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RPOS REGULON MODULATION BY ENVIRONMENTAL SELECTION RPOS REGULON MODULATION BY ENVIRONMENTAL SELECTION By SARAH M. CHIANG, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Sarah M. Chiang, May 2012 DOCTOR OF PHILOSOPHY (2012) McMaster University (Biology) Hamilton, Ontario TITLE: RpoS regulon modulation by environmental selection AUTHOR: Sarah M. Chiang, B.Sc. (Redeemer University College, Ancaster, ON) SUPERVISOR: Dr. Herb E. Schellhorn NUMBER OF PAGES: xi, 257 ii ABSTRACT Regulatory interactions evolve to incorporate new genomic material and contribute to bacterial diversity. These regulatory interactions are flexible and likely provide bacteria with a means of rapid environmental adaptation. In this thesis, the RpoS regulon is used as a model system to investigate the hypothesis that regulon composition and expression are modified according to environmental pressures. Several novel findings are presented, namely the distribution of RpoS homologs in bacteria, the flexibility of the RpoS regulon, and the effect of diverse environmental pressures on RpoS regulon expression. Based on phylogenetic and reciprocal best hits analyses, RpoS was determined to be conserved in γ-, β-, and δ-proteobacteria, likely because it confers a selective advantage in many bacterial niches. Regulon composition, however, was highly flexible. Even between species of the same class, Escherichia coli and Pseudomonas aeruginosa , only 12 of 50 orthologs were regulated in common by RpoS. RpoS regulon flexibility may thus be the result of adaptation to different bacterial habitats. Indeed, mutations in rpoS and differential regulon expression could be identified among environmental E. coli isolates collected from diverse sources. Among environmental E. coli isolates, RpoS mutant frequency was found to be 0.3%, and activity of KatE, a prototypical RpoS regulon member, was undetectable in some isolates despite the presence of functional RpoS. Modulated RpoS regulon expression among environmental E. coli isolates is consistent with environment as a key factor shaping regulatory interactions. Regulon flexibility was similarly apparent in oxidative stress regulons, OxyR and SoxRS, of E. coli . SoxRS regulon function is weakly conserved, possibly due iii to low selective pressure for a superoxide stress response regulon in some bacterial species. Environment, therefore, is a crucial element that defines the dynamics of regulatory networks. iv ACKNOWLEDGEMENTS Pursuing my Ph.D. was an amazing journey, and I am grateful to many people who supported and guided me throughout. Firstly, I would like to thank my supervisor, Herb Schellhorn, for providing direction in my project and giving me opportunities for academic growth. As well, I would like to thank the members of my supervisory committee, Radhey Gupta and Jonathan Dushoff, for their insight into my project and constructive feedback. Thank you to my collaborators, Tao Dong and Tom Edge, who helped to publish the work on environmental E. coli . As well, thanks to Richard Morton for his guidance in the study on RpoS evolution and to Asher Pasha for computer programming. I am also grateful for the friendship of people in the lab. To Tao, it was a pleasure to share the Ph.D. life, and I appreciate your encouragement in research (and your company in procrastination). To Sharmila, I enjoyed having a partner in crime and your style input. To Mirella and Thuraya, thanks for your support during the early years of my graduate education. I would also like to thank the numerous friends and colleagues who made my experience in the lab memorable: Rosemary, Ryan, Shirley, Hamoody, Mahi, Lingzi, Steve, Andrew O., Richard, Jas, Dan, Charlie, Daniel, Vithooshan, Floriana, Nicole, Eva, Ali L., Beckie, Hamza, Philip, Michelle, Cindy, Matthew T., Andrew H., Ali I., Susan, Alvin, and Jennifer. Lastly, I am blessed by the encouragement of Matt, and I am forever grateful to my parents for believing in my ability and cheering me on in my academic pursuits. v PREFACE This thesis is structured in sandwich format and includes three published articles. Chapter 1 (Introduction) provides the necessary background and context for the published articles (Chapters 2-4). Chapter 2, published in Journal of Molecular Evolution in 2010, elucidates the evolution of RpoS and the flexibility of the RpoS regulon between species. Chapter 3, published in Applied and Environmental Microbiology in 2011, demonstrates environmental selection of RpoS mutants and regulon modulation in environmental E. coli isolates. Chapter 4, published in Archives of Biochemistry and Biophysics in 2012, reviews the major oxidative stress regulons of E. coli , OxyR and SoxRS, and their functional conservation in bacteria. Lastly, Chapter 5 (Discussion) discusses how the previous chapters constitute one body of work and support regulon flexibility as a consequence of environmental pressures. S. M. Chiang is the sole contributor of all published articles included in this thesis. Dr. Herb E. Schellhorn supervised and provided direction for Chapters 2-4. Dr. Tao Dong helped in the initial screening for RpoS mutants among environmental E. coli isolates in Chapter 3. Dr. Thomas A. Edge provided environmental E. coli isolates and source details in Chapter 3. The published articles are modified according to thesis format requirements and are printed in this thesis under license. vi TABLE OF CONTENTS Abstract .............................................................................................................................. iii Acknowledgements .............................................................................................................. v Preface ................................................................................................................................. vi Table of Contents .............................................................................................................. vii List of Tables ...................................................................................................................... ix List of Figures ...................................................................................................................... x List of Abbreviations and Symbols ..................................................................................... xi CHAPTER 1: INTRODUCTION ........................................................................................ 1 1.1 Bacterial evolution ..................................................................................................... 2 1.1.1 Regulon development .......................................................................................... 3 1.1.2 Sigma factor conservation .................................................................................. 5 1.2 E. coli sigma factors ................................................................................................... 6 1.2.1 E. coli sigma 70 factors ...................................................................................... 7 1.2.2 E. coli sigma 54 factor ...................................................................................... 10 1.3 RpoS regulon expression ......................................................................................... 10 1.3.1 Induction of RpoS .............................................................................................. 10 1.3.2 RpoS function in stationary phase and stress response .................................... 12 1.4 Mutations in rpoS ..................................................................................................... 15 1.4.1 RpoS functional regions .................................................................................... 15 1.4.2 Environmental selection of RpoS mutants ........................................................ 16 1.5 Thesis overview ....................................................................................................... 17 CHAPTER 2: EVOLUTION OF THE RPOS REGULON: ORIGIN OF RPOS AND THE CONSERVATION OF RPOS-DEPENDENT REGULATION IN BACTERIA.............. 20 2.1 Preface ...................................................................................................................... 21 2.2 Abstract .................................................................................................................... 22 2.3 Introduction .............................................................................................................. 24 2.4 Methods .................................................................................................................... 27 2.5 Results ...................................................................................................................... 30 2.6 Discussion ................................................................................................................ 39 2.7 Conclusions .............................................................................................................. 46 2.8 Acknowledgements .................................................................................................. 47 2.9 References ................................................................................................................ 58 CHAPTER 3: PHENOTYPIC DIVERSITY CAUSED BY DIFFERENTIAL RPOS ACTIVITY AMONG ENVIRONMENTAL ESCHERICHIA COLI ISOLATES
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