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Functional Genomic Examinations of Interactions Between Common Members of the Umh an Gut Microbiota Michael Mahowald Washington University in St

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Functional Genomic Examinations of Interactions Between Common Members of the Umh an Gut Microbiota Michael Mahowald Washington University in St Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) January 2010 Functional Genomic Examinations Of Interactions Between Common Members Of The umH an Gut Microbiota Michael Mahowald Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Mahowald, Michael, "Functional Genomic Examinations Of Interactions Between Common Members Of The umH an Gut Microbiota" (2010). All Theses and Dissertations (ETDs). 222. https://openscholarship.wustl.edu/etd/222 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY Division of Biology and Biomedical Sciences Molecular Microbiology and Microbial Pathogenesis Dissertation Examination Committee: Jeffrey I. Gordon, Chair Douglas E. Berg Michael G. Caparon Daniel E. Goldberg Elaine R. Mardis Clay F. Semenkovich FUNCTIONAL GENOMIC EXAMINATIONS OF INTERACTIONS BETWEEN COMMON MEMBERS OF THE HUMAN GUT MICROBIOTA by Michael Anthony Mahowald A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2010 Saint Louis, Missouri Copyright by Michael Anthony Mahowald 2010 Dedication To my parents, Anthony P. and Mary Briody Mahowald ii ABSTRACT OF THE DISSERTATION Functional genomic examinations of interactions between common members of the human gut microbiota by Michael Anthony Mahowald Doctor of Philosophy in Biology and Biomedical Sciences (Molecular Microbiology and Microbial Pathogenesis) Washington University in St. Louis, 2010 Professor Jeffrey I. Gordon, Chairperson The adult human gut microbiota consists of hundreds to thousands of bacterial spe- cies, the majority belonging to the Bacteroidetes and the Firmicutes. Differences in the bal- ance between these phyla has been linked to obesity in mice and humans. However, little is known about their interactions in vivo. I have used comparative and functional genom- ics, proteomics and biochemical assays to identify the ways they marshal their genomic resources to adapt to life together in the distal gut. I first annotated the complete genome sequences of two human gut Bacteroidetes (Bacteroides vulgatus and Parabacteroides distasonis) and two Firmicutes (Eubacterium rectale and E. eligens). By comparing the genomes of all sequenced gut Bacteroidetes and Firmicutes, I found that gut Bacteroidetes’ genomes contain large groups of genes responsible for (i) sensing, binding, and metabolizing the varied polysaccharides that they encounter in the distal intestine; and (ii) constructing their polysaccharide capsules. These portions of their genomes have been shaped by lateral gene transfer, including phage and conjugative transposons, as well as by gene duplication. By colonizing germ-free mice with B. thetaiotaomicron, or B. vulgatus, or both species together, I documented that B. vulgatus upregulates its unique glycan-degrading enzymes to adapt to the presence of B. iii thetaiotaomicron. In contrast to the Bacteroidetes, the Firmicutes have smaller genomes, a signifi- cantly smaller proportion of glycan-degrading genes, and are suited to degrade a more specialized assortment of dietary carbohydrates. By colonizing germ-free mice with E. rectale and/or B. thetaiotaomicron, I showed that B. thetaiotaomicron, like B. vulgatus, upregulates its unique glycoside hydrolase activities to adapt to the presence of E. rectale, increasing its degradation of host-derived glycans that E. rectale cannot use. In contrast, E. rectale downregulates its polysaccharide degradation genes and upregulates nutrient transporters, likely allowing it to access sugars released by B. thetaiotaomicron’s glycoside hydrolases. These models of the human gut microbiota illustrate niche specialization and functional redundancy within the Bacteroidetes, the adaptable niche specialization that likely underlies the success of Firmicutes in this habitat, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability. iv Acknowledgements Jeffrey Gordon’s lab has been an amazing place to do research. His constant en- couragement and indomitable enthusiasm are incredible to behold, and have been an enor- mous boost at the times when I’ve felt things ought to be going better. I am enormously thankful for the trust and patience he has shown me as I’ve learned my way over the years. The amount of freedom I’ve had to explore scientifically has been wonderful and is clearly quite unique. His attitude makes his lab a fun place to be a student. Without a doubt the best aspect of the lab has been the outstanding group of people he has brought together and continues to renew. I count myself enormously privileged to have had the chance to interact with and learn from everyone who has been a part of his dy- namic group. I could not have accomplished any of this work without more help, expertise, enthusiasm, advice, support, and care from the whole group than I can possibly recount. Nonetheless, a few deserve special mention. Jill Manchester, in addition to her fabulous abilities as a biochemist (without whom virtually none of the biochemical assays reported here would have been done), has been a great lab mom, extremely caring and supportive throughout, in spite of all the forgotten messes I’ve left around the lab (sorry!). Sabrina Wagoner, Dave O’Donnell and Maria Karlsson are all incredibly talented and patient and made all the mouse work, among many other things, possible. It’s hard to imagine how the Gordon lab could function without them. Dr. Janaki Guruge and my classmate Lara Crock started work in the lab on E. rectale, and I am very thankful to them for the groundwork that made my way forward much, much easier. Janaki has been a great friend and source of microbiological advice and laughs. I started my work in the lab with J. F. Rawls, who was as good a mentor, and as kind a person, as I have encountered. Aside from his outstanding science, his ability to plan and v see the big picture outlook for his work were and are an inspiring example and served as a perfect introduction to the lab. Federico Rey and Henning Seedorf have been of particular help with the second half of this work; they have both taught me an enormous amount about bacterial metabo- lism and been a true joy to work with. Their help, enthusiasm and generosity has made this work far better than it could have been otherwise. Eric Martens holds an encyclope- dic knowledge of microbiology in general and the B. theta genome in particular, and has been a model example of technical and scientific rigor and focus – not to mention a fun and kind individual and master brewer. I counted on Buck Samuel as a constant source of technical and grad-student-life advice and generous and careful feedback. The lab would have been a substantially more difficult place to be a student without him. Dan Peterson and Peter Crawford have both been fountains of career advice and encouragement, as well as extremely valuable constructive criticism and perspective, whom I’ll miss a great deal. Swaine Chen has helped deepen my understanding of biostatistics and has also been a great source of critical feedback. Priya Sudarsanam has made a great bay-mate over the last year and helped me keep my lab work in proper perspective even as she’s challenged me to think more deeply about it. Justin Sonnenburg was a valuable glycobiology resource. Ruth Ley offered much of her always creative critique and insight to all matters to do with microbial ecology. Doug Leip taught me much about software design and scripting, and has been a supportive friend. Marios Giannakis and I entered Wash. U. as classmates, and his friendship has been a great blessing over the last six years since we joined the Gordon lab; I hope it will be so for years to come. He was (and remains) the one person I know I can ask for instantaneous recall of anything I might have once known in medical school or college; combining an incredible memory with intellect, enthusiasm, and kindness to match made his influence on me, and I venture to suppose, the lab in general, one that will be missed. He brings the same vi enthusiasm to everything from major league eating to studying chronic atrophic gastritis (although in some ways those two aren’t so unrelated). Pete Turnbaugh entered the lab soon after I did, and his critiques probably have an even larger part of the work shown here than I realize – and that, by the way, is saying quite a lot. His scientific opinion, as well as his taste in movies, has been an inspiration over the years, and hopefully will continue to be. Many other friends within the medical school, especially Vinod Rao, Tina Ling, Chung Lee, Bill Hucker, Bryson Katona, Ram Akilesh and Bill McCoy, have been sup- portive of all my efforts. Many a good time spent over a pitcher, a few rounds of darts, or a good dinner will be fondly remembered. Also, of course, the MSTP staff, Brian Sullivan, Andrew Richards, Christy Durbin and Liz Bayer, have been a huge help with keeping ev- erything going behind the scenes, and more importantly, in setting up, together with the directors, Dan Goldberg and Wayne Yokoyama, such a well-oiled machine of a program. Their work over the years has saved me much trouble (and excessive class time) and made for a much nicer experience. My thesis committee, particularly my chair, Doug Berg, has offered great advice and much of their time to this project, and I thank them for their help. Laura Kyro, Stephanie Amen and Debbie Peterson are the three highly able assistants in the C.G.S., without whom countless tasks, not least of them piecing together this thesis, would be much more difficult.
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