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 ................................................................................................