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Molecular Analysis of Small Rna and Small Protein Regulation of Escherichia Coli Stress Responses

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Molecular Analysis of Small Rna and Small Protein Regulation of Escherichia Coli Stress Responses MOLECULAR ANALYSIS OF SMALL RNA AND SMALL PROTEIN REGULATION OF ESCHERICHIA COLI STRESS RESPONSES BY CHELSEA R. LLOYD DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2018 Urbana, Illinois Doctoral Committee: Associate Professor Carin K. Vanderpool, Chair Professor John E. Cronan Professor William W. Metcalf Professor Peter A. B. Orlean Abstract Small RNA (sRNA) regulators control gene expression throughout all domains of life. In bacteria, they typically affect virulence, metabolism, and stress response genes posttranscriptionally through imperfect antisense pairing with their mRNAs. While most sRNAs are non-coding, a small number act as mRNAs themselves by encoding functional proteins. This study examines the regulatory and physiological effects of both a non-coding sRNA, DicF, and the protein product of a dual-function sRNA, SgrS in Eschericha coli. The sRNA SgrS encodes the small 43-amino acid protein SgrT. Both molecules are expressed during glucose-phosphate stress - a bacteriostatic condition in which phosphosugars accumulate in the cell either because of mutations in glycolysis or because of the transport of non-metabolizable glucose analogs such as αMG or 2DG. While both SgrT and SgrS base pairing can independently mitigate glucose-phosphate stress, they do so through distinct mechanisms. SgrS base pairing destabilizes the mRNA of the respective major and minor glucose transporters PtsG and ManXYZ, thereby inhibiting synthesis of additional glucose permeases and restricting further influx of non- metabolizable sugars. In this study we demonstrate that SgrT acts to specifically inhibit the transport activity of preexisting PtsG transporters, but does not affect ManXYZ. We reveal that by targeting PtsG transport activity SgrT not only prevents influx of non- metabolizable αMG, but also overrides inducer exclusion allowing alternative carbon sources to be transported and metabolized during stress. We also uncover the regions of PtsG that are required for SgrT regulation. ii Although the precise nature of glucose-phosphate stress is not well understood, this work establishes that sugar phosphates are not inherently toxic, but most likely inhibit growth by depleting glycolytic intermediates, as these are expended to transport sugars and not are replenished due to a blockage in glycolysis. Sugar phosphate accumulation is, however, problematic and cells cope by effluxing excess sugars out of the cell. Phosphosugar efflux must be preceded by dephosphorylation and previous studies found that the phosphatase YigL is posttranscriptionally stabilized by SgrS under stress to promote this process. However, we still have yet to identify the efflux pump through which these sugars are flushed out of the cell. Here we provide evidence that the multidrug efflux channel TolC may be involved as tolC mutants exhibit impaired αMG efflux and more impaired growth when combined with an sgrS mutation. While the benefits of SgrS and SgrT to E. coli physiology are clear, the role of the sRNA DicF remains elusive. DicF is produced from the cryptic prophage Qin and while we have yet to identify the conditions under which it is naturally produced, ectopic expression of DicF leads to growth inhibition and filamentation. Previous work has identified a few DicF targets including ftsZ, which causes filamentation, no single target can be attributed to the growth defect we observe. Here we use a combination of approaches to uncover the novel targets ahpC and mdfA. While mutating ahpC has no effect on growth, mdfA (which encodes a proton/sodium/potassium antiporter) mutants partially rescue cell growth. Additionally, under alkaline conditions mdfA mutants expressing DicF are able to outcompete wild type cells. While there are more targets to uncover that contribute to growth inhibition and we have more to learn about DicF regulation and why its maintenance is beneficial, this study provides new insights into E. iii coli physiology during DicF expression and highlights the challenges associated with characterizing sRNA targetomes. iv Acknowledgements First and foremost I want to thank Dr. Cari Vanderpool for being such an inspiring and supportive advisor. She gave me the freedom and opportunity to pursue my passions of teaching and mentorship, and to answer my own scientific questions. She also fostered a fun, energized, and collaborative work environment that made lab feel like a second (or first) home. I also want to acknowledge the past and present members of the Vanderpool lab for being amazing coworkers, friends, travel buddies, Curie sitters, and Secret Santas. I would like to thank my past and present committee members: Dr. John Cronan, Dr. Bill Metcalf, Dr. Peter Orlean, Dr. Jeff Gardner, and the late Dr. Charles Miller for all of their invaluable guidance and advice. Additionally, I would also like to thank Dr. Jim Slauch for his helpful feedback during our group meetings. For their roles in developing and supporting my teaching interests and abilities, I would like to thank Brad Mehrtens, Melissa Reedy, and J.P. Swigart. I am also extremely grateful for Deb LeBaugh, Diane Tsevelekos, and Shawna Smith for all of their help behind the scenes and for letting me nerd-out about fecal bacteria in the microbiology office. I could not have asked for a better, more collaborative department to be a part of. In addition to wonderful research and teaching support I have received at UIUC, I also want to acknowledge the members of Illini Pole Fitness, Carnivale Debauche, and the Defy Gravity community for providing a creative outlet and supportive space for my mind and body. I have made so many personal gains because of those amazing humans. I have been privileged to have a host of teachers and mentors that developed and encouraged my interest in studying science. Thanks to my undergraduate research v advisors at the University of Iowa, Dr. Rich Roller and Dr. John Kirby, for the opportunity to fall in love with research in their labs and for encouraging me to attend graduate school at UIUC. Thanks to my Quincy Senior High School teachers Sarah Stewart, Jackie Stewart, and Dr. Sandra Spalt-Fulte for being inspirational instructors and for encouraging me to pursue a career in STEM; and thanks to Kathi Dooley for shaping me into the person I am today through performance and Dooleycising. I am especially thankful to my 4th grade science teacher Marilyn Meyer for sparking my lifelong love for microbiology by reading graphic excerpts from “The Hot Zone” by Richard Preston to our class. That was a transformative moment when I realized just how powerful microbes could be. Last but not least, I want to thank my family for being so encouraging and supportive during my PhD. Thanks to my Aunt Marilyn, without whom I would not have been able to attend college. Thanks to my late father Ron for his encouragement, curiosity, and for igniting my interest in science. Thanks to my mom Sharon who has always been my biggest cheerleader – who has always been excited about my work and given me unwavering support. She always told me, “Science is like Italian – I love hearing it, but I don’t understand a word of it.” I love her bigger than the universe. Lastly, I thank my amazing partner Jake and cat Curie for their compassion, encouragement, and support through the highs and lows, but mostly I thank them for pretending to be as excited as I am about bacterial physiology. vi This work is dedicated to my family. I love you bigger than the universe. vii Table of Contents Chapter 1: Introduction........................................................................................................1 1.1 Escherichia coli as a model organism ...........................................................................1 1.2 Small RNA (sRNA) regulators ......................................................................................2 1.2.1 Regulatory sRNAs in eukaryotes and archaea....................................................2 1.2.2 Regulatory sRNAs in bacteria ............................................................................4 1.2.3 Dual-function sRNAs..........................................................................................7 1.3 The carbohydrate phosphoenolpyruvate phosphotransferase system (PTS) .................8 1.3.1 The major glucose transporter EIIBCGlc (PtsG)................................................10 1.3.2 The broad substrate transporter EIIABCDMan (ManXYZ) ...............................11 1.3.3 The N-acetylglucosamine transporter EIIBCANag (NagE)................................12 1.4 Glucose-phosphate stress.............................................................................................13 1.4.1 Induction of the stress response by the transcription factor SgrR ....................17 1.4.2 The small RNA SgrS ........................................................................................18 1.4.3 The small protein SgrT .....................................................................................20 1.5 The small RNA DicF ..................................................................................................22 1.5.1 Targets of DicF regulation................................................................................23 1.5.2 Effects of DicF on E. coli physiology...............................................................24 1.6 Aim of the study...........................................................................................................25
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