophilic gas generating kit (BR 56, Oxoid). Further characterization of the atmospheric growth requirements is needed to clarify the true nature of the observed anaerobicity of these isolates as it would be unusual for a Helicobacter sp. to be a true anaerobe. Particularly, information regarding the ability of strains to grow under microaerophilic conditions, and at what oxygen content, as well as determining whether the presence hydrogen (or formate) is needed.

There was no detectable urease activity in any of six isolates. Prior to the presented study, all of the murine helicobacters described possessed urease activity, a common feature of many Helicobacter species. Isolates examined using electron microscopy revealed the unusual feature (for helicobacters) of unsheathed flagella. H. pullorum, which is a urease negative, lower bowel helicobacter isolated from poultry, was the only other species reported to possess an unsheathed flagellum (156),

The level of 16S rRNA gene sequence similarity between the isolates described here and H. pullorum suggested that a novel murine Helicobacter species had been isolated.

6. 5 Comparison to Helicobacter rodentium Simultaneous to the study presented above, a new murine helicobacter, Helicobacter rodentium, was described in July 1997 (149). This new species had a higher sequence similarity (98.2%) to the isolates examined in this study than any of the previously described Helicobacter species. A comparison of H. rodentium with the isolates obtained during this study is presented. All information on H. rodentium was taken from reference ( 149). The closeness of the time frame in which these organisms were identified is highlighted by the GenBank submission dates for both H. rodentium (accession numbers U96296-U96297, 4 April, 1997) and the group H isolates described here (accession numbers AF000221-AF000224, 18 April, 1997). Chapter 6 98

6.5.1 H. rodentium specific PCR A H. rodentium species specific PCR was described by Shen et al. (149) in the description of this new species using primers D86 and D87 (Table 2.1). The target sites for these primers were conserved in the four isolates examined during this investigation. Figure 6.2 highlights the primer target sites for this PCR as well as the nucleotide differences between H. rodentium and the consensus sequence of the four isolates examined.

The H. rodentium specific PCR was used successfully to amplify DNA from the four sequenced isolates with a standard reaction mixture (Chapter 2) and the published thermocycling profile. This consisted of an initial denaturation at 94 °C for 3 minutes followed by 35 cycles of 94 °C for 1 minute, 58°C for 1 minute, 30 seconds and 72°C for 2 minutes. This was followed by an extension step at 72°C for 10 minutes (FfS 320, Corbett Research). This PCR was unable to amplify DNA from other recognized Helicobacter species (Figure 6.3) or from other helicobacter groups identified in this study (Chapters 4 and 5). The PCR was then used to determine how many of the bacteria placed in group H were H. rodentium-like isolates.

All 17 of the isolates classified in group H, which were urease negative and cultured under anaerobic conditions, were positive in the H. rodentium PCR (ABHU=6, CMRl=9, HRl=O, UNSW=2, Table 4.5). These samples were from isolates possessing 16S rDNA with no IVS. All of the DNA samples which contained 16S rDNA polymorphic for the IVS and all isolates classified in bacterial group E (Helicobacter muridarum) were also tested in this PCR to check for the presence of mixed DNA samples. A small number of H. muridarum samples (3/13) were found to be also positive in the H. rodentium PCR indicating that these samples contained DNA isolated from mixed cultures. None of the polymorphic IVS samples were positive in this reaction.

In summary, 17 DNA samples, all extracted from anaerobic bacterial cultures, which were placed in bacterial group H (Chapter 4) were found to be positive in the H. rodentium PCR. Three DNA samples classified in group E (H. muridarum) were also found to be positive in the H. rodentium PCR indicating the presence of a mixed culture. Chapter6 99

Figure 6.2 Comparison of the 4 identical 16S rDNA sequences from the group H isolates determined during this study (UNSW) with Helicobacter rodentium (strain MIT 95-1707).

1 so UNSW •••• AGTGAA CGCTGGCGGC GTGCCTAATA CATGCAAGTC GAACGATGAA MIT TCAGAGTGAA CGCTGGCGGC GTGCCTAATA CATGCAAGTC GAACGATGAA

51 100 UNSW GCTCTAGCTT GCTAGAGTGG ATTAGTGGCG CACGGGTGAG TAATGCATAG MIT GCTCTAGCTT GCTAGAGTGG ATTAGTGGCG CACGGGTGAG TAATGCATAG

101 150 UNSW ATAACATGCC CTTTAGTCTA GGATAGCCAT TGGAAACGAT GATTAATACT MIT GTTATGTGCC.. CTTTAGTCTA GGATAGCCAT TGGAAACGAT GATTAATACT 151 200 UNSW GGATACTCCT TACGAGGGAA AGTTTTTCGC TAAAGGATTG GTCTATGTCC MIT GGATACTCCC TACGGGGGAA AGTTTTTCGC TAAAGGATCA GCCTATGTCC

201 250 UNSW TATCAGCTTG TTGGTGAGGT AATGGCTCAC CAAGGCTATG ACGGGTATCC MIT TATCAGCTTG TTGGTGAGGT AATGGCTCAC CAAGGCTATG ACGGGTATCC

251 300 UNSW GGCCTGAGAG GGTGAACGGA CACACTGGAA CTGAGACACG GTCCAGACTC MIT GGCCTGAGAG GGTGAACGGA CACACTGGAA CTGAGACACG GTCCAGACTC

301 350 UNSW CTACGGGAGG CAGCAGTAGG GAATATTGCT CAATGGGGGA AACCCTGAAG MIT CTACGGGAGG CAGCAGTAGG GAATATTGCT CAATGGGGGA AACCCTGAAG

351 400 UNSW CAGCAACGCC GCGTGGAGGA TGAAGGTTTT CGGATTGTAA ACTCCTTTTG MIT CAGCAACGCC GCGTGGAGGA TGAAGGTTTT CGGATTGTAA ACTCCTTTTC

401 450 UNSW TTAGAGAAGA TAATGACGGT ATCTAACGAA TAAGCACCGG CTAACTCCGT MIT TAAGAGAAGA TTATGACGGT ATCTTAGGAA TAAGCACCGG CTAACTCCGT

451 500 UNSW GCCAGCAGCC GCGGTAATAC GGAGGGTGCA AGCGTTACTC GGAATCACTG MIT GCCAGCAGCC GCGGTAATAC GGAGGGTGCA AGCGTTACTC GGAATCACTG

501 550 UNSW GGCGTAAAGA GCGCGTAGGC GGGATAGCAA GTCAGATGTG AAATCCTATG MIT GGCGTAAAGA GCGCGTAGGC GGGATAGCAA GTCAGATGTG AAATCCTATG

551 600 UNSW GCTTAACCAT AGAACTGCAT TTGAAACTGT TATTCTAGAG TATGGGAGAG MIT GCTTAACCAT AGAACTGCAT TTGAAACTGT TATTCTAGAG TATGGGAGAG Chapter6 100

601 650 UNSW GTAGGTGGAA TTCTTGGTGT AGGGGTAAAA TCCGTAGAGA TCAAGAGGAA MIT GTAGGTGGAA TTCTTGGTGT AGGGGTAAAA TCCGTAGAGA TCAAGAGGAA

651 700 UNSW TACTCATTGC GAAGGCGACC TGCTAGAACA TAACTGACGC TGATGCGCGA MIT TACTCATTGC GAAGGCGACC TGCTGGAACA TAACTGACGC TGATGCGCGA

701 750 UNSW AAGCGTGGGG AGCAAACAGG ATTAGATACC CTGGTAGTCC ACGCCCTAAA MIT AAGCGTGGGG AGCAAACAGG ATTAGATACC CTGGTAGTCC ACGCCCTAAA

751 800 UNSW CGATGAATGC TAGTTGTTGT GGAGCTTGTC TCTGCAGTAA TGCAGCTAAC MIT CGATGAATGC TAGTTGTTGC GAGGCTTGTC CTTGCAGTAA TGCAGCTAAC

801 850 UNSW GCATTAAGCA TTCCGCCTGG GGAGTACGGT CGCAAGATTA AAACTCAAAG MIT GCATTAAGCA TTCCGCCTGG GGAGTACGGT CGCAAGATTA AAACTCAAAG

851 900 UNSW GAATAGACGG GGACCCGCAC AAGCGGTGGA GCATGTGGTT TAATTCGAAG MIT GAATAGACGG GGACCCGCAC AAGCGGTGGA GCATGTGGTT TAATTCGAAG

901 950 UNSW ATACACGAAG AACCTTACCT AGGCTTGACA TTGATAGAAT CCGCTAGAGA MIT ATACACGAAG AACCTTACCT AGGCTTGACA TTGATAGAAT CCGCTAGAGA

951 1000 UNSW TAGTGGAGTG CTAGCTTGCT AGAACTTGAA AACAGGTGCT GCACGGCTGT MIT TAGTGGAGTG CTAGCTTGCT AGAACTTGAA AACAGGTGCT GCACGGCTGT

1001 1050 UNSW CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC MIT CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC

1051 1100 UNSW CCTCGTCCTT AGTTGCTAAC TATTCGGTAG AGCACTCTAA GGAGACTGCC MIT CCTCGTCCTT AGTTGCTAAC TATTCGGTAG AGCACTCTAA GGAGACTGCC ****** ********** ***** 1101 1150 UNSW TTCGCAAGGA GGAGGAAGGT GAGGATGACG TCAAGTCATC ATGGCCCTTA MIT TTCGCAAGGA GGAGGAAGGT GAGGATGACG TCAAGTCATC ATGGCCCTTA

1151 1200 UNSW CGCCTAGGGC TACACACGTG CTACAATGGG AAGCACAAAG AGATGCAATA MIT CGCCTAGGGC TACACACGTG CTACAATGGG AAGTACAAAG AGATGCAATA

1201 1250 UNSW TTGTGAAATG GAGCAAATCT ATAAAACTTC TCTCAGTTCG GATTGTAGTC MIT TTGTGAAATG GAGCAAATCT CAAAAACTTC TCTCAGTTCG GATTGTAGTC ********** ********** Chapter6 101

1251 1300 UNSW TGCAACTCGA CTACATGAAG CTGGAATCGC TAGTAATCGT AAATCAGCTA MIT TGCAACTCGA CTACATGAAG CTGGAATCGC TAGTAATCGT AGATCAGCTA

1301 1350 UNSW TGTTACGGTG AATACGTTCC CGGGTCTTGT ACTCACCGCC CGTCACACCA MIT TGCTACGGTG AATACGTTCC CGGGTCTTGT ACTCACCGCC CGTCACACCA

1351 1400 UNSW TGGGAGTTGT ATTCGCCTTA AGTCGGAATG CCAAACTGGC TACCGCCCAC MIT TGGGAGTTGT ATTCGCCTTA AGTCGGAATG CCAAACTGGC TACCGCCCAC

1401 1423 UNSW GGCGGATGCA GCGACTGGGG TG. MIT GGCGGATGCA GCGACTGGGG TGA

· Nucleotide differences. * H. rodentium specific PCR primer sites (149). Chapter 6 102

M 1234 5 6 7 8 91011

Figure 6.3 PCR products from the Helicobacter rodentium specific reaction using primers D86 and D87 (Table 2.1). DNA separated in 3% agarose/TAE.

lane M Xl 74/Haelll (200 ng) 1 Helicobacter pylori 2 Helicobacter felis 3 Helicobacter mustelae 4 Helicobacter muridarum 5 Helicobacter bi/is 6 Helicobacter hepaticus 7 Helicobacter trogontum 8 ABHU4caesp (group H) 9 CMR2cae (group H) 10 CMRI2liv (group H) 11 UNSWlcaesp (group H) Chapter6 103

6.5.2 Ultrastructural, Physiological and Biochemical Comparisons Ultrastructurally, the description of H. rodentium was similar to that of the isolates investigated during this study. Most importantly, H. rodentium also possessed single, bipolar, unsheathed flagella. Another important similarity between H. rodentium and the isolates here was the lack of detectable urease activity.

There were a number of differences, however, in the biochemical profiles between the six . isolates examined during this study and H. rodentium. Most notable was the lack of detectable catalase and oxidase activities, although these were reported to be "weak" in H. rodentium. Also the ability to grow at 25°C and 42°C differed between H. rodentium and the isolates examined here. A comparison of some of the characteristics determined for the six group H isolates examined with those of H. rodentium is presented in Table 6.2.

Ideally, some of the biochemical and physiological characteristics of both H. rodentium and the isolates described during this study should be reexamined under the same laboratory conditions. Additional analyses, such as cellular fatty acids, total protein profiles and DNA-DNA hybridization studies would clarify whether the described isolates should be classified as H. rodentium or a new closely related species. Chapter 6 104

Table 6.2 Comparison of physiological and biochemical characteristics of the "Helicobacter rodentium-like" group H isolates determined during this study with Helicobacter rodentium.

Characteristic Group H H. rodentium8

Growth microaerophilically + Growth anaerobically + + Urease production - (0/17)b - (0/74) Catalase production - (0/6) + (74/74) weak Oxidase production - (0/6) + (74/74) weak Nitrate reduction - (1/6) + (9/9) Alkaline phosphatase hydrolysis - (0/6) - (0/9) Indoxyl acetate hydrolysis - (0/6) - (0/9) Gamma-2-Glutamyl transpeptidase - (0/6) - (0/9) Growth at 42°C - (0/6) + (7/9) Growth at 25°C + (6/6) - (0/9) Growth with 1 % glycine - (0/6) + (9/9) Sensitivity to Nalidixic acid (30 µg disc) R (6/6) R (74/74) Sensitivity to Cephalothin (30 µg disc) R (6/6) R (74/74) Periplasmic fibers No. of flagella/cell 2 unsheathed 2 unsheathed Distribution of flagella bipolar bipolar

a Data was obtained from reference (149). +, positive reaction;-, negative reaction; R, resistant. b The numbers in parentheses are, numbers of strains positive/number of strains tested. 105

Ribosomal RNA Gene Sequences, Inferred Phylogenies and PCR Identification of Non-Helicobacter, Spiral Bacteria

Chapters 7 and B

Background A significant proportion of the spiral bacteria isolated for this study did not belong to the genus Helicobacter (Chapter 4). These non-helicobacters were divided into two groups (I and J) based on morphological characteristics. Group I organisms, along with a number of the identified helicobacter groups, were found to have morphology type 1 (as defined in Chapter 4 ), being spirillum like organisms. Group J organisms were of morphology type 3 (Chapter 4), having a fat S-shaped morphology. There were no Helicobacter species possessing morphology type 3.

Both of these groups of organisms were cultured on CSA in an anaerobic atmosphere and were urease negative. The appearance of cultures on solid media was of a fine spreading film across the agar, the same as that observed for many helicobacters.

The aim of the studies presented in the following two chapters (Chapters 7 and 8) was to determine the 16S rDNA sequence from a range of isolates representing each of these non-helicobacter groups and, from sequence comparisons, to infer their phylogenetic position within the bacterial domain. From the sequence data obtained and comparisons with 16S rRNA gene sequences from the GenBank and EMBL databases, a further aim was to develop group specific (group I and group J) PCRs for rapid identification of other isolates falling into the same phylogenetic group.

Experimental Design The 16S rDNA was amplified by PCR using the modified consensus primers F27(UFP) and R1492(URP) (Table 2.1) to the conserved 5' and 3' ends of the 16S rRNA gene. The product from several amplification reactions was combined and purified then used as the template DNA in sequencing reactions. Gene sequences for the 16S rRNA were determined using thirteen oligonucleotide primers enabling sequencing of both strands of the 16S rDNA with contiguous overlaps (Table 2.2). DNA amplification and sequencing conditions used were as described in Chapter 5. Sequences were compared to those contained in the GenBank and EMBL databases to reveal the most homologous sequences. Inferred phylogenies for both groups of sequences (I and J) were based on a 106

pairwise distance comparison between the determined sequences and sequences selected from the GenBank database to be included in the phylogenetic analysis.

Uncorrected percentage sequence similarities were calculated from multiple sequence alignments with gaps being included (independent of length), but end gaps excluded. The percentages were calculated based on the number of positions compared between individual sequences (52). Genetic distances corrected for multiple base changes by the method of Jukes and Cantor (73) were calculated from an unambiguous alignment where all positions within the alignment containing ambiguity (insertion, deletion or unknown base) had been removed. The neighbor-joining method (140) was used for tree reconstruction. Bootstrap analysis was performed using 100 resamplings of the data (31 ). The values reported indicate the number of times the species to the right of a node occurred in resampled trees (out of 100 trees). Only those values greater than 50% are reported. Chapter? 107

Chapter 7

Phylogenetic Identification of Bacterial Group J: "fat S"

7.1 Experimental Design Four isolates, representing bacterial group J, were selected for 16S rDNA sequence determination and phylogenetic analysis. These isolates were designated ABHUlSB, ABHUlSBfatS, ABHU2SB and UNSW3caefatS. The determined sequences were then used to design specific PCRs for rapid identification of other isolates falling within this phylogenetic group.

7.2 Results

7.2.1 16S rDNA sequence and phylogenetic analysis Near complete 16S rDNA sequences for the four isolates were determined and have been submitted to the GenBank data base (Table 7.1). A multiple sequence alignment of these 16S rRNA gene sequences is presented in Appendix 2. Comparisons of the determined sequences with those contained in the GenBank and EMBL databases revealed that the most closely related bacterial species were members of the genus Desulfovibrio, which are found in the delta subdivision of the proteobacteria. A multiple sequence alignment of 16S rRNA gene sequences, including the four group J sequences, members of the genus Desulfovibrio and other related organisms, was used as the basis for the phylogenetic analysis. The GenBank accession numbers for sequences included in the phylogenetic tree are listed in Table 7.2. Pairwise comparisons of the 16S rRNA sequences are presented in the similarity matrix as uncorrected percentage sequence similarity values (upper triangle) calculated from the uncorrected alignment of 1638 nucleotide positions, and corrected percentage genetic distances (lower triangle, genetic distances X 100) calculated from a corrected alignment of 1420 nucleotides, after all ambiguous positions had been removed (Table 7.3). The phylogenetic tree was reconstructed from the genetic distance values (Figure 7 .1 ). Chapter 7 108

Table 7.1 GenBank accession numbers for the 16S rRNA gene sequences of non-helicobacter group J isolates determined during this study.

Group Isolate Accession Number

Group J ABHUlSB AB056088 ABHUlSBfatS AF056089 ABHU2SB AF056090 UNSW3caefatS AF056091

Table 7.2 GenBank accession numbers for the 16S rRNA gene sequences of organisms used in phylogenetic analysis.

Organism Accession Referenceb Numbera

Bilophila wadsworthia U82813 (79) Desulfovibrio desulfuricans M34113 (121) Desulfovibrio fairfieldensis U42221 (163) Desulfovibrio halophilus X99237 (201) Desulfovibrio gracilis U53464 Unpublished Desulfovibrio intestinalis Y12254 Unpublished Desulfovibrio longreachii Z24450 (134) Desulfovibrio sp. (ferret MIT 87-599) U07570 (41) Desulfovibrio sp. (strain KH2) X93147 (77) Desulfovibrio sp. (strain KRS 1) X93146 (77) Desulfovibrio termitidis X87409 (77) Escherichia coli 101695 (14) Ileal symbiont intracellularis I U06423 (128) Ileal symbiont intracellularis II Ll5739 (50) Lawsonia intracellularis U30147 (103)

a All sequences are available directly through the GenBank accession numbers. b References for those sequences which have been published are listed. Chapter? 109

Table 7.3 Similarity matrix for species used in phylogenetic reconstruction. Upper triangle values are percentage sequence identities. Lower triangle values are percentage genetic distance measurements corrected for multiple base changes by the method of Jukes and Cantor for sequence positions which were unambiguous for all sequences in the analysis. Isolates whose 16S rDNA sequence was determined during this study are highlighted in bold text.

Sequences are in the order;

1 Desulfovibrio gracilis 11 Desulfovibrio sp. (strain KH2) 2 Desulfovibrio halophilus 12 Desulfovibrio termitidis 3 ABHUlSB 13 Desulfovibrio longreachii 4 ABHUlSBfatS 14 Ileal Symbiont Intracellularis (II) 5 ABHU2SB 15 Lawsonia intracellularis 6 Desulfovibrio fairfieldensis 16 Ileal Symbiont Intracellularis (I) 7 Desulfovibrio desulfuricans 17 Desulfovibrio sp. (MIT 87-599) 8 Desulfovibrio sp. (strain KRS 1) 18 Bilophila wadsworthia 9 Desulfovibrio intestinalis 19 Escherichia coli 10 UNSW3caefatS (") ::r i -.J

2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19

1. Desulfovibrio gracilis 91.2 86.8 86.8 86.8 87.4 88.0 87.7 87.5 86.1 87.8 87.9 87.4 84.6 84.8 84.1 84.8 86.9 77.5 2. Desu/fovibrio halophilus 8.57 85.9 85.9 85.8 86.1 86.8 86.5 87.2 85.6 87. I 87.2 86.7 84.4 84.4 84.1 84.3 85.7 78.1 3. ABHUlSB 12.88 14.31 99.9 99.7 98.0 95.9 95.5 94.4 93.7 90.7 90.8 90.6 88.1 88. 1 87.6 88.3 89.7 77.8 4. ABHUlSBfatS 12.88 14.31 0.00 99.7 98.0 96.0 95.5 94.5 93.7 90.6 90.7 90.6 88.1 88.0 87.5 88.2 89.6 77.8 5. ABHU2SB 12.96 14.40 0.14 0.14 98.1 96.1 95.6 94.4 93.8 90.7 90.8 90.6 88.1 88.1 87.6 88.3 89.7 77.9 6. Desulfovibrio fairfieldensis 12.46 14.48 1.42 1.42 1.42 97.0 96.5 95.3 93.5 91.4 91.5 91.4 88.9 88.8 88.3 88.8 90.9 78.5 1. Desulfovibrio desulfuricans 11.88 13.08 3.46 3.46 3.31 2.65 99.5 96.6 93.1 91.5 91.5 91.2 88.5 88.0 87.6 88.1 91.0 78.7 8. Desulfovibrio sp. (KRS I) 12.04 13. 97 3.83 3.83 3.68 3.02 0.35 96.2 92.7 91.0 91. l 90.6 88.1 87.7 87.3 87.8 90.6 78.4 9. Desulfovibrio intestinalis 12.38 13.29 5.33 5.33 5.33 4.65 3.46 3.83 91.9 90.7 90.7 90.5 87.7 87.4 87.0 87.5 90.4 78.5 10. UNSWJcaefatS 13.72 14.65 5.55 5.55 5.55 5.86 6.31 6.70 7.86 90.8 90.8 90.4 88.0 88.0 87.6 88. l 89.3 77.7 11. Desulfovibrio sp. (KH2) 12.04 13.55 8.73 8.73 8.73 8.02 8.10 8.49 9.05 8.73 99.7 98.2 87.8 87.7 87.2 87.7 91.0 77.5 12. Desulfovibrio termitidis 12.13 13.63 8.81 8.81 8.81 8.10 8.18 8.57 9.12 8.81 0.35 98.2 87.8 87.7 87.2 87.7 91.2 77.7 13. Desulfovibrio longreachii 12.38 13.89 8.65 8.65 8.65 7.86 8.33 8.73 9.20 9.05 1.64 l. 71 87.9 87.7 87.4 87.7 90.7 77.9 14. Ilea! Symbiont lntracellularis 15.69 16.47 11.96 11.96 11.88 11.47 12.29 12.54 13.04 12.21 12.29 12.38 12.21 99.8 98.9 99.8 89.8 78.9 15. Lawsonia intracellularis 15.5116.3011.8011.8011.7111.3012.1312.3812.9612.0412.1312.2112.040.14 99. l 100.0 89.9 78.4 16. Ilea! Symbiont Intracellularis 15.95 16.56 12.29 12.29 12.21 11.80 12.63 12.88 13.46 12.38 12.71 12.79 12.29 0.71 0.57 99.1 89.2 78.4 11.Desulfovibriosp.(MlT81-599) 15.5116.3011.80 11.80 11.7111.3012.13 12.38 12.96 12.04 12.13 12.2112.040.14 0.00 0.57 89.9 78.5 18. Bilophila wadsworthia 13.13 14.74 9.76 9.76 9.68 8.65 8.73 9.05 9.28 10.09 8.57 8.33 8.81 9.92 9.76 10.33 9.76 78.4 19. Escherichia coli 25.01 24.52 25.11 25.11 24.91 24.72 24.72 25.01 24.81 25.1126.0026.20 25.70 24.62 24.42 24.72 24.42 24.42

...... 0 Chapter 7 111

Desulfovibrio termitidis 100 Desulfovibrio sp. KH2 Desulfovibrio longreachii 89 ~----- UNSWJcaefatS 100 Desulfovibrio desulfuricans 100 Desulfovibrio sp. KRS 1

88 L------Desulfovibrio intestinalis Desulfovibrio fairfieldensis 100 83 ABHU2SB ABHUlSB ABHUlSBfatS Lawsonia intracellularis 8 Ileal symbiont intracellularis II

.------1-0-0 Desulfovibrio sp. MIT 52 Ileal symbiont intracellularis I Bilophila wadsworthia

1-----1_00_;------Desulfovibrio gracilis Desulfovibrio halophilus Escherichia coli

5%

Figure 7.1 Phylogenetic tree for organisms belonging to the genus Desulfovibrio and other members of the delta subdivision of the proteobacteria derived from near complete 16S rRNA gene sequences. Genetic distances were calculated by the method of Jukes and Cantor (73) and the tree reconstructed from a pariwise distance matrix by using the neighbor-joining method of Saitou and Nei (140). Scale bar represents a genetic distance between nucleotide sequences of 5%. Bootstrap values (for branches present in more than 50% of 100 resamplings of the data) are indicated at the nodes. Sequences for organisms in coloured, boldface type were determined during this study and represent bacterial group J, as described in the text. Chapter 7 112

The 16S rDNA sequences determined for the isolates originating from the ABHU facility had between 99.7-99.9% identity. The sequence which was most homologous to these isolates was from Desulfovibrio fairfieldensis with similarity values of 98.0-98.1 %. The 16S rDNA sequence obtained from isolate UNSW3caefatS had between 93.7-93.8% sequence identity with the 16S rDNA sequences from the ABHU isolates, and 93.5% identity to D. fairfieldensis. Percentage sequence identities for isolate UNSW3caefatS with Desulfovibrio desulfuricans and Desulfovibrio sp. (KRS 1) were also around 93%.

7.2.2 PCR design The multiple sequence alignment created for the phylogenetic analysis was used to locate oligonucleotides which could be utilized in a PCR for the identification of other isolates of . . this bacterial group. Since the sequences obtained from the isolates in this study were not identical (falling into 2 groups) and had high percentage sequence similarities with some · previously described Desulfovibrio spp., the oligonucleotides chosen were predicted to be able to amplify DNA from other closely related organisms such as D. fairfieldensis, D. desulfuricans, Desulfovibrio intestinalis and Desulfovibrio sp. KRS 1. Candidate primers were identified at positions 225-242 and 733-750 (E. coli numbering), both with a theoretical Tm of 60°C. Comparison of the primer target sequence with sequences contained in GenBank revealed no significant homologies with sequences other than to those Desulfovibrio species which had been used to assist in the primer design. Table 7.4 shows the target region for the primers which was conserved among the sequences determined for this study and a number of other closely related Desulfovibrio species. These primers were synthesized in either the forward (DfairF) or reverse orientation (DfairR) (Table 2.1) and·used to amplify a PCR product of 540 bp. Specific amplification of DNA from the four isolates was achieved with amplification reactions prepared as described previously and a thermocycling profile as follows. An initial denaturation at 94 °C for 3 minutes preceded 30 cycles of 94 °C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute. This was followed by an extension step at 72°C for 10 minutes (FfS 320, Corbett Research). Figure 7 .2 shows the PCR products obtained when the four group J isolates (ABHUlSB, ABHUlSBfatS, ABHU2SB and UNSW3caefatS) and other bacterial species (including bacterial group I isolates and Helicobacter species) were used as template DNA under these conditions. Table 7.4 (i Comparison of bacterial group J specific primer target site with sequences from other bacterial species. .[ !i --.J

Target Sequencea Organism DfairF (225-242) DfairR (733-750) bacterial group J AGGATGAGTCCGCGTCCC GGCCACCTGGACCGGTAT

Desulfovibrio fairfieldensis AGGATGAGTCCGCGTCCC GGCCACCTGGACCGGTAT Desulfovibrio desulfuricans AGGATGAGTCCGCGTCCC GGCCACCTGGACCGGTAT Desulfovibrio intestinalis AGGATGAGTCCGCGGCCC GGCCACCTGGACCGGTAT Desulfovibrio sp. KRS 1 AGGATGAGTCCGCGTCCC GGCCACCTGGACCGGTAT Desulfovibrio longreachii TGGATGAGTCCGCGTCCC GGCCACCTGGACCGATAC Desulfovibrio termitidis TGGATGAGTCCGCGTCCC GGCCACCTGGACCGATAC Lawsonia intracellularis AAGATGAGTCCGCGTCCC GGCCACCTGGACGATAAC Ileal symbiont intracellularis I AAGATGAGTCCGCGTCCC GGCCACCTGGACGATAAC Bilophila wadsworthia AGGATGAGTCCGCGTCCC GGCCACCTGGACGGTAAC Desulfovibrio gracilis AGGATGAACCTGCGTCTC GGCCTGCTGGACAGATAC ***** * *** * * **** ****** *

a Sequences are writen in 5' to 3' orientation and represent positions 225-242 (DfairF) and 733-750 (DfairR), according to E.coli numbering. Asterisks(*) indicate nucleotide positions which are conserved across all species listed.

...... w Chapter 7 114

M 1 2 3 4 5 6 7 8 9 10

Figure 7.2 PCR products from the bacterial group J specific reaction using primers DfairF and DfairR (Table 2.1). DNA separated in 2% agarose/TAE.

lane M Xl 74/Hae III (200 ng) 1 ABHUlSB (group J) 2 ABHUlSBfatS (group J) 3 ABHU2SB (group J) 4 UNSW3caefatS (group J) 5 HRilcae (group I) 6 UNSWRSpl2 (group I) 7 Helicobacter pylori 8 Helicobacter rnuridarurn 9 Helicobacter bilis 10 "no DNA" control Chapter 7 115

All of the non-helicobacter isolates cultured for this study (bacterial groups I and J) were subjected to PCR amplification using primers DfairF and DfairR as described. All isolates which had been classified into bacterial group.J (Chapter 4) gave a positive result in this PCR, with a product of the expected size ( 540 bp) while all of the group I isolates were negative. The total number of isolates confirmed by PCR as being members of group J was 12 (ABHU=lO and UNSW=2). In summary, all of the non-helicobacter isolates classified in bacterial group J were identified as being phylogenetically related, based on a positive reaction in the described PCR. All of these isolates had morphology type 3 and were cultured on CSA in an anaerobic atmosphere. Cultures examined using transmission electron microscopy showed they had a tuft of flagella attached at one end of the cell (Figure 7.3).

7.3 Discussion and Summary

The analysis of 16S rRNA gene sequences obtained during this study of bacterial group J isolates identified them as being members of the genus Desulfovibrio which is phylogenetically placed in the delta subdivision of the proteobacteria. Desulfovibrio are sulfate-reducing bacteria, motile by means of a single polar flagellum or a tuft of polar flagella. They are typically curved (sometimes S shaped or helical) rods (151). These characteristics were fulfilled by the observations presented in this investigation of the group J isolates. Although the production of H2S was not assessed directly, a number of researchers within the School of Microbiology and Immunology, UNSW, confirmed its presence on numerous occasions after incubation jars were opened for subculture purposes (J.L. O'Rourke, M.C. DeUngria, R.A. Bass etc., personal communication)!

The twelve isolates cultured during the present study and classified into bacterial group J (Chapter 4) were confirmed by specific PCR amplification, using primers DfairF and DfairR, as being members of this Desulfovibrio-related group. The PCR was predicted to be capable of amplifying a number of other closely related desulfovibrios. This was done to ensure amplification of all phylogenetically related organisms above the species level, since the 16S rDNA sequences determined here were found to have percentage sequence similarity values outside the range for individual species found within this genus (Figure 7 .1 and Table 7.3 ). Cultures of these most closely related Desulfovibrio species were not obtained for comparative purposes during this study, and therefore the empirical range of bacteria which would give a positive result in the described PCR was not identified. Figure 7.3 Negatively stained preparations of bacterial group J isolates obtained from two different animal facilities showing; (A and B) the straight to curved morphology and tuft of polar flagella, magnification 10 000 and 12 000 respectively, (C and D) higher magnification showing detail of the flagella and the region of insertion of flagella into the cells, magnification 70 000 and 80 000 respectively. A B

•

D C Chapter? 117

The first species of what we now know as Desulfovibrio was probably isolated 100 years ago from a Dutch city canal (175). Sewage contamination caused large amounts of hydrogen sulfide to be liberated from the city canals causing what was described as "a true horror" in the 1895 paper describing the isolation of Spirillum desulfuricans (for a review see reference (175)). Desulfovibrio spp. are commonly found in fresh, marine and brackish waters (151). They are also found in other environments such as oil production plants and other industrial water systems (186). They have been found in the sheep rumen (66) and human intestine (9, 53). Desulfovibrios and related organisms have increasingly been implicated in a number of diseases in both humans and animals. The recently described Desulfovibrio fairfieldensis was isolated and found to be the cause of both a liver abscess and septicemia in a human patient (163). Obligately intracellular bacteria showing a relationship to the genus Desulfovibrio, based on 16S rRNA gene sequences, have been identified as the cause of proliferative enteritis in a wide variety of mammals including the pig (50), hamster, ferret (41), fox, rat, rabbit (139), horse (187) and deer (23). These organisms have been variously referred to as Deal symbiont intracellularis (50), Lawsonia intracellularis (103) and were in some cases initially described as intracellular Campylobacter-lik.e organisms (ICLOs) and intracellular Desulfovibrio organisms (IDOs) (41).

The isolation of these organisms from rodent intestinal tissue is not surprising. The curved and often spiral shape of these bacteria does however, lend further support to the hypothesis that this morphology and characteristic motility is advantageous in the mucus­ associated niches found in the intestinal tract. Chapter 8 118

Chapter 8

Phylogenetic Identification of Bacterial Group I: a new Genus Within the Bacterial Domain with an Unusual Phylogenetic Affiliation

B. 1 Experimental Design Five isolates, representing bacterial group I, were selected for 16S rDNA sequence determination and phylogenetic analysis. Two of these isolates were cultured for this study, and were designated HRilcae and HRI3liv. The remaining three isolates chosen to represent bacterial group I had been cultured previously and were designated UNSWMCSl, UNSW2.6liv and UNSWRSpl2. UNSWMCSl was isolated by the author in 1995 from a caecal scraping from a conventional Q/S mouse housed at the UNSW Microbiology Animal Facility. UNSW2.61iv was isolated by Jani O'Rourke also in 1995 from a liver sample of a conventional Q/S mouse housed at the UNSW Microbiology Animal Facility. UNSWRSp12 was isolated in 1982 from a caecal scraping from a conventional rat. These three isolates were included here since they fitted the description of group I bacteria, being non-helicobacters with a spirillum like morphology (type 1) which grew on CSA under anaerobic conditions. These five sequences were then used to design specific PCRs for rapid identification of other isolates falling within this phylogenetic ·group.

8.2 Results

8.2.1 16S rDNA sequence and phylogenetic analysis Near complete 16S rDNA sequences for the five isolates were obtained using the methods described. The determined l 6S rDNA sequences have been submitted to the GenBank data base under the accession numbers listed in Table 8.1, and multiple sequence alignment of the sequences is presented in Appendix 3. Comparisons of the determined sequences with those contained in the GenBank and EMBL databases revealed that the most closely related bacterial species were those found in the "Flexistipes" group which forms a distinct lineage within the bacterial domain. This group consists of three genera each defined by a single species; Flexistipes sinusarabici (35), Geovibrio ferrireducens (16) and Deferribacter thermophilus (60). The level of sequence similarity of the determined sequences with other bacteria in this group was only moderate, at around 80- Chapter 8 119

85%. A multiple sequence alignment of 16S rRNA gene sequences, including the five group I sequences, the three bacteria making up the "Flexistipes" group and sequences from organisms representing the 11 major lineages within the bacterial domain, was used as the basis for the phylogenetic analysis. The GenBank accession numbers for sequences included in the phylogenetic tree are listed in Table 8.2. Pairwise comparisons of the 16S rRNA sequences are presented in the similarity matrix as uncorrected percentage sequence similarity values (upper triangular half) calculated from the uncorrected multiple alignment of 1677 nucleotide positions, and corrected genetic distances (genetic distances XlOO, lower triangular half) calculated after ambiguous ·nucleotide positions had been removed leaving an alignment of 1202 nucleotides (Table 8.3). The phylogenetic tree was reconstructed using the genetic distance values (Figure 8.1).

The 16S rRNA gene sequences determined for the 5 group I isolates had between 94.7- 99.7% sequence identity. The next most closely related bacterial species was G. ferrireducens, which had between 84.0-84.5% sequence identity with the group I isolates. D. thermophilus and F. sinusarabici had between 82.0-83.0% sequence identity with the group I isolates (Table 8.3). The tree reconstructed from corrected genetic distances confirmed that the "Flexistipes" group (to which the group I isolates were found to belong) forms a separate line of descent from within the bacterial domain.

Table 8.1 GenBank accession numbers for the 16S rRNA gene sequences of non-helicobacter group I isolates determined during this study.

Isolate Accession Number

HRilcae AF059187 HRI3liv AF059188 UNSW2.6liv AF059186 UNSWMCSl AF059189 UNSWRSpl2 AF059190 Chapter 8 120

Table 8.2 GenBank accession numbers for the 16S rRNA gene sequences of organisms used in phylogenetic analysis.

Organism Accession Referenceh Numbera

Cyanobacteria Microcystis aeruginosa U03402 (112) Gram positives Eubacterium barkeri M23927 (184) Heliobacterium chlorum M11212 (194) Clostridium quercicolum M59110 Unpublished Clostridium ramosum M23731 (184) Lactobacillus fennentum M58819 Unpublished Bacillus subtilis X60646 (7) Fusobacteria Fusobacterium nucleatum M58683 Unpublished Proteobacteria Agrobacterium tumefaciens (a) M11223 (200) Chromatium vinosum (13) M26629 Unpublished Escherichia coli (y) J01695 (14) Desulfovibrio desulfuricans (6) M34113 (121) Helicobacter pylori (E) M88157 (157) Flexistipes Group Flexistipes sinusarabici M59231 (35) Defe"ibacter thennophilus U75602 (60) Geovibrio ferrireducens X95744 (16) Cytophagas Bacteroides fragilis M61006 Unpublished Saprospira grandis M58795 Unpublished Thennonema lapsum L11703 (127) Green Sulfur Bacteria Chlorobium limicola M31769 Unpublished Spirochetes Serpulina hyodysenteriae M57741 (158) Treponema pallidum M88726 (125) Leptonema illini M88719 (198) Planctomyces Chlamydia psittaci Ml3769 (182) Green Non Sulfur Bacteria Thennomicrobium roseum M34115 (120) Chlorojlexus aurantiacus M34116 (120) Thermotogales Fervidobacterium nodosum M59177 Unpublished Hydrogeno bacter Aquijlex pyrophilus M83548 (67)

a All sequences are available directly through the GenBank accession numbers. b References for those sequences which have been published are listed. Chapter 8 121

Table 8.3 Similarity matrix for species used in phylogenetic reconstruction. Upper triangle values are percentage sequence identities. Lower triangle values are genetic distance measurements corrected for multiple base changes by the method of Jukes and Cantor for sequence positions which were unambiguous for all sequences in the analysis. Isolates whose 16S rDNA sequence was determined during this study are highlighted in bold text.

Sequences are in the order;

1 Thermonema lapsum 18 Chromatium vinosum 2 Bacteroides fragilis 19 Escherichia coli 3 Saprospira grandis 20 Agrobacterium tumefaciens 4 Chlorobium limicola 21 Desulfovibrio desulfuricans 5 Helicobacter pylori 22 Clostridium quercicolum 6 Treponema pallidum 23 Microcystus aeruginosa 7 Serpulina hyodyserteriae 24 Heliobacterium chlorum 8 Leptonema illini 25 Bacillus subtilis 9 Chlamydia psittaci 26 Lactobacillus fermentum 10 Deferribacter thermophilus 27 Clostridium ramosum 11 Flexistipes sinusarabici 28 Eubacterium barkeri 12 HRilcae 29 Fusobacterium nucleatum 13 HRI3liv 30 Chloroflexus aurantiacus 14 UNSW2.6liv 31 Thermomicrobium roseum 15 UNSWRSp12 32 Aquifex pyrophilus 16 UNSWMCSl 33 Fervidobacterium nodosum 17 Geovibrio ferrireducens -

N N

~

('l)

..... 00

('j

::r

.§

19.89

19.03

19.78

17 18.39

24.13

21.87

27.17 23.56 20.87 26.34 20.87 27.77 25.40

73.8 71.6 82.9 75.0 84.4 84.1 75.3 84.1 74.8 84.0 84.5 75.1

I

I

9.8

I

19.8

16 19.26

27.95

25.92

73.4 71.4 23.26 21.13 22.09 72.2 73.4 73.3 21.02 75.3 21.78 22.09 29.41 72.7 71.8

20.25 27.58

82.3 84.3 95.1 82.0

73.3

96.3

15

19.24 19.78

19.03 19.67

1.34 11.68 11.79

24.47 24.63 23.79

29.36 29.29

25.75

73.2 21.20 74.0 20.54 75.4 21.76 21.76 22.00 72.9 21.87 22.13 73.5 27.53

95.4 95.2 94.9 82.8 94.8 94.7

14

12.07

19013

26.11 20.22 73.4 74.2 20.32 71.0 72.2 23.56 23.33 71.8 73.5 21.98 75.3 21.53 27.77 27.89 22.09 28.99 28.99 29.11 73.1 71.2 71.3 71.3 72.8 20.11

99.5 2.11 82.4 82.2 99.2 2.11

9.13

13

1.86 11.97

73.5 70.9 71.8

82.7 82.9 82.5 73.7 75.2 21.42 21.98 72.5 72.5 73.1 71.5

99.7

72.8 72.8 73.5 73.3

I 3 I I

9.

12

1.85 1.85 1.86

11.97

71.1 20.98 20.98 20.98 72.0 83.0 73.9 21.98 21.98 75.4 24.82 24.82 24.82

28.99 28.99 20.00 20.00 25.75 25.75 20.11 20.11 20.00 20.00 0.00 0.25 0.25

72.9

I I

11

14.58

13.85 14.15 13.85

19.03

18.8 19.24

18.07 19.24

14.35

74.9 73.7 72.5 87.4 82.9 72.4 26.11 76.6 25.99 27.65 27.65 20.54 21.42 25.64 28.87 28.87 72.2 72.7 21.09 21.98 75.2 73.3 72.0 71.6 23.90

21.98 23.33 23.33

20.32 20.87 20.87 20.98

73.5

10

14.15 13.85 14.15 14.45 14.05 13.97 13.65 16.70

17.12 10.14

17.44 17.23

19.57

74.8 72.2

75.7 75.3 74.9 73.9 72.8 75.5 20.00 73.1

21.53

25.05 22.54 23.10 26.46 24.36 25.99 27.77 27.77 27.89

27.89

26.22

25.99 23.22 30.97 27.89 26.58 71.0 26.22 25.17 21.53 21.53 71.3 73.2 29.23 73.3 27.65 73.0 25.29 29.60 24.36 72.8 72.4 27.65 20.43 74.3 25.05 32.24

9 33.66 32.11

76

73.8

24.70 24.70 73.8 24.82 28.26 24.59 28.02 24.47 24.63 27.93 76.0 24.59 25.75 74.1 23.56 26.34 20.22 28.14 28.14 75.3 24.36 24.13 28.38 22. 26.82 26.34 21.65 25.75

22.20

I 3 I

24.24 26.82 28.38 25.99 31.23 30.97 25.17 28.38 32.75 7 8 25.17 24.01 32.37 72.6 25.05 26.11 69.6 72.7 69.8 27.53 26.34 72.7 76.9 78.6

24. 28.02

22.65

24.13 25.29 22.43

26.70 23.10 23.10 23.22 22.88 25.17 71.9 70.9 23.14 25.56 74.2 73.6 23.10 26.82 74.1 22.43 26.94 22.20 25.64 28.74 22.20 22.76 26.46 24.24 22.65 25.75 23.79 26.46 22.20 26.22 23.22

22.54 22.43 25.40 22.20 24.70 24.47 25.40 23.33 22.88

I 3 I

18.60

22.65

27.29 20.98 24.47 21.65

22.54 25.52

74.2 70.5 72.3 71.3 22.76 22.99 73.7 24.59 25.05 24.01 27.77 29.36 28.14

24. 24.82 27.77

22.76 30.22 31.48

24.59

77.7

10

24.24

24.36

22.31 23.79 22.99 26.34 26.34 27.77 22.09 22.43 22.09

24.24 22.54 34.01 76.0 74.2 73.7 23. 24.94 28.02 26.34 22.43 22.43 28.02 4 5 6 21.31 27.65 25.75 24.36 22.76 22.31 22.31 25.17 24.01 23.56 21.09

3 26.94 26.94 27.05 24.59 25.99

28.99 25.87 24.59 26.94 25.52 29.11 26.58 25.40 26.82 28.87 30.35 79.5 29.36 76.5 26.58

28.14 27.77

26.11

30.97

31.73

23.44 78.0 26.94 27.17 31.10 27.83 26.27 24.63 22.47 29.11

27.17 28.74 27.53 2 27.77 27.05 29.73 27.05

34.84 32.88 30.60

29.23

32.75 31.86 33.27 33.92

27.41

26.82

19.24

25.17 27.77 25.17 27.77 25.29 27.89 24.47 27.53 24.36 27.89 25.21 28.87 27.17 23.10 28.14 25.40 25.29 24.13 29.48 22.65 27.05 22.31

21.09

22.88

24.13 28.14 26.58 23.79 21.76

28.99 23.44 23.33 29.23 22.54 25.40 25.75 24.47 28.87 27.29 24.59 28.14 26.22 27.17 24.70 28.62 27.89 25.29 28.14 29.23

roseum

nodosum

chlorum

tumefaciens

nucleatum

thermophilus

fermentum

vinosum

aurantiacus

barkeri

coli

pylori

aeruginosa

lapsum ramosum

quercicolum

sinusarabici

limicola

fragilis illini

pallidum

psittaci

ferrireducens

grandis

hyodysineriae

subtilis

pyrophilus

UNSW2.61iv

HRI31iv

UNSWRSp12

HRilcae

UNSWMCSl

Flexistipes

Chromatium

Bacillus

Escherichia Deferribacter Thermomicrobium Microcystis

Geovibrio Aquifex

Chloroflexus

Fervidobacterium Clostridium Heliobacterium Lactobacillus

Clostridium

Eubacterium Fusobacterium

Leptonema

Chlamydia

Thermonema Bacteroides

Chlorobium

Serpulina

Helicobacter

Treponema

Saprospira

I.

18.

10. 11.

12. 17. 19.

13. 14. 15. 16.

2. 8.

3.

7.

9. 31. 23. 33. 4.

29. 5. 6. 20.Agrobacterium 21.Desulfovibriodesufuricans 22. 30.

24. 32. 25. 26. 27. 28. N

uJ

-

00

0

1-1

:::r .... n

.§

73.3 73.3

71.2 73.3 74.3 76.1

73.7 70.3 73.3 75.3 73.7 71.7

74.3 71.6 74.4 73.0 33 73.4 73.8 71.2 71.0 71.5 70.9

71.6 72.0

73.3

69.2

·

69.2

70.6 70.4 71.9 75.2 73.3

74.3 72.5 32 70.0 75.2 71.1 69.3 73.6 66.1 69.2 68.6 69.0 69.0 69.8 75.0

68.5 71.7 72.0 67.0 73.9 73.4 70.9

69.5 79.5 75.8

73.1 73.4

71.9 70.5 73.6 70.3 75.9 68.2 75.4 72.6 72.0 71.2 71.0 69.9 69.3 73.2 71.3 72.3 76.9 68.3 72.1 70.1 73.4 74.0 31 73.8 73.4 70.6

70.2 70.1

71.8 68.5

74.7 75.1 75.1 71.7 21.53 24.36 24.24 27.77 24.47 22.88

30 73.1 70.2 76.0 75.5 74.3 74.2

76.9 69.7 73.2 70.9 70.8 74.0 70.7 69.8 74.7 75.3 76.3 74.4 73.5 72.8

72.2 71.5 69.2 72.0

72.7 71.4 73.6 73.5

30.72 31.48 31.48

26.94

77.0 71.4 72.7 72.7 72.0 73.5 77.4 72.9 77.2 72.3 76.4 73.4 77.6 78.2 77.1 29 69.7 73.8 72.0 76.1 75.4 74.3 73.9

73.6

75.4

20.32 26.34 24.01 80.4 27.05 22.99

72.8 76.4 73.7 72.3 75.9 72.6 81.6 74.5 76.2 80.7 83.2 76.1 76.4 76.2 73.7 79.5 76.2 73.2 78.2 72.1 79.2 28 76.2 73.8 76.8 79.5 78.4 78.8 77.7

77.1 74.1 72.1

19.57

80.4 21.87 82.9 26.58 27.17 77.6 81.9 29.23 25.75

79.6 75.8 72.3 73.3 75.1 76.0 75.6 74.3 76.3 73.4 27 75.5 74.2 71.9 77.0 77.0 76.5

15.88

17.23

74.0

22.31 80.4 86.3 72.3 77.3 75.7 75.6 73.4 77.5 76.9 24.70 76.1 77.2 75.7 79.9 71.3 73.7 77.5 77.2 71.6 73.8 75.6 76.2 71.4 73.8 75.6 74.2 74.4 74.5 75.3 74.0 74.5 75.1

26 74.1

14.15

11.49

13.95

78.3

77.0 82.2 21.65 75.8 76.2 25.64 27.77 76.9 72.6 79.4 80.0 78.6 78.1 77.2 77.0 73.4 76.8 82.7

76.8 76.9

74.1 25

15.67 18.39 17.23 17.02

24.24 24.24 25.87

80.2

76.7 79.0 76.5 76.4 83.8 76.8 21.09 75.8 75.4 78.1 74.1 25.87 76.6 76.3 77.3 24.59 24.59 74.8 78.9 79.8 79.3 79.4 78.2 73.2 73.3 70.4 71.2

75.7 75.7 74.8

24

19.67

21.31 21.31 22.31 24.01 27.53 27.41 76.4 75.0 28.74 76.0 25.64

73.7 73.5 80.7

73.2 74.5 72.2 23 74.0 74.7 75.7 76.0 73.4 70.1 72.2 73.0 74.7 74.3

17.44 13.85 15.06 16.91 17.44 16.50

76.7 76.5 76.4 73.3

75.5 76.5 26.34 25.52 25.99 23.22 22.99 26.34 76.3 74.3 24.82 77.3 76.6 77.2 76.9 77.1

72.6 76.6 73.9

74.6 22 74.3

72.9 69.3 74.3 75.6

18.49 19.24

19.35

80.8 22.09

77.0 81.4 21.42 21.31 76.8

76.7 75.4 73.9 73.7 73.9 25.40 26.46 27.89 74.7 78.4 20.87 74.3 73.3

77.8

71.3 73.6 76.3 77.4

21

19.35

18.39 19.35

19.35

76.0 77.0 23.56 24.36 20.32 75.5 28.26 26.82 78.0 77.0

79.8 29.60 24.94 25.40 78.3 79.8 83.5 22.76

76.1 21.31 75.8 23.56

77.1 73.7 20 76.1 73.3

77.4 75.3 73.3 72.6 75.0 76.1 75.2

I 0 I

19.35

19

85.7 24.36

75.3 75.9 76.8 75.1 75.8 75.3 27.29 74.8 26.22 26.46 28.62 75.3 23.56 20.32 28.26 72.7 77.0 20.87 76.7 20.76 73.1 71.1 23.10 23. 74.6 21.76 74.5 73.3

71.9

13.05 15.37 18.28

17.96 17.12

18.28

18 18.07

26.11

22.99 78.0 75.8 75.4 21.09 75.6 80.4 20.11 75.5 75.8 22.65 26.58 77.1 75.8 76.8 73.3 76.8 24.36 72.7 21.20 76.6 20.65 25.05 74.4

74.2 71.3 74.0 75.6

roseum

nodosum

eh/arum

tumefaciens

nucleatum

desulfuricans

thermophilus

fermentum

vinosum

aurantiacus

barkeri

coli

aeruginosa

ramosum

/apsum

pylori

quercicolum

sinusarabici

limico/a

fragilis

illini

psittaci

pallidum

ferrireducens

grandis

subtilis

hyodysenteriae

pyrophi/us

UNSW2.6liv

HRI31iv

UNSWRSp12

HRllcae

UNSWMCSl

Bacillus

Chromatium

Flexistipes

Escherichia

Thermomicrobium

Aquifex

Ch/orojlexus Deferribacter Desulfovibrio

Microcystis Fervidobacterium Heliobacterium Lactobacillus

Clostridium Geovibrio Fusobacterium

C/ostridium

Agrobacterium Eubacterium

Leptonema

Chlamydia

Thermonema Bacteroides

Ch/orobium Helicobacter

Treponema

Serpulina

Saprospira

11. 17. 18. 19.

I.

14. 15. 10. 16.

12. 13.

30. 31. 8. 32. 33. 26. 3. 29.

6. 7. 20. 21. 22. 9. 23. 2. 24. 25.

27. 28.

5.

4. Figure 8.1

Phylogenetic tree for organisms representing the major lineages within the domain Bacteria derived from near complete 16S rRNA gene sequences. Genetic distances were calculated by the method of Jukes and Cantor (73) and the tree reconstructed from a pairwise distance matrix by using the neighbor-joining method of Saitou and Nei (140). Scale bar represents a genetic distance between nucleotide sequences of 5%. Bootstrap values (for branches present in more than 50% of 100 resamplings of the data) are indicated at the nodes. Sequences for organisms in coloured, boldface type were determined during this study and represent bacterial group I, as described in the text. Chapter8 124

M,"zcrocystl ·s aeruginosa 5 f Clostridium quercicolum Heliobacterium chlorum Lactobacillus fermentum -60 4 Bacillus subtilis ~ Clostridium ramo sum Eubacterium barkeri ~ Fusobacterium nucleatum Agrobacterium tu mefaciens 1001 I 1001 Chromatium vinosum I Escherichia coli Desulfovibrio desulfurz·cans ~ Helicobacter pylori 6~ HRI31iv 100- HRilcae 100 -UNSW2.6liv CUNSWMCSl - 100 100 89 UNSWRSp12 100 Geovibrio ferrireducens 99 Deferribacter themophi lus Flexistipes sinusarabici Bacteroides fragilis 1001 Thermone ma lapsum 51 Sap rospira grandis .--- Chlorobium lim icola - Leptonema illini 87 Treponema pallidum .... ~ Serp ulina hyodysenteriae Ch lamydia psittaci 81 I Chloroflexus aurantiacus I Thermomicrobium roseum F ervidobacterium nodosum Aquijex pyrophzlus

5% Chapter 8 125

8.2.2 PCR design The multiple sequence alignment created for the phylogenetic analysis was also used to locate unique oligonucleotides which could be used in PCR to identify other bacterial isolates which were members of this previously unrecognized bacterial group. Candidate primers were identified at nucleotide positions 137-158 and 1115-1134 (E. coli numbering). Comparison of the primer target sequence with sequences contained in GenBank and EMBL databases revealed no significant homologies. Table 8.4 shows the target region for the primer and the nucleotide mismatches present in a number of other . bacterial species. These primers were synthesized in either the forward (GreenF) or reverse orientation (GreenR) (Table 2.1) and used to amplify a PCR product of 1010 bp. The theoretical Tm of GreenF and GreenR were 6Q°C and 58°C respectively. Specific amplification of DNA from the five isolates was achieved with amplification reactions prepared as described previously and the following thermocycling profile. An initial denaturation at 94°C for 3 minutes followed by 30 cycles of 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute. This was followed by an extension step at 72°C for 10 minutes (FfS 320, Corbett Research). Figure 8.2 shows the PCR products obtained when the 5 isolates (HRilcae, HRI3liv, UNSW2.6liv, UNSWMCSl and UNSWRSp12) and other bacterial species (including group J isolates and Helicobacter species) were used as template DNA under these conditions.

This PCR was used to confirm the identity of other bacterial isolates cultured for the present study and placed in group I (Chapter 4 ). Template extracted from all of the non­ helicobacter isolates (groups I and J) was subjected to PCR using primers GreenF and GreenR using the above cycling conditions. In addition, all isolates classified in the helicobacter positive groups E or Hand those templates containing a 16S rRNA operons · polymorphic for an IVS were also subjected to the described PCR. This was done to ensure that these samples did not also contain DNA from group I bacteria (since morphologically these isolates were indistinguishable from the helicobacters with a type 1 morphology). Using this PCR, all isolates classified in bacterial group I gave a positive result. In addition, all group J isolates and helicobacter positive samples tested did not amplify in this reaction. Therefore, the total number of isolates cultured for this study and confirmed as being members of group I was 30 (ABHU=5, HRI=21 and UNSW=4). In summary, all non-helicobacter isolates with morphology type 1 were identified as being phylogenetically related based on a positive reaction in the described PCR. All isolates placed in this group had a thin, spirillum like morphology, were highly motile and grew on CSA in an anaerobic atmosphere. Isolates which were examined using transmission electron microscopy had single, bipolar, unsheathed flagella (Figure 8.3). Table 8.4 (") Comparison of bacterial group I specific primer target site with sequences from other bacterial species. f 00

GreenF (137-158) Target Sequencea Organism GreenR (1115-1134) bacterial group I TTTTAGACTGGAACAACTTACC CTATTTCCAGTTGCTAACGG

Geovibrio ferrireducens TCAGAGATTGGGACAACACAGA CTGCTTTTAGTTGCCATCGG Deferribacter thennophilus CCGCAGACCGGGATAACCCATC CTACCCTTAGTTGCCATCGG Flexistipes sinusarabici CATTAGACCTGGATAACCCGGC CTATTCATAGTTGCCATCGG Helicobacter pylori TCTTAGTTTGGGATAGCCATTG CNTTTCTTAGTTGCTAACAG Desulfovibrio desulfuricans CTTATGATCGGGATAACAGTTG CTATGGATAGTTGCCAGCAA Escherichia coli TGATGGAGGGGGATAACTACTG TTATCCTTTGTTGCCAGCGG Lactobacillus fennentum CAGAAGCGGGGGACAACATTTG TTGTTACTAGTTGCCAGCAT Bacteroides fragilis CTTTACTCGGGGATAGCCTTTC TTATCTTTAGTTACTAACAG * * * * * *** * * *

a Sequences are writen in 5' to 3' orientation and represent positions 137-158 (GreenF) and 1115-1134 (GreenR), according to E. coli numbering. Asterisks ( *) indicate nucleotide positions which are conserved across all species listed.

..... N O'I Chapter 8 127

Ml 2 3 4 5 6 7 8 91011

Figure 8.2 PCR products from the bacterial group I specific reaction using primers GreenF and GreenR (Table 2.1). DNA separated in 1% agarose/TAE.

lane M SppUEco Rl(200 ng) 1 HRilcae (group I) 2 HRI3liv (group I) 3 UNSW2.6liv (group I) 4 UNSWMCS 1 (group I) 5 UNSWRSp12 (group I) 6 ABHUlSB(group J) 7 UNSW3caefatS(group J) 8 Helicobacter pylori 9 Helicobacter muridarum 10 Helicobacter bilis 11 "no DNA" control Figure 8.3 Negatively stained preparations of three different group I isolates which possessed a type 1 morphology. (A) isolate HRilcae, magnification 35000, (B) isolate UNSWRSp12, magnification 25 000, (C) isolate UNSWMCSl, magnification 25 000 A

B

C Chapter 8 129

8. 4 Discussion and Summary This study presents the phylogenetic position of a new group of organisms within the bacterial domain based on 16S rRNA gene analysis. The described organisms were most closely related to bacteria from the recently described "Flexistipes" group which currently contains three genera, each being defined by a single species, which were cultured from diverse environments. F. sinusarabici was isolated from hot brine waters of the Red Sea (35), G. ferrireducens from surface sediment of a hydrocarbon-contaminated ditch ( 16) and D. thermophilus was isolated from the production water of an oil field in the North Sea (60). Evidence presented here supports the future description of bacterial group I as a new genus within a possible twelfth "phylum" referred to here as the "Flexistipes" group. In total, thirty isolates cultured for the present study, plus _three previously cultured isolates, were identified as being members of this group using the specific PCR developed here utilizing primers GreenF and GreenR.

The three previously described members of this phylogenetically related group (the "Flexistipes" group) share few phenotypic characteristics. They are, however, all anaerobic (either respiratory or fermentative), straight to curved rods or vibroid in shape. Motility varies with G. ferrireducens being motile by a single polar flagellum, and D. thermophilus being non-motile. In terms of optimal growth temperatures, these bacteria range from being thermophilic (D. thermophilus) to mesophilic (G.ferrireducens). Although growth at different temperatures was not assessed for the group I organisms, their isolation from the rodent intestinal tract would indicate that they are mesophiles, with an optimal growth temperature ·of 37°C.

No analysis of biochemical or physiological characteristics was performed for the group I organisms. Such studies are essential to determine the metabolic capabilities of group I organisms particularly since the most closely related described bacteria originate from such diverse environments, and are only moderately related phylogenetically, based on 16S rRNA gene sequences to bacterial group I. The relatively large number of isolates obtained and confirmed here as being members of this phylogenetically distinct bacterial group (n=33), presents an opportunity to perform a detailed study to clearly define the proposed new genus which may contain multiple species isolated from the same environment. Chapter 9 130

Chapter 9

Analysis of the Distribution of Spiral Bacterial in vivo using Molecular Techniques

9. 1 Background The isolation of Helicobacter species and other similarly fastidious, slow growing, microaerophilic or anaerobic species is not always a straightforward task. The original isolation of Helicobacter pylori occurred only after an accidental extended incubation over a holiday long weekend (101). The existence of Helicobacter species which have yet to be cultured has been well documented (87, 91, 108, 153), and some recently cultured species required laborious and complicated isolation and culture procedures (63, 72).

The isolation of previously undescribed helicobacters has been reported in this study (Chapters 5 and 6), as has the isolation and development of specific PCRs for the detection of two groups of non-helicobacter isolates which made up a significant proportion of the spiral bacteria isolated for this study (Chapters 7 and 8). These results highlighted two issues. Firstly, when investigating the spiral bacteria if the GIT, it is not only Helicobacter species which should be considered. Secondly, the time and effort required to isolate even small numbers of spiral bacteria is very great, and there is a high probability that certain significant organisms will not, or cannot, be cultured using the methods employed. As is being increasingly shown for many natural ecosystems, culture dependent methods for examining bacterial diversity and prevalence in the GIT, while still important, are not ideal.

The aim of the present study was to develop a method for the in vivo detection of the different types of spiral bacteria identified and described in the previous chapters of this thesis (Chapters 4-8). The overall concept was to utilize the range of PCRs developed and/or validated as part of the presented work to detect the presence of different bacterial types in paraformaldehyde fixed, paraffin embedded tissue specimens. This chapter presents a preliminary study to determine the usefulness of this type of PCR detection system, with a view to apply such a method in future to extensive bacterial mapping and localization studies of the GIT. Chapter9 131

9. 2 Experimental Design

9.2.1 Samples For each mouse that was included in this study (ABHU n=6, CMRI n=6, HRI n=8 and UNSW n=3), tissue samples (liver, stomach, small bowel, caecum and large bowel) were fixed in paraformaldehyde and embedded in paraffin blocks as described (Chapter 2) for possible future histological examination. Multiple (approximately 20) 5 µm sections were cut from each block in order for DNA to be extracted from the fixed tissue. Particular care was taken care to avoid contamination between blocks by carefully wiping down the microtome with Histoclear™ and ethanol between sectioning each block and by using a new blade for each block. Sections were deparaffinized and the DNA extracted from the tissue using methods similar to those described by Isola et al. (71) as described (Chapter 2). Four samples were not available for DNA extraction (ABHU caecum, 2 samples; CMRI small bowel, 1 sample; and CMRI large bowel, 1 sample).

9.2.2 PCR design A nested PCR approach was taken for amplification of DNA extracted from paraffin embedded tissue specimens. The experimental design involved an initial primary over­ amplification of the entire array of 16S rDNA molecules present in any sample using low stringency conditions and the consensus primers F27 and R1492 (Table 2.1). The amplification reactions were prepared as described (Chapter 2). Cycling involved an initial denaturation at 94 °C for 5 minutes and was followed by 35 cycles of 94 °C for 10 seconds, 45°C for 20 seconds and 72°C for 2 minutes. This was followed by a hold at 72°C for 7 minutes (2400, Perkin Elmer).

After the primary amplification was completed, samples were taken from each tube and diluted in sterile, distilled water. This diluted, PCR amplified DNA served as the template in the secondary or nested PCR reactions, to determine which of the bacterial species or groups were present in each sample. The "no DNA" control reaction, from the initial 16S rDNA amplification reaction set, was also used as template in the nested reactions to ensure there was no contamination of reactions during the primary PCR. Product from the primary, low stringency, universal bacterial PCR was analyzed in a 1% agarose/fAE gel to ensure successful amplification had been achieved prior to the nested PCRs being performed.

There were up to six nested PCRs carried out on the diluted, primary amplified samples. These reactions were specific for the bacterial groups described in Chapters 4-8 and were carried out using the conditions indicated in these sections. All samples were amplified in Chapter9 132

the generic Helicobacter PCR, using primers H276f and H676r (Table 2.1). Those samples which were found to be positive for the presence of helicobacter DNA were then subjected to PCR amplification specific for Helicobacter bilis (primers C62 and C12, Table 2.1), Helicobacter muridarum (primers H276f and HmurR, Table 2.1) and Helicobacter rodentium (primers D86 and D87, Table 2.1). PCRs specific for Helicobacter hepaticus and Helicobacter trogontum were not carried out since these species were not isolated from any of the mice included in the presented study (Chapter 4 ). All samples were also subjected to the PCRs developed during this study, specific for bacterial groups I (primers GreenF and GreenR, Table 2.1) and J (primers DfairF and DfairR, Table 2.1).

Each set of nested PCRs included a nested negative control which contained 2 µl from the "no DNA" control obtained after low stringency primary PCR amplification of the same PCR set. A standard "no DNA" control containing 2 µl sterile distilled water in the place of template was also included in each reaction set. Positive DNA controls were also included in each nested reaction set.

Particular care was taken to avoid contamination for both primary and secondary DNA amplifications. This involved the use of positive displacement pipettes for all procedures and, except for manipulations involving post amplification 16S rDNA (dilution and addition to prepared nested PCR mixtures), use of a laminar flow cabinet which had not previously been exposed to helicobacter DNA. In addition, PCR reagents (with the exception of Taq polymerase and primers) and disposables were treated in an Ultraviolet Crosslinker (Amersham Life Science, Buckinghamshire, England) for 5 minutes prior to each use.

9.3 Results All DNA samples obtained from extractions of the paraffin embedded tissue specimens were successfully amplified in the primary 16S rDNA amplification reaction. The "no DNA" control reactions consistently showed the presence of a small amount of contaminating DNA when analyzed by agarose gel electrophoresis. The amount of product obtained from amplification of an extracted tissue specimen and a "no DNA" control is presented (Figure 9.1). Nested PCR amplification using these primary "no DNA" controls as template always gave a negative result indicating that the contaminating DNA was from bacteria other than those being tested for. Chapter 9 133

1 2 3 4 5 6 7 8 9 M

Figure 9.1 PCR products from the low stringency bacterial 16S rDNA reaction, using primers F27 and R1492 (Table 2.1). DNA template was extracted from paraformaldehyde fixed tissue samples. DNA sepa­ rated in 1.5% agarose/TAE.

lane 1 no DNA control 2 HRI7cae 3 HRI7LB 4 HRI8stom 5 HRI7liv 6 HRI8cae 7 HRI8SB 8 HRI8LB 9 HRI8liv M Sppl/Eca RI (150 ng) Chapter 9 134

Initially, several primary PCR products were diluted using 10-fold dilutions between 1/10 and 1/10 000 to determine which dilution was necessary for nested PCR amplification using the Helicobacter generic reaction. Helicobacter DNA was successfully amplified in reactions containing primary PCR product diluted 1/10 and 1/100 but not when undiluted, 1/1000 or 1/10 000 diluted product was added as the template. Subsequently, primary amplified template, diluted 1/10, was used as the template DNA in all nested PCRs. Examples of product obtained from nested PCR reactions specific for helicobacters (Figure 9.2) and non-helicobacters (Figure 9.3) are presented.

The results obtained for each of the seven nested PCRs carried out were expressed as; the percentage of all mice, and the percentage of mice from each animal facility giving a positive result (Figures 9.4 and 9.5) and the percentage of each tissue type giving a positive result (Figure 9.6).

All mice (n=23) had at least one sample which was positive in the generic Helicobacter PCR indicating that all animals in this study were infected with helicobacters. In total, 93/111 (84%) samples were positive in the nested Helicobacter genus specific reaction (Figure 9 .6) Thirty-seven of these samples (40% of the total number of helicobacter positive samples) were then found to be negative for all three reactions specific for the various Helicobacter species (H. muridarum, H. bilis and H. rodentium). The tissue type with the most helicobacter negative samples was the small bowel with 7/22 (compared to 2/23 liver, 3/23 stomach, 4/21 caecum and 2/22 large bowel).

Only 3/111 samples were positive in the nested H. bilis PCR, while H. muridarum and H. rodentium were both detected in over half the animals (14/23 and 12/23 respectively). However, H. muridarum was detected in 6/6 animals from CMRI and 7 /8 from HRI, and only 1/6 animals from ABHU and 0/3 UNSW mice. Conversely, H. rodentium was detected in 5/6 animals from ABHU and 3/3 mice from UNSW and only 1/6 and 3/8 mice from CMRI and HRI respectively. Detection of both these species followed the general trend that they were detected in the small bowel samples less often than in other tissue types (Figure 9.6), although, as with the culture rates, numbers of samples were too small to be able to determine if these differences were significant.

Bacterial group J was detected in 100% of mice via PCR using primers DfairF and DfairR (Figure 9.4). This group of organisms was even more widespread in samples than the Helicobacter spp. with only 4/111 samples being negative in this nested PCR. In contrast, bacterial group I was detected in only 2/111 samples. with

).

2.1

2.1).

with

specific

(Table

(Table

specific

control

control

ng)

ng)

D87

C12

rodentium

bilis

and

and

DNA" DNA"

Rl(IOO

Rl(lOO

D86

C62

"no "no

Helicobacter

HRI8SB HRI3cae HR14stom HRI8LB HRI8liv HR14liv ABHUlSB nested primers ABHUlliv Sppl/Eco nested ABHU2stom HRI3liv HRI3SB ABHU2LB primers HRBLB Helicobacter Sppl/Eco ABHUlstom HR14cae HR14LB

D

B

reactions.

1

1

8 3

5 6 7 3 2 9 M 5

2 M 8

4 6 7

9 4

Panel

lane Panel

lane

nested

specific

).

2.1).

2.1

species

PCR

(Table

(Table

and

PCR

genus

specific

H676R

HmurR

and

and

specific

control

control

ng)

ng)

genus

muridarum

H276f

H276f

agaroseffAE.

Helicobacter

DNA"

DNA"

Rl(IOO

Rl(lOO

the

1.5%

"no

"no

primers

primers

in

from

with

nested

Sppl/Eco HRilliv HRilstom HRIISB Helicobacter Sppl/Eco HRI2liv HRI3stom nested with CMRI6cae CMRI6LB CMRI2liv HRI2stom CMRl2SB HRI2cae CMRI6cae HRI2SB CMRI6LB CMRl2LB HRI2LB

Helicobacter

C

A

1

I

separated

3

8 3 2

8 M 5

2 9 M 5 6 7

9 4 6 7

4

products

DNA

Figure 9.2 Figure PCR

Panel

lane Panel

lane

l,.) l,.)

...... V, V,

~ ~

\0 \0

t>:> t>:>

::; ::;

n n

"'O "'O

8 8 9

7 7

6 6

12345 12345

M M 1 2 3 4 5 6 7 8 9

M M

D D

B B

M M

8 8 9

M M

7 7

1 1 2 3 4 5 6 7 8 9

1 1 2 3 4 5 6

C C A A Fi~re 9.3 PCR products from the bacterial groups I and J nested reactions. DNA separated in 1.5% agarose/fAE.

Panel A Bacterial group I specific with primers GreenF and GreenR (Table 2.1). lane 1 ABHU5liv 2 ABHU6cae 3 ABHU6SB 4 ABHU6LB 5 ABHU6liv 6 nested "no DNA" control M M, Sppl/Eco RI(IOO ng) 7 UNSWlcae 8 bacterial group I positive DNA control 9 "no DNA" control respectively

Panel B Bacter ial group J specific with primers DfairF and DfairR (Table 2.1 ). lane 1 ABHUlstom 2 ABHUlcae 3 ABHUlSB 4 ABHUlLB 5 ABHUlliv 6 nested "no DNA" control 7 UNSWlcae M Sppl/Eco Rl(IOO ng) 8 bacterial group J positive DNA control 9 "no DNA" control Chapter 9 136

1 2 3 4 5 6 7 M 8 9 A

1 2 3 4 5 6 7 M 8 9 B Chapter 9 137

100

90

80

70

Q) Cl') 60

+-'= C Q) 50 (.) I,.. Q) Cl. 40

30

20

10

0 I... E E ""') '11 :::, ~ ...... ~ .;2 :::, ~ I... :::, 0 :s ...... 0 C 0 "Cl I... ..0"' I "' '11 C'I I... 0 ·;: "Cl C'I :::, 0 -~ I... ~ E I I I bacterial group

Figure 9.4 Percentage of mice (n=23) in which the bacterial groups represented above were detected using nested PCR on DNA template extracted from paraformaldehyde fixed, paraffin embedded tissue specimens. Chapter 9 138

ABHU CMRI 100 .... 100 - 90 90 - 80 80 - Q) 70 Q) - a Helicobacter Cl) Cl) 70 0:, I- 0:, - • H. bilis +.J 60 +.J 60 • H. muridarum C I- C - Q) so Q) so - u u • H. rodentium I- - I,.. 40 I,.. 40 • g roup I Q) Q) Q. 30 - I- Q. 30 i- • groupJ

20 - I- 20 i-

10 - 10 ~ ,- ,-- ,- 0 + 4- + - 0 : : 4- 4- 4- - bacterial group bacterial group

HRI UNSW

100 ~ 100 ~ 90 -- 90 -- 80 -- 80 - Q) 70 -- Q) 70 Cl) Cl) 0:, 0:, 60 + +.J +.J 60 C C f- - - Q) so Q) so u u I,.. 40 >- - I,.. 40 Q) Q) Q. 30 ~ - Q. 30

20 ~ 20 I- 10 - 10 ,- 0 4- + - 0 - bacterial group bacterial group

Figure 9.5 Percentage of mice, from each animal facility, in which the bacterial groups represented above were detected using nested PCR on DNA template extracted from paraformaldehyde fixed, paraffin embedded tissue specimens.

\0 \0

VJ VJ

,..... ,.....

\0 \0

~ ~

'"1 '"1

i;.) i;.)

O" O"

n n

"d "d

I I

J J

rodentium rodentium

muridarum muridarum

bilis bilis

H. H.

Helicobacter Helicobacter group group

H. H.

H. H.

group group

D D

• •

• •

• •

• •

• •

ious ious

r

va

h h

c

i

e e

Q,) Q,)

-

wh

= =

,:,'I ,:,'I

Q,) Q,)

I,..) I,..)

-.i::i -.i::i

in in

, ,

types

e e

u

~ ~

~ ~

= =

Q,) Q,)

::i ::i

= =

iss

t

R. R.

ifferent ifferent

C

Q,) Q,) e e

) )

m m d

d d P

.Cl .Cl

type type

e

t

O':I O':I

= =

e e

fro

-

--

es

n

tissue tissue

using using

extracted extracted

d d

, ,

e

t

O':I O':I

~ ~ e e

= =

= =

es

...... -= -=

ec

t

pl

e

d

e e

sam

r

A A

we

N

D

ps ps

u

Q,) Q,) I,.. I,..

......

> >

of of

-

.

gro

l l

9.6 9.6

tage tage

n

-

eria

~ ~

t

ce

r

e

ac

0 0

b

P

Figure Figure

10 10

20 20

30 30

so so

40 40

60 60

70 70

90 90 80 80

100 100

Q,) Q,)

I,.. I,..

c. c.

(.) (.) Q,) Q,)

C C

(1:1 (1:1

Cl') Cl') Q,) Q,) ...... Chapter 9 140

Two samples were negative in all nested PCRs. Examination of the product obtained after primary 16S rDNA amplification showed no difference in amplification efficiency between these samples and any of the other primary "over amplified" products. DNA from these samples was reamplified in both primary and nested PCRs with the nested results remaining negative for both samples.

9. 4 Discussion and Summary This study investigated the utility of an in vivo detection system for spiral bacteria in · paraformaldehyde fixed, paraffin embedded tissue specimens. The goal was to determine if this could be used as a culture independent method for determining the presence of different bacterial types. Additionally, how closely culture rates corresponded with PCR detection rates was investigated.

PCR using oligonucleotides complementary to conserved regions of the 16S rRNA gene has been used extensively to amplify small subunit (16S) rRNA genes from various types of total-community genomic DNA in order to perform culture independent analysis of microbial ecosystems (106, 109, 153, 188). Post amplification analysis can involve a number of different methods including nested PCR, Southern hybridization, restriction endonuclease digest analysis and sequencing of amplified DNA either directly or after cloning into appropriate vectors (11, 18, 50, 95, 153). Another approach which increases the initial amount of template DNA is to perform an initial PCR with random hexamer oligonucleotides presumably amplifying the total DNA present in the sample. This amplified DNA is then able to be used in multiple specific PCR analyses (129).

The nested PCR approach was used here since all of the oligonucleotide primers utilized · for specific detection of the various bacterial groups were located within the 16S rRNA gene. This meant that DNA synthesized during a single primary PCR of the entire 16S rDNA, using the universal bacterial consensus primers, served as template in all of the secondary nested reactions.

The consistently PCR positive "no DNA" controls in the primary 16S rDNA PCR, which were negative in all secondary nested reactions, indicated the presence of contaminating DNA originating from a bacterial species other than those being tested for. The most likely origin of the contaminating DNA was from the Taq polymerase, which was extracted and purified from E. coli clones expressing this enzyme. Contamination of commercially available Taq polymerase has been reported previously (136, 146), and the level of contamination observed here was such that, under normal stringency PCR conditions with the same consensus primers, it was below the level of detection. Chapter 9 141

The 2-step generic Helicobacter reaction described in Chapter 3 was not utilized here since the forward primer in this reaction (F27(UFP)) had identical specificity to the forward primer used in the primary 16S rDNA amplification (F27). This reaction would have been only "semi-nested" which may have increased the likelihood of false positives. As a result the presence of an IVS in the 16S rDNA of Helicobacter species was not determined. It is likely, therefore, that some of the 37 unspeciated helicobacter PCR positive samples were representatives of bacterial groups B or D as described in Chapter 4. The H. bilis nested PCR applied here would have also been able to amplify members of bacterial group C, but in the absence of morphological descriptions of the organisms being detected, it was not possible to determine whether the H. bilis positive samples contained DNA from bacterial group A or C. Nested PCR, specific for H. hepaticus and H. trogontum, was not performed since neither of these species were isolated from any of the samples. It is possible, although unlikely, based on the earlier results presented in this thesis (Chapter 4), that some of the unspeciated helicobacter PCR positive samples may have contained DNA from these species. A third possibility was that these samples contained uncultured, and still undescribed helicobacters.

This preliminary study was a first step in an attempt to investigate the localization of mucosa-associated spiral bacteria of the mouse GIT in vivo. The results presented show that it was possible to specifically amplify DNA originating from different bacterial types within the mouse GIT, but there were a number of limitations involved with the methodologies employed. There was little, if any, correspondence between culture rates and PCR detection rates of the bacterial groups which were investigated by both methods. In other words, both the culture methods applied, and detection using the nested PCR method developed here, gave only a limited picture of the range of mucosa-associated spiral bacteria present in the laboratory mice investigated. Further development of a culture independent method for reliable detection of these microorganisms is required in order to study adequately the microbial ecology of the mammalian GIT. There is a need to expand this work using improved fixation techniques and in situ studies in order to elucidate accurately the micro-ecology of these bacteria. Chapter 10 142

Chapter 10

General Discussion

The investigations presented in this thesis were primarily intended to examine the hypothesis that the spiral shaped bacterial of the gastrointestinal tract are phylogenetically related, having evolved with their host to colonize a particular ecological nice. Other related hypotheses examined were concerned with the concept of "autochthonous microbiota" and bacterial distribution in the specific model being examined, that is, laboratory mice. An additional reason for examining the indigenous microorganisms in these animals was the lack of basic knowledge with regard to the types of Helicobacter species present in Australian mice. The significance of laboratory rodents being infected with indigenous Helicobacter species is not fully understood, but a fundamental task must be to determine the presence and type of helicobacters found to naturally infect these animals.

1 O. 1 Phylogeny of spiral shaped bacteria The results presented in this thesis clearly show that there did not exist a single group of phylogenetically related bacteria colonizing the intestinal mucus of the mice examined. Three phylogenetically distinct groups of spiral shaped bacteria were identified, including helicobacters, desulfovibrios and a novel phylogenetic lineage representing a new genus.

10.1.1 Helicobacter species diversity The detailed examination of the Helicobacter species isolated from the limited number of mice included in this study showed that many isolates belonging to this genus did not appear to be from species which had been previously described in the literature. Additionally, some described helicobacters, Helicobacter hepaticus and Helicobacter trogontum, were not isolated from any of the mice investigated in this thesis. As discussed previously, H. hepaticus and Helicobacter bilis infections are widespread in commercial mouse colonies in the United States (42, 137, 148). One of these studies also investigated the presence of Helicobacter muridarum, which was not detected in any rat caecal samples, out of 113 tested, and in less than 1% of 508 mouse caecal samples tested (137). Conversely, in this study H. muridarum was cultured from over 50% of mice from two of the animal facilities, and detected in over 60% of all mice by nested PCR on paraformaldehyde fixed mouse tissue samples. These findings suggest the possibility of major differences between Helicobacter species present in geographically widely separated animals. Chapter 10 143

A new species of Helicobacter was described in this thesis (Chapter 6) which was unusual in that it had no detectable urease activity and possessed unsheathed flagella. A simultaneous study was carried out in the United States which resulted in the description of a novel species, Helicobacter rodentium. This new species was similar to the organisms described in this thesis in terms of 16S rDNA sequence, negative urease activity and morphology. Future studies involving both of these organisms will clarify if they are strains of the same species.

It was considered very probable that other helicobacters isolated during this study represent additional novel species. Bacterial groups C and D were morphologically unlike Helicobacter bilis yet ha~ very high 16S rRNA gene sequence identity to this species. Again, these isolates require further investigation to determine their taxonomic position within the genus. Results presented in this thesis also supplied the first evidence that the SSU ribosomal RNA operon of Helicobacter spp. may be polymorphic for the presence of an intervening sequence in some isolates.

10.1.2 Homology vs. Morphology: implications for Helicobacter taxonomy The present work was an attempt to phylogenetically characterize a morphologically and ecologically defined group of bacteria, that is, spiral shaped bacteria from the gastrointestinal mucosa of laboratory rodents. While morphology is a notoriously uninformative character in terms of bacterial phylogeny and taxonomy, the increased complexity of the spiral shape (above simple rod and cocci forms) means that some classically (morphologically) defined groups of bacteria have been upheld in the light of 16S rRNA sequence analyses (125), while other morphologically defined groups of helical bacteria have been shown to be invalid (193).

The time in history when the first Helicobacter species were isolated has meant that the classification of all members of this genus included comparison of genetic information, particularly 16S rRNA gene sequences. 16S rRNA gene sequence comparisons give strong support to the genus division as a phylogenetically distinct group of organisms, as rRNA homology group III, within rRNA superfamily VI of the proteobacteria (173). Additionally, all Helicobacter species share some common morphological characteristics, being motile, spiral or curved rods. This may lead to the conclusion that morphology within this phylogenetic lineage is a taxonomically relevant character. Chapter 10 144

Difficulties arise for this genus in the definition of the species. Current taxonomic definitions do not specify the degree to which 16S rRNA sequence heterogeneity occurs among the members of species or genera (39). Within the gastric helicobacter group a number of morphologically distinct species have very high 16S rRNA sequence homologies. These species include Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis which differ significantly in their cellular morphologies when examined using electron microscopy (Figure 10.1). Eight different strains representing these 3 species were shown to have rRNA sequence homologies of greater than 98.2% (72). Two 16S rDNA sequences from uncultured "Gastrospirillum hominis" organisms, which themselves have only 96.5% homology (153) are also found within this group of gastric helicobacters and have greater than 98.2% ("G. hominis" 2) or 95.9% ("G. hominis" 1) homology to the 8 H.felis, H. bizzozeronii and H. salomonis strains (72). In the absence of other data, these levels of sequence homology are above those necessary for species discrimination (154). Here, more discriminatory methods were required to determine species delineation, and indeed for these 3 species ( excluding the uncultured "G. hominis") DNA-DNA reassociation values between different species of around 11- 39% support these divisions (72). These species represent a case where morphologic characteristics are a taxonomically valid character assisting discrimination between phylogenetically closely related species.

On the other hand, there exist a number of Helicobacter species which have very similar morphologies, but have 16S rRNA gene sequence similarities below the levels considered to be indicative of a single species. These species are those which have a fusiform rod type morphology and are entwined with periplasmic fibres, and include Helicobacter bilis, Helicobacter trogontum and "Flexispira rappini'' (Figure 10.2). Sequence analysis performed for this study calculated the 16S rRNA gene sequence homologies for H. bilis and H. trogontum to be 96.1 %, H. bilis and "F. rappini" (ATCC 43966, identical in 16S rDNA sequence to ATCC 43879 (26)) to be 98.6% and H. trogontum and "F. rappini" to be 95.6%. Two isolates of "F. rappini'' (ATCC 43968 and ATCC 43879) were, however, shown in another study to have only 95.6% sequence homology (149). Clearly, the taxonomic position of "F. rappini" needs closer attention. Furthermore, the possession of what appears to be a complex morphology type, common to these three organisms, is not a phylogenetically relevant character which allows for the taxonomic clustering of these species, to the exclusion of others, within this genus. In other words, the morphologically similar species are not phylogenetically more closely related to one another than they are to other helicobacters with very different morphologies. Figure 10.1 Examples of gastric Helicobacter spp. which have high 16S rRNA gene sequence similarities but are easily distinguished from one another morphologically. (A) Helicobacter biu.ozeronii, a tight, helical bacterium, magnification 20 000. (B) Helicobacter salomonis, a curved S-shaped bacterium, magnification 20 000 (C) "Gastrospirillum hominis", a tight helical shape, similar to H. bizzozeronii, photographed in ultra-thin sections as this organism has not been cultured in vitro, magnification 30 000. (D) Helicobacter felis, a tight helical bacterium with periplasmic fibres, magnification 16 000 ll. B Figure 10.2 Examples of murine helicobacters which are morphologically very similar but to one another, but represent different species within this genus. All of these bacteria are rod or fusiform in shape, possess multiple, bipolar, sheathed flagella and are entwined with periplasmic fibres. (A and B) "Flexispira rappini''. Bar= 1 µm. Micrograph obtained from reference (145); (C), Helicobacter bilis. Bar= 0.5 µm. Micrograph obtained from reference (48); (D) Helicobacter trogontum. Bar= 0.4 µm. Micrograph obtained from reference ( 104 ). c _ Chapter 10 147

To illustrate this point, two specific examples are described. Firstly, the corrected genetic distances calculated for this thesis placed H. bilis and "F. rappini'' as being most closely related to one another (excluding the sequences for isolates determined in this study), but a different "F. rappini'' isolate was shown to be more closely related to Helicobacter pullorum (149) which is morphologically very different from the fusiform helicobacters. Secondly, this study and others (104, 149) have also shown that the 16S rRNA gene sequence of H. trogontum shares highest sequence homology with H. hepaticus. These two species, like "F. rappini'' and H. pullorum, have very different cell morphologies. These two cases highlight the need to have a phylogenetically based classification from which taxonomically relevant characters can then be determined. These observed characters may or may not include differences in gross morphological characteristics.

This thesis presented a phylogeny, based on 16S rRNA gene sequences, of the Helicobacter genus including 16 sequences determined during the presented study. Some of the groups identified in this phylogenetic reconstruction present similar problems to those described above.

Considering firstly bacterial groups A, C and D, plus H. bilis. The percentage sequence homologies between all of these organisms (n=l 1) was calculated to be between 96.9- 100%. There were 2 distinct morphology types for organisms in these groups (Figure 10.3). However, groups with one morphology type did not necessarily share higher sequence homologies with one other than they did with the groups of organisms having - the other morphology type. For example, the corrected genetic distances between group D organisms which had morphology type 1 (spirillum like) and H. bi/is were smaller then the distances between group A organisms with morphology type 2 (fusiform) and H. bilis.

The Helicobacter rodentium-like bacteria isolated for this study have the converse problem of being morphologically almost identical to H. rodentium, particularly being in possession of unsheathed flagella. The percentage sequence homology between isolates cultures for this study was 98.2%, above the nominal value of 97% for discriminating between species (154). Results from investigation of some basic physiological and biochemical characteristics did, however, reveal some differences, the significance of which is yet to be determined. As illustrated above for some of the gastric helicobacters, however, it is not unknown for different Helicobacter species to have 16S rRNA gene sequence homologies above 98%. Figure 10.3 Negatively stained preparations of two isolates cultured during this study which showed high levels of 16S rDNA sequence homology, but were very different morphologically. (A) fusiform bacterium with multiple flagella and periplasmic fibrils (bacterial group A), (B) spiral bacterium with a single polar flagellum (bacterial group D). A B Figure 10.4 Comparison between an isolate investigated in this study from bacterial group H (CMRI2cae) with Helicobacter rodentium. These two organisms share 98.2% 16S rRNA gene sequence similarity. Both are spiral organisms with single, bipolar, unsheathed flagella. Micrograph (B) obtained from reference (149). Bars= 1 µm and 0.2 µm respectively. A B

- Chapter 10 150

As discussed earlier (Chapters 5 and 6) these groups require further investigation, using more sensitive analysis techniques, in order to resolve the taxonomic position of the isolates investigated during this study.

10.1.3 Helicobacters with polymorphic rRNA operons A number of bacterial species have been shown to possess intervening sequences (NSs) of variable length inserted into their rRNA genes (68). Many of the Helicobacter species isolated during the presented work possessed enlarged 16S rRNA genes. Such isolates had in insertion of approximately 200 bp within the first 300 bp from the 5' end of the gene. Such enlarged genes were concluded to be the result of an inserted IVS. Subsequent sequencing of some of the enlarged genes from bacterial groups A, C and D showed they all possessed an inserted IVS almost identical to that found in H. bilis.

The evolutionary significance of intervening sequences (NS) in ribosomal RN:A operons has been speculated on by a number of authors (48, 68, 70, 169). They have been found to be inserted into a limited number of sites within SSU and LSU rRNA genes and are flanked by a common inverted repeat element causing the formation of a stem structure . which is recognizable by RNA processing enzymes (68). These stem structures are recognized and the NS excised during rRNA maturation resulting in the formation of fragmented rRNA molecules which remain so within the ribosome (169). There is no known function attributed to these inserted sequences. A number of Helicobacter species possess similar IVSs inserted into the 16S or 23S rRNA gene including Helicobacter canis, Helicobacter mustelae, Helicobacter pametensis, Helicobacter cinaedi, Helicobacter fennelliae and Helicobacter muridarum (68, 70, 97). These IVSs are not identical, but differ in size between 93-377 bp and possess regions of sequence similarity characterized by potential insertion/deletion events (68). Hurtado et al. speculate that a single ancestral IVS recombined into both SSU and LSU rRNA genes of different species within the epsilon subdivision of the proteobacteria (68). On the other hand, identical NSs have been detected in the 16S rRNA gene of H. bilis and "F. rappini'' prompting Fox et al. to speculate that the NSs were not present in the common ancestor but were acquired more recently by horizontal transfer, since the NS is more conserved than the genes into which it is inserted (48).

In addition to the existence of NSs inserted into the 16S or 23S rDNAs of Helicobacter species, recent studies have shown that it is possible for the different rRNA operons within a single bacterium to be polymorphic for the presence of an NS. This phenomenon has been demonstrated in both Campylobacter 16S rRNAs (98) and Helicobacter 23S rRNAs (68). Results presented in this thesis also show evidence that Chapter 10 151

polymorphic 16S rRNA genes may exist in Helicobacter species. On a speculative note, it is perhaps significant that the isolates which are potentially polymorphic for the presence of an IVS in their 16S rRNA operons came from animal facilities ABHU and UNSW which had wide Helicobacter species diversity. Conversely, mice from facilities CMRI and HRI each contained only one Helicobacter species, neither of which showed any evidence of possessing polymorphic rRNA operons. If the IVS is able to be horizontally transferred, then within the intestinal environment, an identical IVS may insert into other species.

The investigation of these IVSs is an interesting one warranting further study, particularly from an evolutionary point of view. Are they the product of a single insertion event in a common ancestor which _has then evolved along with the genome into which it was inserted giving rise to the differences observed in various IVSs in different species as speculated by Hurtado et al. (68), or are they a result of recent lateral transfer events as proposed by Fox et al. (48)? This study presented evidence to support the presence of polymorphic 16S rRNA operons in some of the isolates obtained during this study. However, closer examination of these organisms is required before they can be conclusively said to contain such polymorphic rRNA genes. Additionally, investigation of the 23S rDNA operons could provide information on the transfer of IVSs between different genes within the same organism. Future investigations on the nature of the IVS in these isolates could also provide information on the ability of these closely related organisms, both phylogenetically and ecologically, to exchange non-plasmid DNA in situ.

10.1.4 Identification of a novel bacterial genus One of the major findings of this thesis was the identification of the bacterial group I isolates. The 16S rRNA gene sequence comparisons revealed that the bacterial group I isolates did not have any meaningful phylogenetic relationship with other intestinal organisms or even with the class Proteobacteria. These results led to the conclusion that this group represents a previously undescribed bacterial genus.

The 16S rRNA gene sequences showed only a moderate phylogenetic affiliation to bacteria found on a separate, relatively recently described, branch of the bacterial phylogenetic tree, possibly a twelfth "phyla". There were three genera of bacteria located in this phylogenetic group, each containing a single species; Flexistipes sinusarabici (35), Geovibrio ferrireducens (16) and Deferribacter thennophilus (60). The most interesting feature uniting these three species, in terms of the hypotheses of this thesis, is that they all have a spiral or curved morphology. Chapter 10 152

One could speculate that the common ancestor to this phylogenetic group of bacteria had a spiral morphology which, in the case of the organisms isolated and described in this thesis, enabled them to colonize the mammalian gastrointestinal tract in the same way in which the ancestral helicobacters did. This would mean that spiral morphology did in fact give a selective advantage to organisms with this morphological.type for colonization of mucosa! surfaces. The organisms observed today represent a number of different phylogenetic lineages which have evolved similar specialized mechanisms to successfully colonize this ecological niche.

10.2 Microbial Ecology: culture vs. nested PCR detection Significant differences were observed between culture and in vitro nested PCR detection of the different bacterial groups investigated in this thesis. The presence of bacteria belonging to the Helicobacter genus as well as three Helicobacter species, H. muridarum, H. bilis (plus bacterial group C) and H. rodentium, and non-helicobacter groups I and J, was investigated by nested PCR amplification of DNA extracted from paraformaldehyde fixed, paraffin embedded tissue specimens.

Helicobacters were detected in all mice, with 71-90% of all samples from the four animal facilities being positive for helicobacter DNA. A large percentage of the helicobacter positive samples (40%) did not contain DNA from any of the three species being tested for. These results are comparable to those obtained by culture where Helicobacter spp. were isolated from 22/23 mice with a large proportion of these also not belonging to these three species (Chapters 4 and 6). At the level of species detection however, discrepancies were evident between culture rates and number of PCR positive samples. For example, H. bilis (plus bacterial group C) was detected by nested PCR in only a very small number of mice (3/23 or 13 % ) whereas these 2 bacterial groups together were cultured from 13/23 (57%) of mice. Conversely, H. muridarum was detected in 14/23 mice (61 % ) by nested PCR and cultured from only 5/23 mice (22% ).

These discrepancies highlight limitations with both of the methods used to detect the presence of helicobacters. Helicobacter species (some more so than others) are not readily cultured. Indeed, there are gastric helicobacters whose presence has been known for many years which still elude laboratory culture (91, 153). The easily cultured H. bi/is was isolated from almost half of the animals used in this study but detected by nested PCR of DNA from fixed tissue specimens infrequently. On the other hand, the more fastidious H. muridarum was detected in fixed samples more frequently than it was cultured. As well as reflecting a difference between these two organisms in ease of Chapter 10 153

culture, these observations may also suggest something of the ecology of these organisms.

During tissue fixation using conventional methods, much of the mucus covering the epithelium is washed away or dissolved more rapidly than it is stabilized (2). This would result in the loss of bacteria which are found in the mucus blanket layer, while bacteria whose preferred ecological niche is at the bottom of the crypts are maintained within the fixed tissue. The combination in a bacterium of being both difficult to culture and inhabiting epithelial crypts in large numbers, for example H. muridarum (94, 131), resulted in these organisms being detected in higher numbers of samples from fixed tissue specimens than they were from culture. Conversely, a bacterium whose preferred ecological niche was within the mucus blanket would not _be detected via nested PCR on fixed samples. It is possible that H. bilis preferentially colonizes this part of the mucus environment, thus explaining its low level of detection in fixed tissue specimens. Differences in ecological niches may similarly explain the discrepancies between culture and nested PCR detection of the two groups of non-helicobacters investigated in the presented study. Group I organisms were isolated from 13/23 (57%) of all mice included, but detected in only 2/23 (9% ). Group J organisms were isolated from 8/23 (35%) of mice, but detected in 100% of mice via nested PCR.

Improved fixation methods are required to overcome this problem of loss of the mucus blanket layer before such a detection method can provide a better way to determine bacterial diversity without the need to culture.

10.3 Spiral Morphology: a new .hypothesis The original working hypothesis of this thesis was that the spiral bacteria ( of the GIT) are phylogenetically related, having evolved with their host to colonize a particular niche. Results presented in this thesis, particularly the phylogenetic characterization of bacterial groups I and J, did not support this hypothesis. If we hypothesize that spiral morphology in bacteria had evolved, probably in several different ways, prior to the evolution of the gastrointestinal tract, then a slightly different hypothesis seems more appropriate.

The hypothesis that spiral morphology gives a selective advantage to bacteria in the successful colonization of the mucus layer of the GIT remains, after consideration of the results presented here, valid. Consider a prehistoric animal possessing an intestinal tract, with a mucus layer covering the "epithelium", picking up bacteria from the environment. Bacteria which were able to resist being merely passed through would be those most able to penetrate the mucus layer, presumably motile organisms with a spiral morphology. If a Chapter 10 154

number of different phylogenetically distinct bacteria, all with spiral morphologies were able to establish themselves then these would then evolve with their hosts into the groups of bacteria we observe today. There is no reason to assume that the "pre-helicobacters" or "pre-epsilon subdivision bacteria" were the only spiral shaped bacteria able to accomplish this close association and evolve to become "autochthonous microbiota".

A new hypothesis is therefore presented that; • the spiral shaped bacteria of the mammalian gastrointestinal tract are found in phylogenetically unrelated (or distantly related) clusters of phylogenetically closely related organisms which have evolved with their host to colonize specialized ecological niches.

So, the wide phylogenetic diversity found in such bacteria originates from the phylogenetic diversity of the initial colonizing bacteria, while the phylogenetic diversity within bacterial groups comes from the bacterial evolution and adaptation which has occurred, in parallel with host evolution, since the initial colonization of the GIT. Using specific examples from results presented in this thesis, the three phylogenetically distinct groups of bacteria which were isolated from mouse gastrointestinal tissue, helicobacters, desulfovibrio and bacterial group I, had diverged prior to their becoming mucus associated intestinal colonizers. The diversity within each of these three groups has arisen from changes which have occurred since these bacteria formed close relationships with their mammalian hosts. An interesting study would be to investigate the presence of bacterial group I in other host species to see if this group of bacteria has diversified into different species, specific to their host. This would represent a similar situation to the way in which different Helicobacter species have been shown to be specific for particular host species, and if shown also to be the case for bacterial group I, it would further support the hypothesis presented here that diversity within phylogenetically related groups is the result of host-associated evolution.

Interestingly, both of the non-helicobacter groups (I and J) were cultured in anaerobic atmospheres, which indicates that they inhabited slightly different ecological niches in vivo to the Helicobacter spp. isolated, which generally preferred a microaerophilic atmosphere. Investigation, using more sensitive techniques than those employed here, of the intestinal tract are required to determine the ecological micro-niches colonized by each of the bacterial groups described in the presented study. Chapter 10 155

Summary of Major Findings 1. A set of mucus associated spiral bacteria was cultured from mouse gastrointestinal tissue. This represents an important collection of isolates, many of which represent novel bacteria. DNA amplification techniques were developed and used for the identification of cultured isolates and a method developed for detection of bacterial groups in DNA extracted from gastrointestinal tissue samples.

2. Helicobacter species made up a significant proportion of isolates. However, many of these did not appear to belong to previously described species. A description of a new species of Helicobacter was presented (Chapter 6) as well as the first reported evidence of Helicobacter species possessing 16S rRNA operons polymorphic for the presence of an intervening sequence (Chapter 4 ).

3. A new genus of bacteria was identified and shown to have a phylogenetic affiliation with the "Flexistipes" group which is only distantly related to any of the other 11 bacterial phyla. These bacteria were isolated from almost 60% of mice. It is hypothesized that this bacterial group represent another example of a phylogenetically related cluster of bacteria which have evolved specialized mechanisms enabling them to colonize the intestinal mucus layer.

4. Significant differences were observed between the bacterial groups isolated from mice obtained from different animal facilities. Such observations could have important implications in many areas of biomedical research.

A new hypothesis about the phylogenetic relatedness of the mucosa associated, spiral bacteria is proposed. • The spiral bacteria of the mammalian gastrointestinal tract are found in phylogenetically unrelated (or distantly related) clusters of phylogenetically closely related organisms which have evolved with their host to colonize specialized ecological niches. Chapter 10 156

Future Work

It remains clear from the results presented in this thesis and previous studies by others that within the complex microbial ecology of the mammalian gastrointestinal tract, spiral bacteria play an important role in the mucus layer covering the epithelium. Studies investigating microbial diversity and ecology in these complex ecosystems should continue.

Specifically related to the results presented in this thesis, the bacterial groups isolated and phylogenetically examined, require further investigation before being taxonomically classified. These studies, as discussed earlier, would involve whole genome studies and detailed biochemicq]_ and physiological examination.

This thesis represents a preliminary study using a limited number of mice to examine not only the original working hypothesis but additionally, the concept of an "autochthonous microbiota". The results obtained and presented in this thesis have meant a new hypothesis regarding phylogenetic diversity of spiral shaped bacteria has been proposed. However, limitations of mouse numbers and some of the methodologies employed meant that the specific questions regarding the mouse "autochthonous microbiota" could not be adequately answered. Improved methods for the detection of organisms without the need for culture are required to determine conclusively the presence of particular organisms. In order to precisely determine which bacterial types are present in particular regions of the gastrointestinal tract (macroscale), and where the preferred ecological niches for different bacterial types, within the mucus milieu, are located (microscale), a two-part investigation is proposed.

Extraction of DNA from fresh tissue specimens would prevent the loss of mucosa associated bacteria which are only found in the surface mucus and not inside the epithelial crypts. Mapping studies using a combination of DNA extraction from fresh tissue and nested PCR detection of different bacterial types could be performed. Taking the entire gastrointestinal tract and dividing it into many small sections, a few centimeters each, would provide a detailed picture, on a macroscale, of the location within the GIT of the different bacterial types. This would however, still tell us very little about the microscale location of these bacteria in association with the structure of the epithelium and mucus lining. Furthermore, without any form of tissue fixation, specimens could not be examined histologically.

Improved methods for tissue fixation which maintain the integrity of the mucus layer have recently been developed (2). Such methods would result in those bacteria, which seemed Chapter 10 157

to be lost during the fixation procedure employed for this study, being maintained within the fixed tissue. Using such methods, in conjunction with in situ hybridization techniques, would facilitate detection of individual cells in association with the mucus layer and epithelial structure. A combination of mapping and microlocalization studies would reveal the ecological niches inhabited by these bacteria and would represent an important study in microbial ecology.

Finally, an important ecological study should include an investigation of the microbial diversity of mucosa-associated spiral bacteria in natural animal populations, for example in wild mice. From an Australian perspective, this could also include comparisons between the microbiota of introduced species verses that found in indigenous small marsupial "mice" which _occupy the same environmental niches. References 158

References

1. Akkermans, A. D. L., Mirza, M. S., Harmsen, H. J. M., Blok, H. J., Herron, P. R., Sessitsch, A. and Akkermans, W. M. 1_994. Molecular ecology of microbes: a review of promises, pitfalls and true progress. FEMS Microbiol Rev. 15: 185-194. 2. Allan-Wojtas, P., Farnworth, E. R., Modler, H. W. and Carbyn, S. 1997. A solvent-based fixative for electron microscopy to improve retention and visualization of the intestinal mucus blanket for probiotics studies. Microscop Res Tech. 36: 390-399. 3. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. 1990. Basic local alignment search tool. J Mol Biol. 215: 403-410. 4. Amann, R. I., Krumholz, L. and Stahl, D. A. 1990. Fluorescent­ oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 172: 762-770. 5. Amann, R. I., Ludwig, W. and Schleifer, K. H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 59: 143-169. 6. Archer, J. R., Romero, S., Ritchie, A. E., Hamacher, M. E., Steiner, B. M., Bryner, J. H. and Schell, R. F. 1988. Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis. J Clin Microbiol. 26: 101-105. 7. Ash, C., Farrow, J. A. E., Wallbanks, S. and Collins, M. D. 1991. Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small subunit ribosomal RNA sequences. Lett Appl Microbiol. 13: 202-206. 8. Battles, J. K., Williamson, J. C., Pike, K. M., Gorelick, P. L., Ward, J.M. and Gonda, M.A. 1995. Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene. J Clin Microbiol. 33: 1344-1347. 9. Beerens, E. J. and Romond, H. 1977. Sulfate-reducing anaerobic bacteria in human faeces. Am J Clin Nutr. 30: 1770-1776. 10. Bizzozero, G. 1893. Ueber die schauchformigen drusen des magendarmk.anals und die bezeihungan ihres epithels zu dem oberflachenepithel der schleimhaut. Arch Mikr Anast. 42: 82. 11. Britschgi, T. B. and Fallon, R. D. 1994. PCR-amplification of mixed 16S rRNA genes from an anaerobic, cyanide-degrading consortium. FEMS Microbiol Ecol. 13: 225-231. 12. Brock, T. D. 1966. Principles of microbial ecology, p. Pages. Prentice-Hall, Englewood Cliffs, New Jersey. 159

13. Bronsdon, M. A., Goodwin, C. S., Sly, L. I., Chilvers, T. and Schoenknecht, F. D. 1991. Helicobacter nemestrinae sp. nov., a spiral bacterium found in the stomach of a pigtailed macaque (Macaca nemestrina ). Int J Syst Bacteriol. 41: 148-153. 14. Brosius, J., Palmer, M. L., Kennedy, P. J. and Noller, H. F. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci USA. 75: 4801-4805. 15. Burnens, A. P., Stanley, J., Schaad, U. B. and Nicolet, J. 1993. Novel Campylobacter-like organism resembling Helicobacter fennelliae isolated from a boy with gastroenteritis and from dogs. J Clin Microbiol. 31: 1916-1917. 16. Caccavo Jr, F., Coates, J. D., Rossello-Mora, R. A., Ludwig, W., Schleifer, K. H., Lovley, D. R. and Mcinerney, M. J. 1996. Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe(ill)-reducing bacterium. Arch Microbiol. 165: 370-376. 17. Cano, R. J., Borucki, M. K., Higby-Schweitzer, M., Poinar, H. N., Poinar Jr, G. 0. and Pollard, K. J. 1994. Bacillus DNA is fossil bees: an ancient symbiosis? Appl Environ Microbiol. 60: 2164-2167. 18. Cooper, D. M., Swanson, D. L., Barns, S. M. and Gebhart, C. J. 1997. Comparison of the 16S ribosomal DNA sequences from the intracellular agents of proliferative enteritis in a hamster, deer, and ostrich with the sequence of a porcine isolate of Lawsonia intracellularis. lnt J Syst Bacteriol. 47: 635-639. 19. Davis, C. P., Mulcahy, D., Takeuchi, A. and Savage, D. C. 1972. Location and description of spiral-shaped microorganisms in the normal rat cecum. Infect lmmun. 6: 184-192. 20. DeLey, J. 1992. The Proteobacteria: ribosomal RNA cistron similarities and bacterial taxonomy, p. 2111-2140. In A. Balows, H. G. Truper, M. Dworkin, W. Harder and K. H. Schleifer (ed.), The prokaryotes, 2. Springer-Verlag, New York. 21. Dewhirst, F. E., Seymour, C., Fraser, G. J., Paster, B. J. and Fox, J. G. 1994. Phylogeny of Helicobacter isolates from bird and swine feces and description of Helicobacter pametensis sp. nov. Int J Syst Bacteriol. 44: 553-560. 22. Doenges, J. L. 1939. Spirochetes in the gastric glands of Macacus rhesus and of man without related disease. Arch Pathol Lab Med. 27: 469-477. 23. Drolet, R., Larochelle, D. and Gebhart, C. J. 1996. Proliferative enteritis in white-tailed deer. J Vet Diagn Invest. 8: 250-253. 24. Dobos, R., Schaedler, R. W., Costello, R. and Hoet, P. 1965. Indigenous, normal, and autochthonous flora of the gastrointestinal tract. J Exp Med. 122: 67-76. References 160

25. Eaton, K. A., Dewhirst, F. E., Paster, B. J., Tzellas, N., Coleman, B. E., Paola, J. and Sherding, R. 1996. Prevalence and varieties of Helicobacter species in dogs from random sources and pet dogs: an_imal and public health implications. J Clio Microbiol. 34: 3165-3170. 26. Eaton, K. A., Dewhirst, F. E., Radin, M. J., Fox, J. G., Paster, B. J., Krakowka, S. and Morgan, D. R. 1993. Helicobacter acinonyx sp.nov., isolated from cheetahs with gastritis. Int J Syst Bacteriol. 43: 99-106. 27. Eckloff, B. W., Podzorski, R. P., Kline, B. C. and Cockerill, F. R. I. 1994. A comparison of 16S ribosomal DNA sequences from five isolates of . Helicobacter pylori. Int J Syst Bacteriol. 44: 320-323. 28. Edwards, U., Rogall, T., Blocker, H., Emde, M. and Bottger, E. C. 1989. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 17: 7843- 7853. 29. Erlandsen, S. L. and Chase, D. G. 1972. Paneth call function: phagocytosis and intracellular digestion of intestinal microorganisms II. Spiral microorganisms. J Ultrastruct Res. 41: 319-333. 30. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol. 17: 368-376. 31. Felsenstein, J. 1985. Confidence limits on phylogenetics: an approach using the bootstrap. Evolution. 39: 783-791. 32. Felsenstein, J. 1989. PHYLIP. Phylogeny interface package. Cladistics. 5: 164- 166. 33. Fennell, C. L., Totten, P. A., Quinn, T. C., Patton, D. L., Holmes, K. K. and Stamm, W. E. 1984. Characterization of Campylobacter-like organisms isolated from homosexual men. J Infect Dis. 149: 58-66. 34. Ferrero, R. L. and Lee, A. 1988. Motility of Campylobacter jejuni in a viscous environment: comparison with conventional rod-shaped bacteria. J Gen Microbiol. 134: 53-59. 35. Fiala, G., Woese, C. R., Langworthy, T. A. and Stetler, K. 0. 1990. Flexistipes sinusarabici, a novel genus and species of eubacteria occurring in the Atlantis II deep brines of the Red Sea. Arch Microbiol. 154: 120-126. 36. Fitch, W. M. 1971. Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool. 20: 406-416. 37. Fitch, W. M. and Margoliash, E. 1967. Construction of phylogenetic trees. Science. 155: 279-284. 38. Fox, G. E., Pechman, K. R. and Woese, C. R. 1977. Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics. lot J Syst Bacteriol. 27: 44-57. 161

39. Fox, G. E., Wisotzkey, J. D. and Jurtshuk Jr, P. 1992. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. lnt J Syst Bacteriol. 42: 166-170. 40. Fox, J. C., Correa, P., Taylor, N. S., Zavala, D., Fontham, E., Janney, F., Rodriquez, E., Hunter, F. and Diavolitsis, S. 1989. Campylobacter pylori associated gastritis and immune response in a population at increased risk of gastric carcinoma. Am J Gastroenterol. 89: 775-781. 41. Fox, J. G., Dewhirst, F. E., Fraser, G. J., Paster, B. J., Shames, B. and Murphy, J.C. 1994. Intracellular Campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a Desulfovibrio sp. J Clin Microbiol. 32: 1229-1237. 42. Fox, J. G., Dewhirst, F. E., Tully, J. G., Paster, B. J., Yan, L., Taylor, N. S., Collins, M. J., Gorelick, P. L. and Ward, J. M. 1994. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol. 32: 1238-1245. 43. Fox, J. G., Drolet, R., Higgins, R., Messier, R., Yan, L., Coleman, B. E., Paster, B. J. and Dewhirst, F. E. 1996. Helicobacter canis isolated from a dog liver with multifocal necrotizing hapatitis. J Clin Microbiol. 34: 2479-2482. 44. Fox, J. G., Paster, B. J., Dewhirst, F. E., Taylor, N. S., Yan, L. L., Macuch, P. J. and Chmura, L. M. 1992. Helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmision of a gastric helicobacter. Infect Immun. 60: 606-611. 45. Fox, J. G., Taylor, N. S., Edmonds, P. and Brenner, D. J. 1988. Campylobacter pylori subsp. mustelae subsp. nov. isolated from the gastric mucosa of ferrets (Mustelae putorius furo ), and an emended description of Campylobacter pylori. Int J Syst Bacteriol. 38: 367-370. 46. Fox, J. G. and Wang, T. C. 1997. Helicobacter and liver disease. ltal J Gastroenterol Hepatol. 29: 5-12. 47. Fox, J. G., Yan, L., Shames, B., Campbell, J., Murphy, J. C. and Li, X. 1996. Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Infect Immun. 64: 3673-3681. 48. Fox, J. G., Yan, L. L., Dewhirst, F. E., Paster, B. J., Shames, B., Murphy, J. C., Hayward, A., Belcher, J. C. and Mendes, E. N. 1995. Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice. J Clin Microbiol. 33: 445-454. References 162

49. Franklin, C. L., Beckwith, C. S., Livingston, R. S., Riley, L. K., Gibson, S. V., Besch-Williford, C. L. and Hook Jr, R. R. 1996. Isolation of a novel Helicobacter species, Helicobacter cholecystus sp. nov., from the gallbladders of Syrian hamsters with cholangiofibrosis and centrilobular pancreatitis. J Clin Microbiol. 34: 2952-2958. 50. Gebhart, C. J., Barns, S. M., McOrist, S., Lin, G. and Lawson, C. H. K. 1993. Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a realtionship to Desulfovibrio species. Int J Syst Bacteriol. 43: 533- 538. 51. Gebhart, C. J., Fennell, C. L. and Murtaugh, M. P. 1989. Campylobacter cinaedi is normal intestinal flora in hamsters. J Clin Microbiol. 27: 1692-1694. 52. 53. Gibson, G. R., Cummings, J. H. and MacFarland, G. T. 1988. Competition of hydrogen between sulfate-reducing bacteria and methanogenic bacteria from the humanlarge intestine. J Appl Bacteriol. 54. Giovannoni, S. J., Britschgi, T. B., Moyer, C. L. and Field, K. G. 1990. Genetic diversity in Sargasso Sea bacterioplankton. Nature. 345: 60-63. 55. Giovannoni, S. J., DeLong, E. F., Olsen, G. J. and Pace, N. R. 1988. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 170: 720-726. 56. Gobel, U. B. 1995. Phylogenetic amplification for the detection of uncultured bacteria and the analysis of complex microbiota. J Microbiol Meth. 23: 117-128. 57. Goodwin, C. S., Armstrong, J. A., Chilvers, T., Peters, M., Collins, M. D., Sly, L., McConnell, W. and Harper, W. E. 1989. Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter pylori gen. nov. and Helicobacter mustelae comb. nov. respectively. Int J Syst Bacteriol. 39: 397-405. 58. Gordon, J. H. and Dobos, R. 1970. The anaerobic bacterial flora of the mouse cecum. J Exp Med. 131: 251-260. 59. Graham, D. 1989. Campylobacter pylori and peptic ulcer disease. Gastroenterology. 96: 615-625. 60. Greene, A. C., Patel, B. K. and Sheehy, A. J. 1997. Deferribacter thermophilus gen. nov., sp. nov., a novel thermophilic manganese- and iron-reducing bacterium isolated from a petroleum reservoir. lnt J Syst Bacteriol. 47: 505-509. 61. Gustafsson, B. E. and Maunsbach, A. B. 1971. Ultrastructure of the enlarged cecum in germfree rats. Z Zellforsch. 120: 555-578. 62. Gutell, R. R., Larsen, N. and Woese, C. R. 1994. Lessons from an evolving rRNA: 16S and 23S rRNA structures frona comparative perspective. Microbiol Rev. 58: 10-26. 163

63. Hanninen, M. L., Happonen, I. and Saari, S. 1996. Culture and characteristics of Helicobacter biu,ozeronii, a new canine gastric Helicobacter sp. Int J Syst Bacteriol. 46: 160-166. 64. Hazell, S. L., Lee, A., Brady, L. and Hennessy, W. 1986. Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. J Infect Dis. 153: 658-663. 65. Henrikson, R. C. 1973. Ultrastructural aspects of mouse cecal epithelium. Z Zellforsch. 140: 445-449. 66. Howard, B. H. and Hungate, R. E. 1976. Desulfovibrio of the sheep rumen. Appl Environ Microbiol. 32: 589-602. 67. Huber, R., Wilharm, T., Huber, D., Trincone, A., Burggraf, S., Koenig, H., Rachel, R., Rockinger, I., Fricke, H. and Stetter, K. 0. 1992. Aquifex pyrophilus new-genus new-species represents a novel group of marine hyperthermophilic hydrogen-oxidizing bacteria. Syst Appl Microbiol. 15: 340-351. 68. Hurtado, A., Clewley, J. P., linton, D., Owen, R. J. and Stanley, J. 1997. Sequence similarities between large subunit ribosomal RNA gene intervening sequences from different Helicobacter species. Gene. 194: 69-75. 69. Hurtado, A. and Owen, R. J. 1997. A molecular scheme based on 23S rRNA gene polymorphisms for rapid identification of Campylobacter and Arcobacter species. J Clin Microbiol. 35: 2401-2404. 70. Hurtado, A. and Owen, R. J. 1997. A rapid identification scheme for Helicobacter pylori and other species of Helicobacter based on 23S rRNA gene polymorphisms. System Appl Microbiol. 20: 222-231. 71. Isola, J., DeVries, S., Chu, L., Ghazvini, S. and Waldman, F. 1994. Analysis of changes in DNA sequence copy number by comparative genomic hybridization in aechival paraffin-embedded tumor samples. Am J Pathol. 145: 1301- 1308. 72. Jalava, K., Kaartinen, M., Utriainen, M., Happonen, I. and Hanninen, M.-L. 1997. Helicobacter salomonis sp. nov., a canine gastric Helicobacter sp. related to Helicobacter felis and Helicobacter biu,ozeroni. Int J Syst Bacteriol. 47: 975-982. 73. Jukes, T. H. and Cantor, C. R. 1969. Evolution of protein molecules, p. 21- 132. In H. N. Munro (ed.), Mammalian protein metabolism, 3. Academic Press, Inc., New York. 74. Kimura, M. 1987. Molecular evolutionary clock and the neutral theory. J Mol Evol. 26: 24-33. References 164

75. Kirkbride, C. A., Gates, C. E., Collins, J. E. and Ritchie, M. S. 1985. Ovine abortion associated with an anaerobic bacterium. J Am Vet Med Assoc. 186: 789- 791. 76. Kolmer, W. and Wagner, R. J. 1916. Uber eine im magenfundus des hundes vorkomrnende saprophytische spirochate. Centralbl f Bakt (Abt I). 78: 382-383. 77. Kuhnigk, T., Branke, J., Krekeler, D., Cypionka, H. and Konig, H. 1996. A reasible role of sulfate-reducing bacteria in the termite gut. Syst Appl Microbiol. 19: 139-149. 78. Lane, D. L., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. and Pace, N. R. 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA. 82: 6955-6959. 79. Laue, H., Denger, K. and Cook, A. M. 1997. Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU. Appl Environ Microbiol. 63: 2016- 2021. 80. Leach, W. D. 1977. MSc. UNSW, Sydney. 81. Leach, W. D., Lee, A. and Stubbs, R. P. 1973. localization of bacteria in the gastrointestinal tract: a possible explanation of intestinal spirochaetosis. Infect Imrnun. 7: 961-972. 82. Lee, A. 1980. Normal flora of animal intestinal surfaces, p. 145-173. In G. Bitton and K. C. Marshall (ed.), Adsorption of microorganisms to surfaces, Wiley, New York. 83. Lee, A. 1985. Neglected niches: the microbial ecology of the gastrointestinal tract, p. 115-162. In K. C. Marshall (ed.), Advances in Microbial Ecology, 8. Plenum Press, New York. 84. Lee, A. 1989. Campylobacter pylori and CLO in animals: overview of mucus­ colonizing organisms, p. 246-260. In B. J. Rathbone and R. V. Heatley (ed.), Campylobacter pylori and gastroduodenal disease, Blackwell Scientific Publications, Oxford. 85. Lee, A. 1991. Spiral organisms: what are they? A microbiologic introduction to Helicobacter pylori. Scand J Gastroenterol. 26 Supplement 187: 9-22. 86. Lee, A., Chen, M. H., Coltro, N., O'Rourke, J., Hazell, S., Hu, P. J. and Li, Y. Y. 1993. Long term infection of the gastric mucosa with Helicobacter species does induce atrophic gastritis in an animal model of Helicobacter pylori infection. Zbl Bakt (lnt J Med Microbiol). 280: 38-50. 87. Lee, A., Eckstein, R. P., Fevre, D. I., Dick, E. and Kellow, J. E. 1989. Non Campylobacter pylori spiral organisms in the gastric antrum. Aust NZ J Med. 19: 156-158. 88. Lee, A., Fox, J. and Hazell, S. 1993. Pathogenicity of Helicobacter pylori - a perspective. Infect Imrnun. 61: 1601-1610. 165

89. Lee, A., Gordon, J. and Dobos, R. 1968. Enumeration of the oxygen sensitive bacteria usually present in the intestine of healthy mice. Nature (London). 220: 1137-1139. 90. Lee, A., Hazell, S. L. and O'Rourke, J. L. 1988. Isolation of a spiral­ shaped bacterium from the cat stomach. Infect Iminun. 56: 2843-2850. 91. Lee, A. and O'Rourke, J. 1993. Gastric bacteria other than Helicobacter pylori. Gastroenterol Clinics Nth Am. 22: 21-42. 92. Lee, A. and O'Rourke, J. 1996. The Helicobacter felis mouse model, p. 188- 203. In A. Lee and F. Megraud (ed.), Helicobacter pylori: techniques for clinical and basic research, WB Saunders, London. 93. Lee, A., O'Rourke, J., DeUngria, M. C., Robertson, B., Daskalopolous, G. and Dixon, M. F. 1997. A standardardized mouse model of Helicobacter pylori infection: Introducing the Sydney strain. Gastroenterology. 112: 1386-1397. 94. Lee, A., Phillips, M. W., O'Rourke, J. L., Paster, B. J., Dewhirst, F. E., Fraser, G. J., Fox, J. G., Sly, L. I., Romaniuk, P. J., Trust, T. J. and Kouprach, S. 1992. Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. Int J Syst Bacteriol. 42: 27-36. 95. Li, C., Ha, T., Ferguson, D. A., Chi, D. S., Zhao, R., Patel, N. R., Krishnaswamy, G. and Thomas, E. 1996. A newly developed PCR assay of H. pylori in gastric biopsy, saliva and feces. Digest Dis Sci. 41: 2142-2149. 96. Lim, R. K. S. 1920. A prrasitic spiral organism in the stomach of the cat. Parasitology. 12: 108-112. 97. Linton, D., Clewley, J. P., Burnens, A., Owen, R. J. and Stanley, J. 1994. An intervening sequence (IVS) in the 16S rRNA gene of the eubacterium Helicobacter canis. Nucleic Acids Res. 22: 1954-1958. 98. Linton, D., Dewhirst, F. E., Clewley, J. P., Owen, R. J., Burnens, A. P. and Stanley, J. 1994. Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterisation of the intervening sequence found in some strains. Microbiology. 140: 847-855. 99. Loy, J. K., Dewhirst, F. E., Weber, W., Frelier, P. F., Garbar, T. L., Tasca, S. I. and Templeton, J. W. 1996. Molecular phylogeny and in situ detection of the etiologic agent of necrotizing hepatopancreatitis in shrimp. Appl Environ Microbiol. 62: 3439-3445. 100. Ludwig, W. and Schleifer, K. H. 1994. Bacterial phylogeny based on 16S and 23S rRNA sequence analysis. FEMS Microbiol Rev. 15: 155-173. References 166

101. Marshall, B. J., Royce, H., Annear, D. I., Goodwin, C. S., Pearman, J. W., Warren, J. R. and Armstrong, J. A. 1984. Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbiol Lett. 25: 83-88. 102. Marshall, B. J. and Warren, J. R. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1: 1311-1314. 103. McOrist, S., Gebhart, C. J., Boid, R. and Barns, S. M. 1995. Characterization of I...awsonia intracellularis gen.nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy. Int J Syst Bacteriol. 45: 820-825. 104. Mendes, E. N., Queiroz, D. M. M., Dewhirst, F. E., Paster, B. J., . Moura, S. B. and Fox, J. G. 1996. Helicobacter trogontum sp. nov., isolated from the rat intestine. Int J Syst Bacteriol. 46: 916-921. 105. Mitchell, H. M. 1993. The epidemiology of Helicobacter pylori infection and its relation to gastric cancer, p. 95-114. In C. S. Goodwin and B. W. Wormsley (ed.), Helicobacter pylori: biology and clinical practice, CRC Press, Florida. 106. Monstein, H., Kihlstrom, E. and Tiveljung, A. 1996. Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes. APMIS. 104: 451-458. 107. Moore, W. E. C. and Holdeman, L. V. 1974. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol. 27: 961-979. 108. Morris, A., Ali, M. R., Thomsen, L. and Hollis, B. 1990. Tightly spiral shaped bacteria in the human stomach: another cause of active chronic gastritis? Gut. 31: 139-143. 109. Moyer, C. L., Dobbs, F. C. and Karl, D. M. 1994. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Appl Environ Microbiol. 60: 871-879. 110. Mullis, K. B. and Faloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaaction. Methods Enzymol. 155: 335-350. 111. Murray, R. G. E., Brenner, D. J., Colwell, R. R., De Vos, P., Goodfellow, M., Grimont, P. A. D., Pfennig, N., Stackebrandt, E. and Zavarzin, G. A. 1990. Report of the ad hoe committee on approaches to taxonomy within the Proteobacteria. Int J Syst Bacteriol. 40: 213-215. 112. Neilan, B. A., Cox, P. T., Hawkins, P. R. and Goodman, A. E. 1994. 16S ribosomal RNA gene sequence and phylogeny of toxic Microcystis sp. (Cyanobacteria). DNA Sequence. 4: 333-337. 167

113. Neilan, B. A., Jacobs, D., Del Dot, T., Blackall, L. L., Hawkins, P. R., Cox, P. T. and Goodman, A. E. 1997. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int J Syst Bacteriol. 47: 693-697. 114. O'Rourke, J., Lee, A. and MeNeill, J. 1988. Differences in the gastrointestinal microbiota of specific pathogen free mice: an often unknown variable in biomedical research. Lab Animals. 22: 297-303. 115. Olsen, G. J., Lane, D. L., Giovannoni, S. J. and Pace, N. R. 1986. Microbial ecology and evolution: a ribosomal RNA approach. Ann Rev Microbiol. 40: 337-365. 116. Olsen, G. J., Larsen, N. and Woese, C. R. 1991. The ribosomal RNA database project. Nucleic Acids Res. 19 (Suppl.): 2017-2018. 117. Olsen, G. J., Overbeek, R., Larsen, N., Marsh, T. L., Mccaughey, M. J ., Maciukenas, M. A., Kuan, W., Macke, T. J ., Xing, Y. and Woese, C.R. 1992. The ribosomal database project. Nucleic Acids Res. 20 (Suppl.): 2199-2200. 118. Olsen, G. J. and Woese, C. R. 1993. Ribosomal RNA: a key to phylogeny. FASEB J. 7: 113-123. 119. On, S. L. W. 1996. Identification methods for campylobacters, helicobacters, and related organisms. Clin Microbiol Rev. 9: 405-422. 120. Oyaizu, H., DeBrunner-Vossbrink, B. A., Mandelco, L., Studier, J. A. and Woese, C. R. 1987. The green non-sulfur bacteria: a deep branching in the eubacterial line of descent. Syst Appl Microbiol. 9: 47-53. 121. Oyaizu, H. and Woese, C. R. 1985. Phylogenetic relationships among the sulfate respiring bacteria, myxobacteria and purple bacteria. Syst Appl Microbiol. 6: 257- 263. 122. Pace, N. R., Stahl, D. A., Lane, D. J. and Olsen, G. J. 1986. The analysis of natural microbial populations by ribosomal RNA sequences, p. 1-55. In K. C. Marshall (ed.), Advances in Microbial Ecology, 9. Plenum Press, New York. 123. Palys, T., Nakamura, L. K. and Cohan, F. M. 1997. Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. Int J Syst Bacteriol. 47: 1145-1156. 124. Parsonnet, J., Friedman, G. D., Vandersteen, D. P., Chang, J. H., Vogelman, J. H., Orentriech, N. and Sibley, R. K. 1991. Helicobacter pylori infection and the risk of gastric adenocarcinoma. N Engl J Med. 325: 1127-1131. 125. Paster, B. J., Dewhirst, F. E., Weisburg, W. G., Tordoff, L. A., Fraser, G. J., Hespell, R. B., Stanton, T. B., Zablen, L., Mandelco, L. and Woese, C.R. 1991. Phylogenetic analysis of spirochetes. J Bacteriol. 173: 6101-6109. References 168

126. Paster, B. J., Lee, A., Fox, J. G., Dewhirst, F. E., Tordoff, L. A., Fraser, G. J., O'Rourke, J. L., Taylor, N. S. and Ferrero, R. 1991. Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria. Int J Syst Bacteriol. 41: 31-38. 127. Patel, B. K. C., Saul, D. S., Reeves, R. A., Williams, L. C., Cavanagh, J.-E., Nichols, P. D. and Bergquist, P. L. 1994. Phylogeny and lipid composition of Thermonema lapsum, a therophilic gliding bacterium. FEMS Microbiol Lett. 115: 313-317. 128. Peace, T. A., Brock, K. V. and Stills, H. F. 1994. Comparative analysis of the 16S rRNA gene sequence of the putative agent of proliferative ileitis of hamsters. lot J Syst Bscteriol. 44: 832-835. 129. Peng, H. Z., Isaacson, P. G., Diss, T. C. and Pan, L. X. 1994. Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification. J Clio Pathol. 47: 605-608. 130. Phillips, M., Lee, A. and Leach, W. D. 1978. The mucosa-associated microflora of the rat intestine. AJEBAK. 56: 649-662. 131. Phillips, M. W. and Lee, A. 1983. Isolation and characterization of a spiral. bacterium from the crypts of rodent gastrointestinal tracts. Appl Environ Microbiol. 45: 675-683. 132. Queiroz, D. M. M., Contigli, C., Coimbra, R. S., Nogueira, A. M. M. F., Mendes, E. N., Rocha, G. A. and Moura, S. B. 1992. Spiral bacterium associated with gastric, ileal and caecal mucosa of mice. Lab Anim. 26: 288- 294. 133. Rappin, J.P. 1881. Contra l'etude de bacterium de la bouche a l'etat normal, p. 68. In Q. by, R. S. Breed, E. G. D. Murray and A. P. Hitchens (ed.), Bergey's manual of determinative bacteriology, Williams & Wilkins Co., Baltimore. 134. Redburn, A. C. and Patel, B. K. 1993. Phylogenetic analysis of Desulfotomaculum thermobenzaicum using polymerase chain reaction-amplified 16S rRNA-specific DNA. FEMS Microbiol Lett. 113: 81-86. 135. Reiman, D. A. 1993. The identification of uncultured microbial pathogens. J Infect Dis. 168: 1-8. 136. Reiman, D. A., Loutit, J. S., Schmidt, T. M., Falkow, S. and Tomkins, L. S. 1990. The agent of bacillary angiomatosis: an approach to the isentification of uncultured pathogens. N Engl J Med. 323: 1573-1580. 137. Riley, L. K., Franklin, C. L., Hook Jr, R. R. and Besch-Williford, C. 1996. Identification of murine helicobacters by PCR and restriction enzyme analyses. J Clio Microbiol. 34: 942-946. 169

138. Romero, S., Archer, J. R., Hamacher, M. E., Bologna, S. M. and Schell, R. F. 1988. Case report of an unclassified microaerophilic bacterium associated with gastroenteritis. J Clin Microbiol. 26: 142-143. 139. Rowland, A. C. and Lawson, G. H. K. 1992. Porcine proliferative enteropathies, p. 560-569. In A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire and D. J. Taylor (ed.), Diseases of swine, Iowa State University Press, Ames. 140. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4: 406-425. 141. Salomon, H. 1896. Ueber das spirillum des saugetiermagens und sen verhalten zu den Belegzellen. Zentralbl Bakteriol. 19: 422-441. 142. Savage, D. C. and Blumershine, R. V. H. 1974. Surface-surface associations in microbial communities populating epithelial habitats in the murine gastrointestinal ecosystem: scanning electron microscopy. Infect Immun. 10: 240-250. 143. Savage, D. C., Dobos, R. and Schaedler, R. W. 1968. The gastrointestinal epithelium and its autochthonous bacterial flora. J Exp Med. 127: 67-76. 144. Savage, D. C., McAllister, J. S. and Davis, C. P. 1971. Anaerobic bacteria on the mucosa! epithelium of the murine large bowel. Infect Immun. 4: 492-502. 145. Schauer, D. B., Ghori, N. and Falkow, S. 1993. Isolation and characterization of "Flexispira rappini" from laboratory mice. J Clin Microbiol. 31: 2709- 2714. 146. Schmidt, T. M., Pace, B. and Pace, N. R. 1991. Detection of DNA contamination in Taq polymerase. Biotechniques. 11: 176-177. 147. Seymour, C., Lewis, R. G., Kim, M., Gagnon, D. F., Fox, J. G., Dewhirst, F. E. and Paster, B. J. 1994. Isolation of Helicobacter strains from wild bird and swine feces. Appl Environ Microbiol. 60: 1025-1028. 148. Shames, B., Fox, J. G., Dewhirst, F., Yan, L., Shen, Z. and Taylor, N. S. 1995. Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay. J Clin Microbiol. 33: 2968-2972. 149. Shen, Z., Fox, J. G., Dewhirst, F. E., Paster, B. J., Foltz, C. J., Yan, L., Shames, B. and Perry, L. 1997. Helicobacter rodentium sp. nov., a urease-negative Helicobacter species isolated from laboratory mice. Int J Syst Bacteriol. 47: 627-634. 150. Shomer, N. H., Dangler, C. A., Schrenzel, M. D. and Fox, J. G. 1997. Helicobacter bi/is-induced inflammatory bowel disease in scid mice with defined flora. Infect Immun. 65: 4858-4864. 151. Singleton, P. and Sainsbury, D. 1994. Dictionary of microbiology and molecular biology, p. Pages. John Wiley and Sons Ltd, Chichester. References 170

152. Sly, L. I., Bronsdon, M. A., Bowman, J. P., Holmes, A. and Stackebrandt, E. 1993. The phylogenetic position of Helicobacter nemestrinae. Int J Syst Bacteriol. 43: 386-387. 153. Solnick, J. V., O'Rourke, J., Lee, A., Paster, B. J., Dewhirst, F. E. and Tompkins, L. S. 1993. An uncultured gastric spiral organism is a newly identified helicobacter in humans. J Infect Dis. 168: 379-385. 154. Stackebrandt, E. and Goebel, B. M. 1994. A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bact. 44: 846-849. 155. Stackebrandt, E. and Woese, C. R. 1984. The phylogeny of prokaryotes. Microbiol Sci. 1: 117-122. 156. Stanley, J., Linton, D., Burnens, A. P., Dewhirst, F. E., On, S. L. W., Porter, A., Owen, R. J. and Costas, M. 1994. Helicobacter pullorum sp. nov. -genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis. Microbiology-UK. 140: 3441-3449. 157. Stanley, J., Linton, D., Burnens, A. P., Dewhirst, F. E., Owen, R. J., Porter, A., On, S. L. W. and Costas, M. 1993. Helicobacter canis sp. nov., a new species from dogs: an integrated study of phenotype and genotype. J Gen Micorbiol. 139: 2495-2504. 158. Stanton, T. B., Jensen, N. S., Casey, T. A., Tordoff, L. A., Dewhirst, F. E. and Paster, B. J. 1991. Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innoces comb. nov. Int J Syst Bacteriol. 41: 50- 58. 159. Steffan, R. J. and Atlas, R. M. 1991. Polymerase chain reaction: applications in environmental microbiology. Ann Rev Microbiol. 45: 137-161. 160. Steinbrueckner, B., Haerter, G., Pelz, K., Weiner, S., Rump, J.-A., Deissler, W., Bereswill, S. and Kist, M. 1997. Isolation of Helicobacter pullorum from patients with enteritis. Scand J Infect Dis. 29: 315-318. 161. Stills, H. F., Hook, R. R. and Kinden, D. A. 1989. Isolation of Campylobacter-like organisms from healthy Syrian hamsters (Mesocricetus auratus). J Clin Microbiol. 27: 1497-2501. 162. Talley, N. J. and Noack, K. B. 1993. The worldwide prevalence of Helicobacter pylori: asymptomatic infection and clinical states associated with infection in adults, p. 15-35. In C. S. Goodwin and B. W. Worsley (ed.), Helicobacter pylori Biology and Clinical Practice, CRC Press, Boca Raton. 163. Tee, W., Dyall-Smith, M., Woods, W. and Eisen, D. 1996. Probable new species of Desulfovibrio isolated from a pyogenic liver abscess. J Clin Microbiol. 34: 1760-1764. 171

164. Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalities and weight matrix choice. Nucleic Acids Res. 22: 4673-4680. 165. Thompson, L. M. I., Smibert, R. M., Johnson, J. L. and Krieg, N. R. 1988. Phylogenetic study of the genus Campylobacter. Int J Syst Bacteriol. 38: 190- 200. 166. Totten, P. A., Fennel, C. L., Tenover, F. C., Wezenberg, J. M., Perine, P. L., Stann, W. E. and Holmes, K. K. 1985. Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.); two new Campylobacter species associated with enteric disease in homosexual men. J Infect Dis. 151: 131-139. 167. Trexler, P. C. 1987. Animals of defined microbiological status, p. 85-98. In T. Poole (ed.), The UF AW handbook on the care and management of laboratory animals, Langman Scientific and Technical, Harlow. 168. Trivett-Moore, N. L., Rawlinson, W. D., Yuen, M. and Gilbert, G. L. 1997. Helicobacter westmeadii sp. nov., a new species isolated from bloos cultures of two AIDS patients. J Clin Microbiol. 35: 1144-1150. 169. Trust, T. J., Logan, S. M., Gustafson, C. E., Romaniuk, P. J., Kim, N. W., Chan, V. L., Ragan, M. A., Guerry, P. and Gutell, R. R. 1994. Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp. J Bacteriol. 176: 4597-4609. 170. Ursing, J.B., Lior, H; and Owen, R. J. 1994. Proposal of minimal standards for describing new species of the family Campylobacteraceae. Int J Syst Bacteriol. 44: 842-845. 171. Van de Peer, Y., Caers, A., De Rijk, P. and De Wachter, R. 1998. Database on the structure of small ribosomal subunit RNA. Nucl Acids Res. 26: 179- 182. 172. Vandamme, P. and DeLey, J. 1991. Proposal for a new family, Campylobacteraceae. Int J Syst Bacteriol. 41: 451-455. 173. Vandamme, P., Falsen, E., Rossau, R., Hoste, B., Segers, P., Tytgat, R. and J., D. 1991. Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int J Syst Bacteriol. 41: 88-103. 17 4. Vandamme, P ., Pot, B., Gillis, M., Devos, P ., Kersters, K. and Swings, J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev. 60: 407-438. References 172

175. Voordouw, G. 1995. The genus Desulfovibrio: the centennial. Appl Environ Microbiol. 61: 2813-2819. 176. Ward, D. M., Bateson, M. M., Weller, R. and Ruff-Roberts, A. L. 1992. Ribosomal RNA analysis of microorganisms as they occur in nature, p. 219-286. In K. C. Marshall (ed.), Advances in microbial ecology, 12. Plenum Press, New York. 177. Ward, J. M., Anver, M. R., Haines, D. C. and Benveniste, R. E. 1994. Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am J Pathol. 145: 959-968. 178. Ward, J. M., Fox, J. G., Anver, M. R., Haines, D. C., George, V. C., Collins Jr, M. J., Gorelick, P. L., Nagashima, K., Gonda, M. A., Gilden, R. V., Tully, J. G., Russel, R. J., Benveniste, R. E., Paster, B. J., Dewhirst, F. E., Donovan, J. C., Anderson, L. M. and Rice, J. M. 1994. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst. 86: 1222-1227. 179. Warren, J. R. and Marshall, B. J. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. i: 1273-1275. 180. Wayne, L. G., Brenner, D. J., Colwell, R. R., Girmont, P. A. D., Kandler, 0., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E., Stackebrandt, E., Starr, M. P. and Truper, H. G. 1987. Report of the ad hoe committee on reconciliation of approaches to bacterial systematics. lot J Syst Bacteriol. 37: 463-464. 181. Weisburg, W. G., Barns, S. M., Pelletier, D. A. and Lane, D. J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 173: 697- 703. 182. Weisburg, W. G., Hatch, T. P. and Woese, C. R. 1986. Eubacterial origin of chlamydiae. J Bacteriol. 167: 570-574. 183. Weisburg, W. G., Oyaizu, Y., Oyaizu, H. and Woese, C. R. 1985. Natural relationships between Bacteroides and Flavobacteria. J Bacteriol. 164: 320-236. 184. Weisburg, W. G., Tully, J. G., Rose, D. L., Petzel, J. P., Oyaizu, H., Yang, D., Mandelco, L., Sechrest, J., Lawrence, T. G., VanEtten, J. L., Maniloff, J. and Woese, C. R. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J Bacteriol. 171: 6455-6467. 185. Wesley, I. V., Schroeder-Tucker, L., Baetz, A. L., Dewhirst, F. E. and Paster, B. J. 1995. Arcobacter-specific and Arcobacter butzleri-specific 16S rRNA-based DNA probes. J Clio Microbiol. 33: 1691-1698. 186. Widdell, F. and Bak, F. 1992. Gram negative mesophilic sulfate reducing bacteria, p. 3352-3378. In A. Balows, H. G. Truper, M. Dworkin, W. Harder and K. H. Schleifer (ed.), The prokaryotes, 4. Springer-Verlag, New York. 173

187. Williams, N. M., Harrison, L. and Gebhart, C. J. 1996. Proliferative enteropathy ina foal caused by Lawsonia intracellularis-like bacterium. J Vet Diagn Invest. 8: 254-256. 188. Wilson, K. H. 1994. Detection of culture-resistant bacterial pathogens by amplification and sequencing of ribosomal DNA. Clio Infect Dis. 18: 958-962. 189. Winker, S. and Woese, C. R. 1991. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics. System Appl Microbiol. 14: 305-310. 190. Woese, C. R. 1987. Bacterial evolution. Microbiol Rev. 51: 221-271. 191. Woese, C.R. 1991. The use of ribosomal RNA in reconstructing evolutionary relationships among bacteria., p. 1-24. In R. K. Selander, A. G. Clark and T. S. Whittam (ed.), Evolution at the molecular level., Sinauer Associates Inc., Sunderland, Massachusetts. 192. Woese, C.R. 1994. There must be a prokaryote somewhere: microbiology's search for itself. Microbiol Rev. 58: 1-9. 193. Woese, C. R., Blanz, P., Hespell, R. B. and Hahn, C. M. 1982. Phylogenetic relationships among various helical bacteria. Curr Microbiol. 7: 119-124. 194. Woese, C. R., DeBrunner-Vossbrink, B. A., Oyaizu, H., Stackebrandt, E. and Ludwig, W. 1985. Gram positive bacteria: possible photosynthetic ancestry. Science. 229: 762-765. 195. Woese, C.R. and Fox, G. E. 1977. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci USA. 74: 5088-5090. 196. Woese, C. R., Gutell, R., Gupta, R. and Noller, H. F. 1983. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev. 47: 621-669. 197. Woese, C. R., Kandler, 0. and Wheelis, M. L. 1990. Towards a natural system of organisms: proposal for the domains Aechaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 87: 4576-4579. 198. Woese, C. R., Mandelco, L., Yang, D., Gherna, R. and Madigan, M. T. 1990. The case for the relationship of the flavobacteria and their relatives to the green sulfur bacteria. Syst Appl Microbiol. 13: 258-262. 199. Wotherspoon, A. C., Doglioni, C., Diss, T. C., Langxing, P ., Moschini, A., de Boni, M. and Isaacson, P. G. 1993. Regression of primary low-grade B-cell gastric lymphoma of mucosa associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet. 342: 575-577. 200. Yang, D., Oyaizu, Y., Oyaizu, H., Olsen, G. J. and Woese, C. R. 1985. Mitochondrial origins. Proc Natl Acad Sci USA. 82: 4443-4447. References 174

201. Zhilina, T. N., Zavarzin, G. A., Rainey, F. A., Pikuta, E. N., Osipov, G. A. and Kostrikina, N. A. 1997. Desulfonatronovibrio hydrogenovorans gen. nov., sp. nov., an alkaliphilic, sulfate-reducing bacterium. Int J Syst Bacteriol. 47: 144-149. 202. Zuckerkandl, E. and Pauling, L. 1965. Evolutionary divergence and convergence in proteins, p. 97-166. In V. Bryson and H.J. Vogel (ed.), Evolving genes and proteins, Academic Press, New York. Appendix 175

Appendix 1 Multiple sequence alignment of 16S rDNA sequences determined in this study from Helicobacter species representing bacterial groups A, C, D, E and H as described in the text HRI3caefr AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC HRI6caefr AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC HRI7stomfr AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC ABHU3cae AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC UNSW3 . llcae ------ATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC ABHU4stomsp AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC UNSW3SBsp AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC UNSWMCSpl AGTGAACGCTGGCGGC-TGCCTAATACATGCAAGTCGAGCGATGAGGC'ITCTAGACTTGC UNSWl. ?col -----ACTCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAGGC'ITCTAG-CTTGC UNSW1.7sp AGTGAACGCTGGCGGCGTG-CTAATACATGCAAGTCGAACGATGAAGC'ITCTAG-CTTGC UNSW1.6cae AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITITAG-CTTGC UNSWl. 7st AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC'ITITAG-CTTGC CMRI2cae AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC-TCTAG-CTTGC UNSWlcaesp AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC-TCTAG-CTTGC CMRI2liv AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC-TCTAG-CTTGC ABHU4caesp AGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGATGAAGC-TCTAG-CTTGC *************** ****** ** * *** *****

HRI3caefr TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC HRI6caefr TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC HRI7stomfr TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC ABHU3cae TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC UNSW3.llcae TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC ABHU4stomsp TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC UNSW3SBsp TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC UNSWMCSpl TAGAAAGTGGATTAGTGGCGCACGGGTGAAGTAATGCATAGGTTATGTGCCCTTTAGGTC UNSWl. ?col TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC UNSW1.7sp TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC UNSW1.6cae TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC UNSWl. 7st TAG-AAGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGGTTATGTGCCCTTTA-GTC CMRI2cae TAG--AGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGATAACATGCCCTTTA-GTC UNSWlcaesp TAG--AGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGATAACATGCCCTTTA-GTC CMRI2liv TAG--AGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGATAACATGCCCTTTA-GTC ABHU4caesp TAG--AGTGGATTAGTGGCGCACGGGTG-AGTAATGCATAGATAACATGCCCTTTA-GTC *** *********************** ************ * * ********* ***

HRI3caefr TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT HRI6caefr TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT HRI7stomfr TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT ABHU3cae TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT UNSW3 . llcae TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT ABHU4stomsp TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT UNSW3SBsp TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAGGGGCTT UNSWMCSpl TGGGATAGCCACTGGAAACGGTGATTAATACTGGATA'ITCCCTACGGGGGAAAGGGGCTT UNSWl. ?col TGGGATAGCCACTGGAAACGGTGATTAATACTGGATA'ITCCCTACGGGGGAAAGGGGCTT UNSWl. 7sp TGGGATAGCCACTGGAAACGGTGATTAATACTGGATA'ITCCCTACGGGGGAAAGGGGCTT UNSW1.6cae TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAG-----­ UNSWl. 7st TGGGATAGCCACTGGAAACGGTGATTAATACTGGATACTCCCTACGGGGGAAAG-----­ CMRI2cae TAGGATAGCCATTGGAAACGATGATTAATACTGGATACTCCTTACGAGGGAAAG-----­ UNSWlcaesp TAGGATAGCCATTGGAAACGATGATTAATACTGGATACTCCTTACGAGGGAAAG-----­ CMRI2liv TAGGATAGCCATTGGAAACGATGATTAATACTGGATACTCCTTACGAGGGAAAG-----­ ABHU4caesp TAGGATAGCCATTGGAAACGATGATTAATACTGGATACTCCTTACGAGGGAAAG------* ********* ******** **************** *** **** ******* Appendix 176

HRI3caefr TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG HRI6caefr TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG HRI7stomfr TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG ABHU3cae TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG UNSW3.llcae TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG ABHU4stomsp TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG UNSW3SBsp TCAATAAAGAA'ITI'CTCT'ITITAGTGCTITGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG UNSWMCSpl TCAATAAAGAA'ITI'CTCT'ITITAGTGTITI'GTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG UNSW1.7col TCAATAAAGAA'ITI'CTCT'ITITAGTG'ITI'GGTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG UNSW1.7sp TCAATAAAGAA'ITI'CTCT'ITITAGTGTGTI'GTGTI'GTI'GGCACAAMTI'CTAGTA'ITI'GG UNSW1.6cae UNSW1.7st CMRI2cae UNSWlcaesp CMRI2liv ABHU4caesp

HRI3caefr AATGAGAAATI'GATGTl'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC HRI6caefr AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC HRI7stomfr AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC ABHU3cae AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC UNSW3.llcae AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC ABHU4stomsp AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC UNSW3SBsp AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC UNSWMCSpl AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC UNSW1.7col AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC UNSW1.7sp AATGAGAAATI'GATGTI'GTGAAGCAATl'TGTGCGGAGACTAGACTI'AGTGTCTGTCGCAC UNSW1.6cae UNSWl. 7st CMRI2cae UNSWlcaesp CMRI2liv ABHU4caesp

HRI3caefr AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C HRI6caefr AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C HRI7stomfr AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C ABHU3cae AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C UNSW3.llcae AAGCAAATI'GCGAACTCATCGATI'TATCG-CCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C ABHU4stomsp AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C UNSW3SBsp AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C UNSWMCSpl AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C UNSWl. 7col AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C UNSW1.7sp AAGCAAATI'GCGAACTCATCGATI'TATCGTCCAAAGACGAATI'TI'TI'ATI'GAAAGCCTI'C UNSW1.6cae ------TI'T------Tl'C UNSWl. 7st ------TI'T------Tl'C CMRI2cae ------TI'T------Tl'C UNSWlcaesp ------TI'T------Tl'C CMRI2liv ------TI'T------Tl'C ABHU4caesp ------TI'T------Tl'C *** *** Appendix 177

HRI3caefr GCTAAAGGATCAGCCTATGTCCTATCAGC'ITG'ITGG'IGAGGTAATGGCTCACCAAGGCTA HRI6caefr GCTAAAGGATCAGCCTATGTCCTATCAGC'ITG'ITGG'IGAGGTAATGGCTCACCAAGGCTA HRI7stomfr GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA ABHU3cae GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSW3.llcae GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA ABHU4stomsp GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSW3SBsp GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSWMCSpl GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSWl. 7col GCTAAAGGATCAGCCTATGTCCTATCAGC'ITG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSW1.7sp GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSW1.6cae GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTTACCAAGGCTA UNSWl. 7st GCTAAAGGATCAGCCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTTACCAAGGCTA CMRI2cae GCTAAAGGA'ITGGTCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA UNSWlcaesp GCTAAAGGA'ITGGTCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA CMRI2liv GCTAAAGGA'ITGGTCTATGTCCTATCAGC'ITG'ITGG'IGAGGTAATGGCTCACCAAGGCTA ABHU4caesp GCTAAAGGA'ITGGTCTATGTCCTATCAGCTTG'ITGG'IGAGGTAATGGCTCACCAAGGCTA ********** * *********************************** **********

HRI3caefr 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAACTGAGACACGGTCCAGAC HRI6caefr 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC HRI7stomfr 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC ABHU3cae 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAACTGAGACACGGTCCAGAC UNSW3.llcae 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC ABHU4stomsp 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC UNSW3SBsp 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC UNSWMCSpl 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC UNSW1.7col 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAACTGAGACACGGTCCAGAC UNSW1.7sp 'IGACGGGTATCCGGCC'IGAGAGGG'IGATCGGACACACTGGAAC'IGAGACACGGTCCAGAC UNSW1.6cae 'IGACGGGTATCCGGCC'IGAGAGGGTGAACGGACACACTGGAAC'IGAGACACGGTCCAGAC UNSW1.7st 'IGACGGGTATCCGGCC'IGAGAGGGTGAACGGACACACTGGAAC'IGAGACACGGTCCAGAC CMRI2cae 'IGACGGGTATCCGGCC'IGAGAGGGTGAACGGACACACTGGAAC'IGAGACACGGTCCAGAC UNSWlcaesp 'IGACGGGTATCCGGCC'IGAGAGGGTGAACGGACACACTGGAAC'IGAGACACGGTCCAGAC CMRI2liv 'IGACGGGTATCCGGCC'IGAGAGGGTGAACGGACACACTGGAAC'IGAGACACGGTCCAGAC ABHU4caesp 'IGACGGGTATCCGGCC'IGAGAGGGTGAACGGACACACTGGAAC'IGAGACACGGTCCAGAC *************************** ********************************

HRI3caefr TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C HRI6caefr TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C HRI7stomfr TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C ABHU3cae TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C UNSW3.llcae TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C ABHU4stomsp TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C UNSW3SBsp TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C UNSWMCSpl TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCATCAA-C UNSWl. 7col TCCTACGGGAGGCAGCAGTAGGGAATATTGCTC-ATGGGGGAAACCCTGAAGCAGCAACC UNSWl. 7sp TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C UNSW1.6cae TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGCGAAAGCCTGAAGCAGCAA-C UNSW1.7st TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGCGAAAGCCTGAAGCAGCAA-C CMRI2cae TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C UNSWlcaesp TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C CMRI21iv TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C ABHU4caesp TCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAA-C ********************************* ***** **** ********* *** * Appendix 178

HRI3caefr GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCCTTITGTAAGAGAAGATI'A'ffiAC HRI6caefr GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITGTAAGAGAAGATI'A'ffiAC HRI7stomfr GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITGTAAGAGAAGATI'A'ffiAC ABHU3cae GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCCTI'Tl'GTAAGAGAAGATI'A'ffiAC UNSW3.llcae GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITGTAAGAGAAGATI'A'ffi-C ABHU4stomsp GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCCTI'Tl'GTAAGAGAAGATI'A'ffiAC UNSW3SBsp GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCCTI'Tl'GTAAGAGAAGATI'A'ffiAC UNSWMCSpl GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITGTGAGAGAAGATAA'ffiAC UNSWl. 7col GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITG'ffiAGAGAAGATAA'ffiAC UNSW1.7sp GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCCTI'Tl'G'ffiAGAGAAGATAA'ffiAC UNSW1.6cae GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITGTI'AGAGAAGATAA'ffiAC UNSWl. 7st GCCGCGTGGAGGATGAAGG'ITITAGGATI'GTAAACTCC'ITITGTI'AGAGAAGATAA'ffiAC CMRI2cae GCCGCGTGGAGGATGAAGGTI'TI'CGGATI'GTAAACTCC'ITITGTI'AGAGAAGATAA'ffiAC UNSWlcaesp GCCGCGTGGAGGATGAAGGTI'TI'CGGATI'GTAAACTCC'ITITGTI'AGAGAAGATAA'ffiAC . CMRI2liv GCCGCGTGGAGGATGAAGGTI'TI'CGGATI'GTAAACTCC'ITITGTI'AGAGAAGATAA'roAC ABHU4caesp GCCGCGTGGAGGATGAAGGTI'TI'CGGATI'GTAAACTCCTI'Tl'GTI'AGAGAAGATAA'ffiAC *********************** ******************** ********* *** *

HRI3caefr GGTATCTI'ACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG HRI6caefr GGTATCTI'ACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG HRI7stomfr GGTATCTI'ACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG ABHU3cae GGTATCTI'ACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSW3 . llcae GGTATCTI'ACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG ABHU4stomsp GGTATCTI'ACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSW3SBsp GGTATCTI'A-GAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSWMCSpl GGTATCTCACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSWl. 7col GGTATCTCRCGAATAAGCACCGGC-TAACTCCGTGCCAGCA-CCGCGGTAATACGGAGGG UNSW1.7sp GGTATCTCACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSW1.6cae GGTATCTAACGAATAAGCACCGGCTI'AACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSWl. 7st GGTATCTAACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG CMRI2cae GGTATCTAACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG UNSWlcaesp GGTATCTAACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG CMRI2liv GGTATCTAACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG ABHU4caesp GGTATCTAACGAATAAGCACCGGC-TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGG ******* ************** **************** ******************

HRI3caefr TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA HRI6caefr TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA HRI7s tomfr TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA ABHU3cae TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA UNSW3.llcae TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA ABHU4stomsp TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA UNSW3SBsp TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGAGAGTAAGTCAGA UNSWMCSpl TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGCGGTCAAGTCAGA UNSWl. 7col TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGCGGTCAAGTCAGA UNSW1.7sp TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGCGGTCAAGTCAGA UNSW1.6cae TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGTGCGCAGGCGGGCTAATAAGTCAGA UNSWl. 7st TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGTGCGCAGGCGGGCTAATAAGTCAGA CMRI2cae TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGATAGCAAGTCAGA UNSWlcaesp TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGATAGCAAGTCAGA CMRI2liv TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGATAGCAAGTCAGA ABHU4caesp TGCAAGCGTI'ACTCGGAATCACTGGGCGTAAAGAGCGCGTAGGCGGGATAGCAAGTCAGA *********************************** *** ******* ******** Appendix 179

HRI3caefr TGTGAAA'ICCTGTAGCTl'AACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG HRI6caefr TGTGAAA'ICCTGTAGCTTAACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG HRI7stomfr TGTGAAA'ICCTGTAGCTTAACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG ABHU3cae TGTGAAA'ICCTGTAGCTTAACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG UNSW3.llcae TGTGAAA'ICCTGTAGCTTAACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG ABHU4stomsp TGTGAAA'ICCTGTAGCTTAACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG UNSW3SBsp TGTGAAA'ICCTGTAGCTTAACTACAGAACTGCATITGAAACTACTTI'TCTAGAGTATGGG UNSWMCSpl TGTGAAA'ICCTGTAGC'ICAACTACAGAACTGCATITGAAACTGACCA'ICTAGAGTATGGG UNSWl. ?col TGTGAAA'ICCTGTAGC'ICAACTACAGAACTGCATITGAAACTGACCA'ICTAGAGTATGGG UNSWl. 7sp TGTGAAA'ICCTGTAGC'ICAACTACAGAACTGCATITGAAACTGACCA'ICTAGAGTATGGG UNSW1.6cae TGTGAAA'ICCTATAGCTTAACTATAGAACTGCATITGAAACTATTAG'ICTAGAGTGTGGG UNSWl. 7st TGTGAAA'ICCTATAGCTTAACTATAGAACTGCATITGAAACTATTAG'ICTAGAGTGTGGG CMRI2cae TGTGAAA'ICCTATGGCTTAACCATAGAACTGCATITGAAACTGTTAT'ICTAGAGTATGGG UNSWlcaesp TGTGAAA'ICCTATGGCTTAACCATAGAACTGCATITGAAACTGTTAT'ICTAGAGTATGGG CMRI2liv TGTGAAA'ICCTATGGCTTAACCATAGAACTGCATITGAAACTGTTAT'ICTAGAGTATGGG ABHU4caesp TGTGAAA'ICCTATGGCTTAACCATAGAACTGCATITGAAACTGTTAT'ICTAGAGTATGGG *********** * *** *** * ****************** ******** ****

HRI3caefr AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA HRI6caefr AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA HRI7stomfr AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA ABHU3cae AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSW3.llcae AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA ABHU4stomsp AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSW3SBsp AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSWMCSpl AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSWl. ?col AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSW1.7sp AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSW1.6cae AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSWl. 7st AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA CMRI2cae AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA UNSWlcaesp AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA CMRI2liv AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA ABHU4caesp AGAGGTAGGTGGAAT'ICTTGGTGTAGGGGTAAAA'ICCGTAGAGA'ICAAGAGGAATACTCA ************************************************************

HRI3caefr TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA HRI6caefr TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA HRI7stomfr TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA ABHU3cae TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSW3 . llcae TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA ABHU4stomsp TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSW3SBsp TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSWMCSpl TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSWl. ?col TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSW1.7sp TTGCGAAGGCGACCTGCTGGAACATTACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSW1.6cae TTGCGAAGGCGACCTACTGGAACATTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAA UNSWl. 7st TTGCGAAGGCGACCTACTGGAACATTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAA CMRI2cae TTGCGAAGGCGACCTGCTAGAACATAACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA UNSWlcaesp TTGCGAAGGCGACCTGCTAGAACATAACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA CMRI2liv TTGCGAAGGCGACCTGCTAGAACATAACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA ABHU4caesp TTGCGAAGGCGACCTGCTAGAACATAACTGACGCTGATGCGCGAAAGCGTGGGGAGCAAA *************** ** ****** ********* **** ******************* Appendix 180

HRI3caefr CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCC'mCT HRI6caefr CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT HRI7stomfr CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT ABHU3cae CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT UNSW3 . llcae CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT ABHU4stomsp CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT UNSW3SBsp CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT UNSWMCSpl CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT UNSWl. 7col CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT UNSW1.7sp CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCCTGCT UNSW1.6cae CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCTI'GCT UNSWl. 7st CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GCCTI'GCT CMRI2cae CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GTGGAGCT UNSWlcaesp CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GTGGAGCT CMRI2liv CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GTGGAGCT ABHU4caesp CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATGCTAGTI'GTI'GTGGAGCT ***************************************************** ***

HRI3caefr TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG HRI6caefr TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG HRI7stomfr TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG ABHU3cae TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSW3 . llcae TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG ABHU4stomsp TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSW3SBsp TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSWMCSpl TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSWl. 7col TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSW1.7sp TGTCAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSW1.6cae TGACAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSWl. 7st TGACAGGGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG CMRI2cae TGTCTCTGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG UNSWlcaesp TGTCTCTGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG CMRI2liv TGTCTCTGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG ABHU4caesp TGTCTCTGCAGTAATGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAG ** * *****************************************************

HRI3caefr ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC HRI6caefr ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC HRI7stomfr ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC ABHU3cae ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSW3.llcae ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC ABHU4stomsp ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSW3SBsp ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSWMCSpl ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSWl. 7col ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSW1.7sp ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSW1.6cae ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSWl. 7st ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC CMRI2cae ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC UNSWlcaesp ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC CMRI2liv ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC ABHU4caesp ATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTI'AATTC ************************************************************ Appendix 181

HRI3caefr GAAGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro HRI6caefr GAAGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro HRI7stomfr GAAGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro ABHU3cae GAGGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCTA-CTAGAGATAG'ro UNSW3.llcae GAGGATACGCGAAGAACCTTACCTAGGC'ITGA,CATI'GATAGAATCTA-CTAGAGATAG'ro ABHU4stomsp GAGGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCTA-CTAGAGATAG'ro UNSW3SBsp GAGGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCTA--TAGAGATAG'ro UNSWMCSpl GAGGCTACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCTA-CTAGAGATAG'ro UNSW1.7col GAGGCTACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAG-ATCTA-CTAGAGATAG'ro UNSWl. 7sp GAGGCTACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCTA-CTAGAGATAG'ro UNSW1.6cae GAAGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCT-ATAGAGATATGG UNSW1.7st GAAGATACGCGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCT-ATAGAGATATGG CMRI2cae GAAGATACACGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro UNSWlcaesp GAAGATACACGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro CMRI2liv GAAGATACACGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro ABHU4caesp GAAGATACACGAAGAACCTTACCTAGGCTI'GACATI'GATAGAATCCG-CTAGAGATAG'ro ** * *** ******************************** *** ******** *

HRI3caefr GAGTGCTGGCTTGCCAGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC HRI6caefr GAGTGCTGGCTTGCCAGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC HRI7stomfr GAGTGCTGGCTTGCCAGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC ABHU3cae GAGTGC--CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC UNSW3.llcae GAGTGC--CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC ABHU4stomsp GAGTGC--CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC UNSW3SBsp GAGTGC--CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roT-G-TCAGCTCG'roTC UNSWMCSpl GAGTGC--CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC UNSW1.7col GAGTGC--CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC UNSWl. 7sp GAGTGC- -CCTTCGGGGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC UNSW1.6cae GAGTGCCACTTC'ro'roGAGCTTGAAAACAGGTGCTGCACGGC'roTCGCTCAGCTCG'roTC UNSWl. 7st GAGTGCCACTTC'ro'roGAGCTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCGTGTC CMRI2cae GAGTGCTAGCTTGCTAGAACTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC UNSWlcaesp GAGTGCTAGCTTGCTAGAACTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC CMRI2liv GAGTGCTAGCTTGCTAGAACTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC ABHU4caesp GAGTGCTAGCTTGCTAGAACTTGAAAACAGGTGCTGCACGGC'roTCG-TCAGCTCG'roTC ****** * ** ************************** * ************

HRI3caefr G'ro1.GA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG HRI6caefr G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG HRI7stomfr G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG ABHU3cae G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG UNSW3. llcae G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG ABHU4stomsp G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG UNSW3SBsp G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-C-ACCCTCGTCCTTAGTTGCTAGCAG UNSWMCSpl G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCGACAACCCTCGTCCTTAGTTGCTAGCAG UNSWl. 7col G'roAGA'roTTGGGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG UNSW1.7sp G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG UNSW1.6cae G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG UNSWl. 7st G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAGCAG CMRI2cae G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAACTA UNSWlcaesp G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACC-TCGTCCTTAGTTGCTAACTA CMRI2liv G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAACTA ABHU4caesp G'roAGA'roTT-GGGTTAAGTCCCGCAACGAGCG-CAACCCTCGTCCTTAGTTGCTAACTA ********** ********************** * *** **************** * Appendix 182

HRI3caefr TK:G-GCTGA-GCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC HRI6caefr TK:G-GCTGA-GCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC HRI7stomfr TK:G-GCTGA-GCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC ABHU3cae TK:G-GCTGA-GCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC UNSW3.llcae TK:G-GCTGA-GCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC ABHU4stornsp TK:G-GCTGA-GCACTCTAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC UNSW3SBsp TK:G-GCTGA-GCACTCTAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC UNSWMCSpl TK:G-GCTGAGGCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC UNSWl. 7col TK:G-GCTGA-GCACTCTAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC UNSW1.7sp TK:G-GCTGA-GCACTCTAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC UNSW1.6cae TTTAAGCTGA-GCAC'K:TAAGGAGACTGCCTK:GTACAGGAGGAGGAAGGTGAGGACGAC UNSWl. 7st TTTAAGCTGA-GCAC'K:TAAGGAGACTGCCTK:GTA-AGGAGGAGGAAGGTGAGGACGAC CMRI2cae -TK:GGTAGA-GCAC'K:TAAGGAGACTGCCTK:GCA-AGGAGGAGGAAGGTGAGGATGAC UNSWlcaesp -TK:GGTAGA-GCAC'K:TAAGGAGACTGCCTK:GCA-AGGAGGAGGAAGGTGAGGATGAC CMRI21iv -TK:GGTAGA-GCACTCTAAGGAGACTGCCTK:GCA-AGGAGGAGGAAGGTGAGGATGAC ABHU4caesp -TK:GGTAGA-GCAC'K:TAAGGAGACTGCCTK:GCA-AGGAGGAGGAAGGTGAGGATGAC * * ** *********************** * ******************* ***

HRI3caefr G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGGCATACAA HRI6caefr G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGGCATACAA HRI7stomfr G'ICAAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGGCATACAA ABHU3cae G'ICAAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAA UNSW3.llcae G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAA ABHU4stornsp G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAA UNSW3SBsp G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAA UNSWMCSpl G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAA UNSWl. 7col G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAC UNSWl. 7sp G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGACATACAA UNSW1.6cae G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGGCGCACAA UNSWl. 7st G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGGCGCACAA CMRI2cae G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGAAGCACAA UNSWlcaesp G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGAAGCACAA CMRI21iv G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGAAGCACAA ABHU4caesp G'K:AAGTCATCATGGCCCTTA-CGCCTAGGGCTACACACGTGCTACAATGGGAGGCACAA ********************* ****************************** ***

HRI3caefr TGAGGAGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATGTC'K:TCAGTK:GGATTGTAG HRI6caefr TGAGGAGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATG'K:'K:TCAGTK:GGATTGTAG HRI7stomfr TGAGGAGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATG'K:'K:TCAGTK:GGATTGTAG ABHU3cae AAAGATGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATG'K:'K:TCAGTK:GGATTGTAG UNSW3.llcae AAAGATGCAATACCGCGAGGTGGAGCAAATC'K:TAAAATGTC'K:TCAGTK:GGATTGTAG ABHU4stornsp AAAGATGCAATACCGCGAGGTGGAGCAAATC'K:TAAAATGTC'K:TCAGTK:GGATTGTAG UNSW3SBsp AAAGATGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATG'K:'K:TCAGTK:GGATTGTAG UNSWMCSpl AAAGATGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATGTCTCTCAGTK:GGATTGTAG UNSWl. 7col AAAGATGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATG'K:TCTCAG-TCGGATTGTAG UNSW1.7sp AAAGATGCAATACCGCGAGGTGGAGCAAA'K:'K:TAAAATG'K:'K:TCAGTK:GGATTGTAG UNSW1.6cae AGAGGAGCAATA'K:GTGAGGTGGAGCAAA'K:'K:TAAAACG'K:'K:TCAGG'K:GGATTGTAG UNSWl. 7st AGAGGAGCAATA'K:GTGAGGTGGAGCAAA'K:'K:TAAAACG'K:'K:TCAGTK:GGATTGTAG CMRI2cae AGAGATGCAATATTGTGAAATGGAGCAAATCTATAAAACTK:'K:TCAGTK:GGATTGTAG UNSWlcaesp AGAGATGCAATATTGTGAAATGGAGCAAATCTATAAAACTK:'K:TCAGTK:GGATTGTAG CMRI21iv AGAGATGCAATATTGTGAAATGGAGCAAATCTATAAAACTK:'K:TCAGTK:GGATTGTAG ABHU4caesp AGAGATGCAATATGGTGAAATGGAGCAAA'K:TATAAAACTK:'K:TCAGTK:GGATTGTAG ** ****** * ** ************ ***** ******** *********** Appendix 183

HRI3caefr TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGCAAATCAGCAATGTTGCGG HRI6caefr TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGCAAATCAGCAATGTTGCGG HRI7stomfr TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGCAAATCAGCAATGTTGCGG ABHU3cae TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG UNSW3.llcae TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG ABHU4stomsp TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG UNSW3SBsp TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG UNSWMCSpl TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG UNSWl. 7col TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG UNSW1.7sp TCTGCAACTCGACTACATAAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCAATGTCACGG UNSW1.6cae TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCCATGTCACGG UNSWl. 7st TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCG'ffiAATCAGCCATGTCACGG CMRI2cae TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGTAAATCAGCTATGTTACGG UNSWlcaesp TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGTAAATCAGCTATGTTACGG CMRI2liv TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGTAAATCAGCTATGTTACGG ABHU4caesp TCTGCAACTCGACTACA'ffiAAGC'roGAATCGCTAGTAATCGTAAATCAGCTATGTTACGG ****************** ********************** ******* **** ***

HRI3caefr 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG HRI6caefr 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG HRI7stomfr 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG ABHU3cae 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSW3.llcae 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG ABHU4stomsp 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSW3SBsp 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSWMCSpl 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSW1.7col 'ffiAATACGTTCCCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSW1.7sp 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSW1.6cae 'ffiAATACGTT-CCCGGGTGCTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSWl. 7st 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG CMRI2cae 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG UNSWlcaesp 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG CMRI2liv 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG ABHU4caesp 'ffiAATACGTT-CCCGGGT-CTTGTACTCACCGCCCGTCACACCATGGGAGTTGTATT-CG ********** ******* ************************************** **

HRI3caefr CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG HRI6caefr CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG HRI7stomfr CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG ABHU3cae CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG UNSW3.llcae CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG ABHU4stomsp CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG UNSW3SBsp CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG UNSWMCSpl CCTTAAGTCGGGATACTAAATTGGTTACC-CCCACGGGGGATGCAGCGACTGGGGTG UNSWl. 7col CCTTAAGTCGGGATACTACATT-GTTACCGCCCACGCCCGATGCAGCGACT-----­ UNSWl. 7sp CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG UNSW1.6cae CCTTAAGTCGGGACACTAAATT-GTTACCGCCCACGGCGGATGCAGC------­ UNSW1.7st CCTTAAGTCGGGATACTAAATTGGTTACCGCCCACGGCGGATGCAGCGACTGGGGTG CMRI2cae CCTTAAGTCGGAATGCCAAACTGGCTACCGCCCACGGCGGATGCAGCGACTGGGGTG UNSWlcaesp CCTTAAGTCGGAATGCCAAACTGGCTACCGCCCACGGCGGATGCAGCGACTGGGGTG CMRI2liv CCTTAAGTCGGAATGCCAAACTGGCTACCGCCCACGGCGGATGCAGCGACTGGGGTG ABHU4caesp CCTTAAGTCGGAATGCCAAACTGGCTACCGCCCACGGCGGATGCAGCGACTGGGGTG *********** * * * * * * **** ****** ********

* indicate nucleotide positions which are conserved for all sequences represented in the alignment. Appendix 184

Appendix 2 Multiple sequence alignment of 16S rDNA sequences determined in this study representing bacterial group J as described in the text..

ABHUlSB A'ITGAACGCTGGCGGCGTGCTI'AACACATGCAAGTCGAACGCGAAAGCGGC'ITCGGCCG­ ABHUlSBfatS A'ITGAACGCTGGCGGCGTGC'ITAACACATGCAAGTCGAACGCGAAAGCGGC'ITCGGCCGT ABHU2SB A'ITGAACGCTGGCGGCGTGCTI'AACACATGCAAGTCGAACGCGAAAGCGGC'ITCGGTCGC UNSW3caefats A'ITGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGCGAAAGGGGC'ITCGGCCCC ******************** ************************** ******** *

.ABHUlSB GAGTAGAGTGGCGCACGGGTGAGTAACGCG'ffiGACAATCTGCCCTCATGACCGGGATAAC ABHUlSBfatS GAGTAGAGTGGCGCACGGGTGAGTAACGCG'ffiGACAATCTGCCCTCATGACCGGGATAAC ABHU2SB GAGTAGAGTGGCGCACGGGTGAGTAACGCG'ffiGACAATCTGCCCTCATGACCGGGATAAC UNSW3caefatS GAGTAAAG'K:GCGCACGGGTGAGTACCGCG'ffiGATAATCTGCCTTCAAGATGGGGATAAC ***** ******************* ******** ******** *** ** ********

ABHUlSB AGT'ffiGAAACGACTGCTAATACCGGATACGCTC'ffiGATGAACAT'IT--GGAGGAAAGACG ABHUlSBfatS AGT'ffiGAAACGACTGCTAATACCGGATACGCTC'ffiGATGAACAT'IT--GGAGGAAAGACG ABHU2SB AGT'ffiGAAACGACTGCTAATACCGGATACGCTC'ffiGATGAACAT'IT--GGAGGAAAGACG UNSW3caefatS AGT'ffiGAAACGACTGCTAATACCGAATACGCTCACAATAATCA'IT'ITGTGGGGAAAGATG ************************ ******** ** * ***** * ******* *

ABHUlSB. GCCTCTGCATGCAAGCTGTCGTATGAGGATGAGTCCGCGTCCCA'ITAGCTTG'ITGGTGGG ABHUlSBfatS GCCTCTGCATGCAAGCTGTCGTATGAGGATGAGTCCGCGTCCCA'ITAGCTTG'ITGGTGGG ABHU2SB GCCTCTGCATGCAAGCTGTCGTATGAGGATGAGTCCGCGTCCCA'ITAGCTTG'ITGGTGGG UNSW3caefatS GCCTCTGCAT--ATGCTG'ITGC'ITGAAGATGAGTCCGCGTCCCA'ITAGCTTG'ITGGCGGG ********** * ***** * *** ***************************** ***

ABHUlSB GTAA-GGCCTACCAAGGCGACGATGGGTAGCCGATCTGAGAGGATGATCGGCCACACTGG ABHUlSBfatS GTAA-GGCCTACCAAGGCGACGATGGGTAGCCGATCTGAGAGGATGATCGGCCACACTGG ABHU2SB GTAA'K:GCCTACCAAGGCGACGATGGGTAGCCGATCTGAGAGGATGATCGGCCACACTGG UNSW3caefatS GTAAAGGCCCACCAAGGCGACGATGGGTAGCCGATCTGAGAGGATGATCGGCCACACTGG **** **** **************************************************

ABHUlSB AACTGAAACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA'ITGCGCAATGGGC ABHUlSBfatS AACTGAAACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA'ITGCGCAATGGGC ABHU2SB AACTGAAACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA'ITGCGCAATGGGC UNSW3caefats AACTGAAACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA'ITGCGCAATGGGC ************************************************************

ABHUlSB GAAAGCCTGACGCAGCGACGCCGCGTGAGGGATGAAGG'IT'ITCGGATCGTAAACCTCTGT ABHUlSBfatS GAAAGCCTGACGCAGCGACGCCGCGTGAGGGATGAAGG'IT'ITCGGATCGTAAACCTCTGT ABHU2SB GAAAGCCTGACGCAGCGACGCCGCGTGAGGGATGAAGG'IT'ITCGGATCGTAAACCTCTGT UNSW3caefatS GAAAGCCTGACGCAGCGACGCCGCGTGAGGGATGAAGGTC'ITCGGATCGTAAACCTCTGT *************************************** ******************** Appendix 185

ABHUlSB CAGAAGGGAAGAAAGTGCG'ffiGTGCTAATCAGCCGCGTATTGACGGTACCTTCAAAGGAA ABHUlSBfatS CAGAAGGGAAGAAAGTGCG'ffiGTGCTAATCAGCCGCGTATTGACGGTACCTTCAAAGGAA ABHU2SB CAGAAGGGAAGAAAGTGCG'ffiGTGCTAATCAGCCGCGTATTGACGGTACCTTCAAAGGAA UNSW3caefatS CAGGAGGGAAGAAGTTATACGGTGCTAATCAGCCGTATATTGACGGTACCTCCAAAGGAA *** ********* * *************** ************** ********

ABHUlSB GCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGA ABHUlSBfatS GCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGA ABHU2SB GCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGA UNSW3caefatS GCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGA ************************************************************

ABHUlSB ATTACTGGGCGTAAAGCGCACGTAGGCTGTTGTGTAAGTCAGGGGTGAAATCCCACGGCT ABHUlSBfatS ATTACTGGGCGTAAAGCGCACGTAGGCTGTTGTGTAAGTCAGGGGTGAAATCCCACGGCT ABHU2SB ATTACTGGGCGTAAAGCGCACGTAGGCTGTTGTGTAAGTCAGGGGTGAAATCCCACGGCT UNSW3caefatS ATCACTGGGCGTAAAGCGCACGTAGGCTGTTTTGTAAGTCAGAGGTGAAAGCCCACGGCT ** **************************** ********** ******* *********

ABHUlSB CAACCGTGGAACTGCCCTTGATACTGCATGACTGGAATCCGGGAGAGGGTGGCGGAATTC ABHUlSBfatS CAACCGTGGAACTGCCCTTGATACTGCATGACTGGAATCCGGGAGAGGGTGGCGGAATTC ABHU2SB CAACCGTGGAACTGCCCTTGATACTGCATGACTGGAATCCGGGAGAGGGTGGCGGAATTC UNSW3caefatS TAACCGTGGAACTGCCTTTGATACTGCTTGACTAGAATCCGGGAGAGGGTGGCGGAATTC *************** ********** ***** **************************

ABHUlSB CAGGTGTAGGAGTGAAATCCGTAGATATCTGGAGGAACATCAGTGGCGAAGGCGGCCACC ABHUlSBfatS CAGGTGTAGGAGTGAAATCCGTAGATATCTGGAGGAACATCAGTGGCGAAGGCGGCCACC ABHU2SB CAGGTGTAGGAGTGAAATCCGTAGATATCTGGAGGAACATCAGTGGCGAAGGCGGCCACC UNSW3caefatS CAGGTGTAGGAGTGAAATCCGTAGATATCTGGAGGAACATCAGTGGCGAAGGCGGCCACC ************************************************************

ABHUlSB TGGACCGGTATTGACGCTGAGGTGCGAAAGCG'ffiGGGAGCAAACAGGATTAGATACCCTG ABHUlSBfatS TGGACCGGTATTGACGCTGAGGTGCGAAAGCG'ffiGGGAGCAAACAGGATTAGATACCCTG ABHU2SB TGGACCGGTATTGACGCTGAGGTGCGAAAGCG'ffiGGGAGCAAACAGGATTAGATACCCTG UNSW3caefatS TGGACCGGTATTGACGCTGAGGTGCGAAAGCG'ffiGGGAGCAAACAGGATTAGATACCCTG ************************************************************

ABHUlSB GTAGTCCACGCTGTAAACGATGGATGCTAGATGTCGGGGAGTATTCTTCGGTGTCGTAGT ABHUlSBfatS GTAGTCCACGCTGTAAACGATGGATGCTAGATGTCGGGGAGTATTCTTCGGTGTCGTAGT ABHU2SB GTAGTCCACGCTGTAAACGATGGATGCTAGATGTCGGGGAGTATTCTTCGGTGTCGTAGT UNSW3caefatS GTAGTCCACGCTGTAAACGATGGATGCTAGGTGTTGGGGAGTATTCCTCGGCGCCGTAGT ****************************** *** *********** **** * ******

ABHUlSB TAACGCGTTAAGCATCCCGCC'ffiGGGAGTACGGTCGCAAGGCTGAAACTCAAAGAAATTG ABHUlSBfatS TAACGCGTTAAGCATCCCGCC'ffiGGGAGTACGGTCGCAAGGCTGAAACTCAAAGAAATTG ABHU2SB TAACGCGTTAAGCATCCCGCC'ffiGGGAGTACGGTCGCAAGGCTGAAACTCAAAGAAATTG UNSW3caefatS TAACGCGTTAAGCATCCCGCC'ffiGGGAGTACGGTCGCAAGGCTGAAACTCAAAGAAATTG ************************************************************ Appendix 186

ABHUlSB ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTITAA'ITCGATGCAACGCGAAGAACCTT ABHUlSBfatS ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTITAA'ITCGATGCAACGCGAAGAACCTT ABHU2SB ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTITAA'ITCGATGCAACGCGAAGAACCTT UNSW3caefatS ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTITAA'ITCGATGCAACGCGAAGAACCTT ************************************************************

ABHUlSB ACCTGGGTTTGACATCTGGGGAATCCTCCCGAAAAGGAGGAGTGCCC'ITCGGGGAGCCCC ABHUlSBfatS ACCTGGGTTTGACATCTGGGGAATCCTCCCGAAAAGGAGGAGTGCCC'ITCGGGGAGCCCC ABHU2SB ACCTGGGTTTGACATCTGGGGAATCCTCCCGAAAAGGAGGAGTGCCC'ITCGGGGAGCCCC UNSW3caefatS ACCTGGGTTTGACATCCGGAGAACCCTCCCGAAAAGGAGGGG-GCCCCTCGGGGAGCTCC **************** ** *** **************** * **** ********* **

ABHUlSB AAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC ABHUlSBfatS AAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC ABHU2SB AAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC UNSW3caefatS GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC . ***********************************************************

ABHUlSB AACGAGCGCAACCCCTATGCATAGTTGCCAGCGAGTGAAGTCGGGCACTCTATGCAGACT ABHUlSBfatS AACGAGCGCAACCCCTATGCATAGTTGCCAGCGAGTGAAGTCGGGCACTCTATGCAGACT ABHU2SB AACGAGCGCAACCCCTATGCATAGTTGCCAGCGAGTGAAGTCGGGCACTCTATGCAGACT UNSW3caefats AACGAGCGCAACCCCTGTTCATAGTTGCCATCAAGTGAAGTTGGGCACTCTATGGAGACC **************** * *********** * ******** ************ ****

ABHUlsB· GCCCGGGTTAACCGGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACACCCA ABHUlSBfatS GCCCGGGTTAACCGGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACACCCA ABHU2SB GCCCGGGTTAACCGGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACACCCA UNSW3caefatS GCCCGGGTCAACCGGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACACCCA ******** ******************** ******************************

ABHUlSB GGGCTACACACGTACTACAATGGCGCGCACAAAGGGGAGCGAGACCGCGAGGTGGAGCCA ABHUlSBfatS GGGCTACACACGTACTACAATGGCGCGCACAAAGGGGAGCGAGACCGCGAGGTGGAGCCA ABHU2SB GGGCTACACACGTACTACAATGGCGCGCACAAAGGGGAGCGAGACCGCGAGGTGGAGCCA UNSW3caefatS GGGCTACACACGTACTACAATGGCGCACACAAAGGGCAGCGAAGCCGCGAGGTGGAGCTA ************************** ********* ***** ************** *

ABHUlSB ATCCCAAAAAACGCGTCCCAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGA ABHUlSBfatS ATCCCAAAAAACGCGTCCCAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGA ABHU2SB ATCCCAAAAAACGCGTCCCAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGA UNSW3caefatS ATCCCAAAAAATGCGTCTCAG'ITCGGA'I'rGGAGTCTGCAACTCGACTCCATGAAGTCGGA *********** ***** **** ******* **************** ************

ABHUlSB ATCGCTAGTAA'ITCGAGATCAGCATGCTCGGGTGAATGCG'ITCCCGGGCCTTGTACACAC ABHUlSBfatS ATCGCTAGTAA'ITCGAGATCAGCATGCTCGGGTGAATGCG'ITCCCGGGCCTTGTACACAC ABHU2SB ATCGCTAGTAA'ITCGAGATCAGCATGCTCGGGTGAATGCG'ITCCCGGGCCTTGTACACAC UNSW3caefatS ATCGCTAGTAA'ITCCGGATCAGCATGCCGGGGTGAATGCG'ITCCCGGGCCTTGTACACAC ************** *********** ******************************* Appendix 187

ABHUlSB CGCCCG'K:ACACCACGAAAGTCGGTI'ITACCCGAAGCCGGTGAGCCAACCAGTAATGGAG ABHUlSBfatS CGCCCG'K:ACACCACGAAAGTCGGTI'ITACCCGAAGCCGGTGAGCCAACCAGTAATGGAG ABHU2SB CGCCCG'K:ACACCACGAAAGTCGGTI'ITACCCGAAGCCGGTGAGCCAACCAGCAATGGAG UNSW3caefatS CGCCCG'K:ACACCACGAAAGTCGGTI'ITACCCGAAGCCGGTGAGCTAAC'IGGCAACAGAA ********************************************* *** * ** **

ABHUlSB GCATCCGTCTACGGTAGGGCCGATGATTGGGGTG ABHUlSBfatS GCATCCGTCTACGGTAGGGCCGATGATTGGGGTG ABHU2SB GCATCCGTCTACGGTAGGGCCGATGATTGGGGTG UNSW3caefatS GCAGCCGTCTACGGTAGGGCCGATGATTGGGGTG *** ******************************

* indicate nucleotide positions which are conserved for all sequences represented in the alignment. Appendix 188

Appendix 3 Multiple sequence alignment of 16S rDNA sequences determined in this study representing bacterial group I as described in the text.

UNSW2.6liv AACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCAGGGAG-AAAGTCT-'ITI'GGGGAT HRI3liv AACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCAGGGAGCAAAGTCTCTTCGGGGAT HRilcae AACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCAGGGAG-AAAGTCTCTTCGGGGAT UNSWRSp12 ------AACACATGCAAGTCAGGGAGWAAAGTTTCTTCGGGACG UNSWMCSl AGACGNYGANCTGCGSGTGCTTAACACATGCAAGTCAGGGAGNAAAGN'K:CATTTGGAGC ******************** **** * **

UNSW2.6liv GATTAAACCGGCGCACGGGTGAGTAACACGTGAGTGACCTGCCTITI'AGACTGGAACAAC HRI3liv GATTAAACCGGCGCACGGGTGAGTAACACGTGAGTGACCTGCCTITI'AGACTGGAACAAC HRilcae GATTAAACCGGCGCACGGGTGAGTAACACGTGAGTGACCTGCCTITI'AGACTGGAACAAC UNSWRSpl2 AGNTAAACCGGCGCACGGGTGAGTAACACGTGAGTAACCTGCCTITI'AGACTGGAACAAC UNSWMCSl GAGTAAACCCGCGCACGGGTGAGTAACATGTGAGTAACCTGCCTITI'AGACTGGAACAAC ****** ****************** ****** ************************

UNSW2.6liv TTACCGAAAGGTGAGCTAATGCCGGATAAGTTATATAAGTGCATGTITATATAGGAAAAG HRI3liv TTACCGAAAGGTGAGCTAATGCCGGATGAGTTATATAAGTGCATGTITATATAGGAAAAG HRilcae TTACCGAAAGGTGAGCTAATGCCGGATGAGTTATATAAGTGCATGTITATATAGGAAAAG UNSWRSp12 TTACCGAAAGGTGAGCTAATGCCGGATGAATTATGTAACTGCATGGTTATATAGAAAAAG UNSWMCSl TTACCGAAAGGTGAGCTAATGCCGGATGAATTATRTATCTGCATGGGTATATAGAAAAAG *************************** * **** ** ****** ******* *****

UNSW2.6liv TTGGGGAGACCTGACGCTGAAAGATGGACTCGCGTCCCATTAGCTAGTTGGGAGGGTAAT HRI3liv TTGGGGAGACCTGACGCTGAAAGATGGACTCGCGTCCCATTAGCTAGTTGGTAGGGTAAT HRilcae TTGGGGAGACCTGACGCTGAAAGATGGACTCGCGTCCCATTAGCTAGTTGGTAGGGTAAT UNSWRSp12 CTGGGGCGACCTGGTGCTAAAAGAT-GACTCGCGTCCCATTAGCTAGTTGGTGGGGTAGA UNSWMCSl CTGGGGNRACCTGACGCTGAGAGATGGACTCGCGTCCCATTAGCTAGTTGGTGGGGTAGT ***** ***** *** * **** ************************* *****

UNSW2.6liv -GGCCTACCAAGGCGACGATGGGTAGCCGGCCTGAGAGGGTGGCCGGCCACACTGGGACT HRI3liv -GGCCTACCAAGGCGACGATGGGTAGCCGGCCTGAGAGGGTGGCCGGCCACACTGGGACT HRilcae -GGCCTACCAAGGCGACGATGGGTAGCCGGCCTGAGAGGGTGGCCGGCCACACTGGGACT UNSWRSp12 -AGCCTACCAAGGCGACGATGGGTAGCCGGCCTGAGAGGGTGGCCGGCCACACTGGGACT UNSWMCSl AGGCCTACCAAGGCGACGATGGGTAGCCGGCCTGAGAGGGTGGCCGGCCACACTGGGACT **********************************************************

UNSW2 .6liv GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATITTGCGCAATGCTCGTAA HRI3liv GAGACAC-GCCCAGACT-CTACGGGAGGCAGCAGTGGGGAATITTGCGCAATGCTCGTAA HRilcae GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATITTGCGCAATGCTCGTAA UNSWRSpl2 GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATITTGCGCAATGCTCGAGA UNSWMCSl GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATITTGCGCAATGCTCGAAA ******* ********* *************************************** *

UNSW2.6liv GAGTGACG-CAGCGACGCCGCGTGAATGACGAAGGCCTTCGGGTCGTAAAGTTCTTTCGA HRI3liv GAGTGACGCCAGCGACGCCGCGTGAATGACGAAGGCCTTCGGGTCGTAAAGTTCTTl'CGA HRilcae GAGTGACG-CAGCGACGCCGCGTGAA'mACGAAGGCCTTCGGGTCGTAAAGTTCTTl'CGA UNSWRSpl2 GAG'mACG-CAGCGACGCCGCGTGAA'mACGAAGGCCTTCGGGTCGTAAAGTTCTTTCGA UNSWMCSl GAG'mACG-CAGCGACGCCGCGTGAA'mACGAAGGCCTTCGGGTCGTAAAGTTCTTTCGA ******** *************************************************** Appendix 189

UNSW2.61iv CAGGGAAGAA-AATGCCTATAAGTAACTGTGTATGTATl'GACGGTACCTGTATAAGCAGC HRI31iv CAGGGAAGAA-AATGCCTATAAGTAACTGTGTATGTATl'GACGGTACCTGTATAAGCAGC HR.Ilcae CAGGGAAGAA-AATGCCTATAAGTAACTGTGTATGTATl'GACGGTACCTGTATAAGCAGC UNSWRSp12 CAGGGAAGAA-TGTGTATGGTAGTAACTGACTATACAGTGACGGTACCTGTATAAGCAGC UNSWMCSl CAGGGAAGAATTGTGTATANNAGTAACTGGCTATATATl'GACGGTACCTGTATAAGCAGC ********** ** * ******** *** * **********************

UNSW2.6liv CCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCGAGCGTTGTI'CGGAGT HRI31iv CCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCGAGCGTTGTI'CGGAGT HRilcae CCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCGAGCGTTGTI'CGGAGT UNSWRSp12 CCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCGAGCGTTGTI'CGGAGT UNSWMCSl CCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCGAGCGTTGTI'CGGAGT ************************************************************

UNSW2.6liv GACTGGGCGTAAAGAGCACGTAGGCGGTGTTGTAAGTCATTAGTCAAAGACTAGAGCTCA HRI31iv GACTGGGCGTAAAGAGCACGTAGGCGGTGTTGTAAGTCATTAGTCAAAGACTAGAGCTCA HR.Ilcae GACTGGGCGTAAAGAGCACGTAGGCGGTGTTGTAAGTCA.TTAGTCAAAGACTAGAGCTCA UNSWRSpl2 GACTGGGCGTAAAGAGCACGTAGGCGGTGTTGTAAGTCATTAGTCAAAGGCTAGAGCTCA UNSWMCSl GACTGGGCGTAAAGAGCACGTAGGCGGTGTTGTAAGTCATTAGTCAAAGACTAGAGCTCA ************************************************* **********

UNSW2.61iv ACTTTAGTAAGGCTAGTGATACTATAATACTAGAGTATCAGAGAGGATTGCAGAATI'CCT HRI3liv ACTTTAGTAAGGCTAGTGATACTATAATACTAGAGTATCAGAGAGGATTGCAGAATI'CCT HRilcae ACTTTAGTAAGGCTAGTGATACTATAATACTAGAGTATCAGAGAGGATTGCAGAATI'CCT UNSWRSpl2 ACTTTAGTAAGGCTAGTGATACTATAATACTAGAGTATCAGAGAGGATTGCAGAATI'CCT UNSWMCSl ACTTTAGTAAGGCTAGTGATACTATAGTACTAGAGTATCAGAGAGGATTGCAGAATI'CCT ************************** *********************************

UNSW2.61iv GGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAATACCGTTAGCGAAGGCGGCAATCTG HRI3liv GGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAATACCGTTAGCGAAGGCGGCAATCTG HRilcae GGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAATACCGTTAGCGAAGGCGGCAATCTG UNSWRSpl2 GGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAATACCGGTAGCGAAGGCGGCAATCTG UNSWMCSl GGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAATACCATTAGCGAAGGCGGCGATCTG *************************************** ************* *****

UNSW2.61iv GCTGGAAACTGACGCTGA-GTGCGAAAGCGT-GGTAGC-AACAGGATTAGATACCCTGGT HRI31iv GCTGGAAACTGACGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGT HR.Ilcae GCTGGAAACTGACGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGT UNSWRSpl2 GCTGGAAACTGACGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGT UNSWMCSl GCTGGAAACTGACGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGT ********~********* ************ ****** *********************

UNSW2.6liv AGTCCACGCTGTAAACGATGGATGCTAGGTGTTGGGCTTTTAAGTTCAGTGCCGCAGCAA HRI3liv AGTCCACGCTGTAAACGATGGATGCTAGGTGTTGGGCTTTTAAGTTCAGTGCCGCAGCAA HRilcae AGTCCACGCTGTAAACGATGGATGCTAGGTGTTGGGCTTTTAAGTTCAGTGCCGCAGCAA UNSWRSpl2 AGTCCACGCTGTAAACGATGGATGCTAGGTGTTGGGCTAATAAGTTCAGTGCCGCAGCAA UNSWMCSl AGTCCACGCTGTAAACGATGGATGCTAGGTGTTGGGCTATTAAGTTCAGTGCCGCAGCAA ************************************** ********************

UNSW2.61iv ACGCGATAAGCATCCCGCCTGGGGAGTACGTTTGCAAGAATGAAACTCAAAGGAATTG HRI3liv ACGCGATAAGCATCCCGCCTGGGGAGTACGTTTGCAAGAATGAAACTCAAAGGAATTG HR.Ilcae ACGCGATAAGCATCCCGCCTGGGGAGTACGTTTGCAAGAATGAAACTCAAAGGAATTG UNSWRSpl2 ACGCGATAAGCATCCCGCCTGGGGAGTACGTTTGCAAGAATGAAACTCAAAGGAATTG UNSWMCSl ACGCGATAAGCATCCCGCCTGGGGAGTACGTTTGCAAGAATGAAACTCAAAGGAATTG ********************************************************** Appendix 190

UNSW2.6liv ACGGGGGCCCGCACAAGCGGTGGAGCACGTGGTITAATTCGATGCTAACCGAAGAACCTT HRI3liv ACGGGGGCCCGCACAAGCGGTGGAGCACGTGGTTTAATTCGATGCTAACCGAAGAACCTT HRilcae ACGGGGGCCCGCACAAGCGGTGGAGCACGTGGTTTAATTCGATGCTAACCGAAGAACCTT UNSWRSp12 ACGGGGG-CCGCACAAGCGGTGGAGCACGTGG'ITI'AATTCGATGCTAACCGAAGAACCTT UNSWMCSl ACGGGGGNCCGCACAAGCGGTGGAGCACGTGGTTTAATTCGATGCTAACCGAAGAACCTT ******* ****************************************************

UNSW2.61iv ACCTGGGTTTGACATCCACAGAAGGCGTTAGAGATAATGC'ffiTGCCTGATTTATCAGGA HRI3liv ACCTGGGTTTGACATCCACAGAAGGCGTTAGAGATAATGC'ffiTGCCTGATTTATCAGGA HRilcae ACCTGGGTTTGACATCCACAGAAGGCGTTAGAGATAATGC'ffiTGCCTGATTTATCAGGA UNSWRSp12 ACCTGGGTTTGACATCCACAGAATACTATAGAGATATGGTAGAGCCTGATTTATCAGGA UNSWMCSl ACCTGGGTTTGACATCCACAGAATACTATAGAGATATGGTAGTGCCTGGTTTACCAGGA *********************** * ******** * * ***** **** *****

UNSW2.6liv GC'ffiTGAGACAGGTGC'ffiCA'ffiGC'ffiTCGTCAGCTCGTGCCGTGAGG'ffiTTGGGTTAAGT HRI3liv GC'ffiTGAGACAGGTGC'ffiCA'ffiGC'ffiTCGTCAGCTCGTGCCGTGAGG'ffiTTGGGTTAAGT HRilcae GC'ffiTGAGACAGGTGC'ffiCA'ffiGC'ffiTCGTCAGCTCGTGCCGTGAGG'ffiTTGGGTTAAGT UNSWRSp12 AC'ffiTGAGACAGGTGC'ffiCA'ffiGC'ffiTCGTCAGCTCGTGCCGTGAGG'ffiTTGGGTTAAGT UNSWMCSl AC'ffiTGAGACAGGTGC'ffiCATGGC'ffiTCGTCAGCTCGTGCCGTGAGG'ffiTTGGGTTAAGT ***********************************************************

UNSW2.6liv CCCGCAACGAGCGCAACCCCTATTTCCAGTTGCTAACGGTTGAAGCTGAGCACTCTGGAG HRI3liv CCCGCAACGAGCGCAACCCCTATTTCCAGTTGCTAACGGTTGAAGCTGAGCACTCTGGAG HRilcae CCCGCAACGAGCGCAACCCCTATTTCCAGTTGCTAACGGTTGAAGCTGAGCACTCTGGAG UNSWRSpl2 CCCGCAACGAGCGCAACCCCTATTTCCAGTTGCTAACGGGTAGAGCTGAGCACTCTGGAG UNSWMCS1 CCCGCAACGAGCGCAACCCCTATTTCCAGTTGCTAACGGGTTAAGCTGAGCACTCTGGAG *************************************** * *****************

UNSW2.6liv AGACTGCCAGCGATAAGCTGGAGGAAGGTGGGGACGA'ffiTCAAGTCATCA'ffiGCCCTTAT HRI3liv AGACTGCCAGCGATAAGCTGGAGGAAGGTGGGGACGACGTCAAGTCATCA'ffiGCCCTTAT HRilcae AGACTGCCAGCGATAAGCTGGAGGAAGGTGGGGACGACGTCAAGTCATCA'ffiGCCCTTAT UNSWRSp12 AGACTGCCAGCGATAAGCTGGAGGAAGGTGGGGACGACGTCAAGTCATCA'ffiGCCCTTAT UNSWMCSl GGACTGCCAGCGATAAGCTGGAGGAAGGTGGGGACGACGTCAAGTCATCA'ffiGCCCTTAT ************************************ **********************

UNSW2.6liv GTCCAGGGCTACACACGTGCTACAA'ffiGCATAATCAGAGGGAAGCATCTCCGCAAGGATA HRI3liv GTCCAGGGCTACACACGTGCTACAA'ffiGCATAATCAGAGGGAAGCATCTCCGCAAGGATA HRilcae GTCCAGGGCTACACACGTGCTACAA'ffiGCATAATCAGAGGGAAGCATCTCCGCAAGGATA UNSWRSp12 GTCCAGGGCTACACACGTGCTACAA'ffiGCATAATCAGAGGGAAGCAACTCCGAGAGGATA UNSWMCSl GTCCAGGGCTACACACGTGCTACAA'ffiGCATAATCAGAGGGAAGCAGCTCCGAGAGGATA ********************************************** ***** ******

UNSW2.6liv AGCGAATCTCATAAATTA'ffiTCTCAGTTCAGATTGCAGTC'ffiCAACTCGAC'ffiCATGAAG HRI3liv AGCGAATCTCATAAATTA'ffiTCTCAGTTCAGATTGCAGTC'ffiCAACTCGAC'ffiCATGAAG HRilcae AGCGAATCTCATAAATTA'ffiTCTCAGTTCAGATTGCAGTC'ffiCAACTCGAC'ffiCATGAAG UNSWRSp12 AGCGAATCTCATAAAGTA'ffiTCTCAGTTCAGATTGCAGTC'ffiCAACTCGAC'ffiCATGAAG UNSWMCSl AGCGAATCTCAGAAAGTA'ffiTCTCAGTTCAGATTGCAGTC'ffiCAACTCGAC'ffiCATGAAG *********** *** ********************************************

UNSW2.61iv TCGGAATCGCTAGTAATCGCAGATCAGCAAAGCTGCGGTGAATACGTTCCCGGGCCT'ffi HRI3liv TCGGAATCGCTAGTAATCGCAGATCAGCAAAGCTGCGGTGAATACGTTCCCGGGCCT'ffi HRilcae TCGGAATCGCTAGTAATCGCAGATCAGCAAAGCTGCGGTGAATACGTTCCCGGGCCT'ffi UNSWRSpl2 TCGGAATCGCTAGTAATCGCAGATCAGCAAAGCTGCGGTGAATACGTTCCCGGGCCT'ffi UNSWMCSl TCGGAATCGCTAGTAATCGCAGATCAGCAAAGCTGCGGTGAATACGTTCCCGGGCCT'ffi *********************************************************** Appendix 191

UNSW2.6liv TACACACCGCCCGTCACACCACGGGAGTCGGTCGCGCCTGAAGCCGGTGGCCTATCAG HRI3liv TACACACCGCCCGTCACACCACGGGAGTCGGTCGCGCCTGAAGCCGGTGGCCTATCAG HRilcae TACACACCGCCCGTCACACCACGGGAGTCGGTCGCGCCTGAAGCCGGTGGCCTATCAG UNSWRSpl2 TACACACCGCCCGTCACACCACGGGAGTCGGTCGCGCCTGAAGCCGGTGGCCTACCCG UNSWMCSl TACACACCGCCCGTCACACCACGGGAGTCGGTCGCGCCTGAAGCCGGTGGCCTATCAG ****************************************************** * *

UNSW2.6liv TAATGGGGGAGCCGTCTATGGCGAGATTGGTAACTGGGGTG HRI3liv TAATGGGGGAGCCGTCTATGGCGAGATTGGTAACTGGGGTG HR.Ilcae TAATGGGGGAGCCGTCTATGGCGAGATTGGTAACTGGGGTG UNSWRSpl2 TTATGAGGGANCCGTCTATGGCGAGATTGGTAACTGGGGTG UNSWMCSl T-AT-GGGGAACC-TCTATGGCGAGATTGGTAACTGGGGTG * ** **** ** ***************************

* indicate nucleotide positions which are conserved for all sequences represented in the alignment. A good friend once asked me over a beer at the Royal.. ...

"When are you going to stop using the primer and do the final coat?"

Well, I guess this is it.. ...