University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2014 Comparative Genomics of Microbial Chemoreceptor Sequence, Structure, and Function Aaron Daniel Fleetwood University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Bacteriology Commons, Biochemistry Commons, Bioinformatics Commons, Computational Biology Commons, Environmental Microbiology and Microbial Ecology Commons, Genomics Commons, Microbial Physiology Commons, Molecular Biology Commons, Organismal Biological Physiology Commons, Other Biochemistry, Biophysics, and Structural Biology Commons, Pathogenic Microbiology Commons, Structural Biology Commons, and the Systems Biology Commons Recommended Citation Fleetwood, Aaron Daniel, "Comparative Genomics of Microbial Chemoreceptor Sequence, Structure, and Function. " PhD diss., University of Tennessee, 2014. https://trace.tennessee.edu/utk_graddiss/3125 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Aaron Daniel Fleetwood entitled "Comparative Genomics of Microbial Chemoreceptor Sequence, Structure, and Function." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Igor B. Jouline, Major Professor We have read this dissertation and recommend its acceptance: Robert Hettich, Elias Fernandez, Elizabeth Howell Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Comparative Genomics of Microbial Chemoreceptor Sequence, Structure, and Function A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Aaron Daniel Fleetwood December 2014 Copyright © by Aaron Daniel Fleetwood All rights reserved. ii Dedication To my Wife, Dr. Ellen Ann Fleetwood To my Family, Dan, Betsy, Zach, and Nathan To The Messengers, Mark, Theresa, and Paul And To Friends Cougar, Ketra, Nash, Ginger, and Piece iii Acknowledgments I would like to express sincere and profound appreciation to my mentor, Dr. Igor B. Zhulin, who has been an unwavering source of support and guidance in addition to expanding and sharpening my scientific worldview. I would also like to thank the Genome Science and Technology Program for taking a chance on what on paper might have appeared to be an economist and linguist, not a biologist. In particular, Dr. Albrecht von Arnim has been a tremendous resource and advisor over the years. I am ever grateful to my committee members, Dr. Robert Hettich, Dr. Elias Fernandez, and Dr. Elizabeth Howell. They, along with Dr. Zhulin, are responsible for fostering and nurturing my passion for genome and protein science, as well as shaping my scientific philosophy at every stage and level of my graduate experience. Additionally, many outstanding faculty, including Dr. Jerome Baudry and Dr. Tim Sparer, were instrumental in both my academic education and my growth as a scientist and professional. Thank you also to my lab mates Amit Uphadyay, Ogun Adebali, Dr. Kirill Borziak, and Dr. Davi Ortega for wonderful meetings, discussions, and camaraderie. I also owe a debt of gratitude to past members of the Zhulin Lab whose past and current work have served as a foundation and inspiration for my research (Drs. Kristen Wuichet, Luke Ulrich, Roger Alexander, and Brian Cantwell). Finally, thank you to our experimental collaborators and advisors (Drs. Brian Crane, Victoria Korolik, Caroline Harwood, and Harry Mobley) for bringing our ideas to vivo. I would not be here today without tremendous support and funding from the Genome Science and Technology Program, the J. Wallace and Katie Dean Multi-Year Graduate Fellowship, the UTK Graduate School, the Microbiology Department, Oak Ridge National Laboratory and the National Institutes of Health. iv Abstract Microbial chemotaxis receptors (chemoreceptors) are complex proteins that sense the external environment and signal for flagella-mediated motility, serving as the GPS of the cell. In order to sense a myriad of physicochemical signals and adapt to diverse environmental niches, sensory regions of chemoreceptors are frenetically duplicated, mutated, or lost. Conversely, the chemoreceptor signaling region is a highly conserved protein domain. Extreme conservation of this domain is necessary because it determines very specific helical secondary, tertiary, and quaternary structures of the protein while simultaneously choreographing a network of interactions with the adaptor protein CheW and the histidine kinase CheA. This dichotomous nature has split the chemoreceptor community into two major camps, studying either an organism’s sensory capabilities and physiology or the molecular signal transduction mechanism. Fortunately, the current vast wealth of sequencing data has enabled comparative study of chemoreceptors. Comparative genomics can serve as a bridge between these communities, connecting sequence, structure, and function through comprehensive studies on scales ranging from minute and molecular to global and ecological. Herein are four works in which comparative genomics illuminates unanswered questions across the broad chemoreceptor landscape. First, we used evolutionary histories to refine chemoreceptor interactions in Thermotoga maritima, pairing phylogenetics with x-ray crystallography. Next, we uncovered the origin of a unique chemoreceptor, isolated only from hypervirulent strains of Campylobacter jejuni, by comparing chemoreceptor signaling and sensory regions from Campylobacter and Helicobacter. We then selected the opportunistic human pathogen Pseudomonas aeruginosa to address the question of assigning multiple chemoreceptors to multiple chemotaxis pathways within the same organism. We assigned all P. aeruginosa receptors to pathways using a novel in silico approach by incorporating sequence information spanning the entire taxonomic order Pseudomonadales and beyond. Finally, we surveyed the chemotaxis systems of all environmental, commensal, v laboratory, and pathogenic strains of the ubiquitous Escherichia coli, where we discovered an ancestral chemoreceptor gene loss event that may have predisposed a well-studied subpopulation to adopt extra-intestinal pathogenic lifestyles. Overall, comparative genomics is a cutting edge method for comprehensive chemoreceptor study that is poised to promote synergy within and expand the significance of the chemoreceptor field. vi Preface In the following dissertation, comparative genomics methods were used to study microbial chemoreceptor sequences, structures, and functions. Microbial in this case refers primarily to Bacteria, though chemoreceptors are found in Archaea as well (hence the broader term in the title). The first chapter will be an introduction, in which I provide a brief overview of the motivations behind this work before diving into the scientific discipline (computational biology), methods (comparative genomics), and tools (bioinformatics) that are utilized throughout. This section covers aspects ranging from the basic (core tenets of biology) to the technical (pitfalls and nuances of comparative work). I will then introduce the field of chemotaxis with a special emphasis on chemoreceptors. This introductory “review” will be further subdivided into two major sections corresponding to the two major communities that study chemoreceptors: structure-focused signal transduction work and microbiology-based behavioral/physiological studies. The motivations of these two fields are quite different, with the former pursuing more fundamental and mechanistic understanding and the latter seeking applied connections, such as links to pathogenicity. After the introductory chapter, the main body of the dissertation is divided into four additional chapters which correspond to full peer-reviewed publications, manuscripts submitted for review, or featured aspects of manuscripts in preparation, all of which have been produced during my graduate work. Biology today (especially computational work) is a collaborative and highly interdisciplinary endeavor, so for each chapter I provide the contributions of myself and the other authors, as well as contributions outside of authorship but still deserving of mention. Each chapter will consist of an abstract, introduction, results, and discussion section, and any materials and methods specific to that paper or published in the manuscript will accompany these chapters as well. Chapter 3 (Assigning Chemoreceptors to Pathways in Pseudomonas aeruginosa) represents my most significant independent effort as a graduate student. Beginning vii with the first chapter, the studies increase in organizational complexity from studying a handful of proteins in a limited set of organisms to comparing entire sets of chemotaxis systems across broad taxonomic divisions. This serves to illustrate the power of comparative genomics to adapt to the scale of the biological problem, and for proteins like chemoreceptors,