Graduate Theses, Dissertations, and Problem Reports 2005 Stationary phase induction of RpoS in enteric bacteria Matthew Louis Hirsch West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Hirsch, Matthew Louis, "Stationary phase induction of RpoS in enteric bacteria" (2005). Graduate Theses, Dissertations, and Problem Reports. 2243. https://researchrepository.wvu.edu/etd/2243 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Stationary Phase Induction of RpoS in Enteric Bacteria Matthew Louis Hirsch Dissertation submitted to the School of Medicine at West Virginia University In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology, Immunology and Cell Biology Thomas Elliott Ph.D, Chair Nyles Charon Ph.D Lisa Salati Ph.D. Rosana Schafer Ph.D. Dave Yelton Ph.D. Department of Microbiology, Immunology and Cell Biology Morgantown, West Virginia 2005 Keywords: RpoS, ppGpp, Fis, translational regulation, Salmonella, Escherichia Copyright 2005 Matthew L. Hirsch Abstract: Stationary Phase Induction of RpoS in Enteric Bacteria Matthew Louis Hirsch In enteric bacteria, stress adaptation is mediated by the RpoS protein, one of several sigma-factors that in association with RNA polymerase, collectively allow a tailored transcriptional response to environmental cues. Stress stimuli including low temperature, osmotic shock, and starvation all result in a substantial increase in RpoS abundance. Perhaps the most pronounced affect is observed during growth to stationary phase (SP) in rich medium. The mechanism of regulation depends on the specific signal, but may occur at the level of transcription, translation, protein activity or targeted proteolysis. In both Escherichia coli and Salmonella enterica cultured in rich undefined medium, the RpoS protein is barely detectable during exponential growth and increases >30-fold as cells enter SP. Under these conditions, SP induction depends on transcriptional and translational control with proteolysis affecting basal levels but not regulation per se. The transiently expressed Fis protein, whose abundance inversely correlates to that of RpoS, binds just upstream of the primary rpoS promoter and represses transcription nearly 10-fold specifically during exponential growth. SP induction at the translational level relies on a novel form of genetic control dependent on the 24 nucleotides preceding the rpoS initiation codon (ribosome-binding sequence, RBS). The RNA secondary structure of the rpoS RBS is necessary and sufficient for a nearly 10-fold translational increase during SP. Control at this level is not a result of differential transcript stability, nor does it involve the known rpoS regulators ppGpp, DksA, HU, Hfq or the small regulatory RNAs, DsrA and RprA. The environmental stimuli that trigger RBS-mediated SP induction of rpoS translation also remain unknown, but similar to transcriptional control, regulation is only seen in rich undefined media. Collectively, transcriptional repression by Fis and RBS-mediated induction at the translational level account for approximately 95% of the overall SP induction of RpoS. Acknowledgments It’s strange how life guides you to different places, for instance, six years ago I would have guessed that I would currently be in a medical residency program. Instead, I am finishing up my foundation as a scientific investigator. I do not question my current position because it led me to the great classic geneticist Tom Elliott, figuratively, among the last of a dying breed. By always “telling me what I needed to hear” (often repeatedly) he has provided me with the understanding and confidence to succeed in research. Additionally, I appreciate the evening conversations with Nyles Charon and Dave Yelton and the technical support of Adam Goodwill. Socially, my friends and family have weaved a strong web of support and believe something fruitful will come of my work. Specific thanks to Jackie Bower, mom and dad, sister Shel, Aaron Marshall and Dev Jana, and the newcomers Bijal Patel, James Cunningham, and Amy Jones. You guys inspire me. And of course, my acknowledgment is not complete without recognition of the creature that probably has taught me the most in the least time, Neo the Wonderdog. iv Table of Contents Abstract.......................................................................................................................................... ii Acknowledgments ........................................................................................................................ iv Table of Contents .......................................................................................................................... v List of Tables ..............................................................................................................................viii List of Figures............................................................................................................................... ix Chapter 1 Literature Review ......................................................................................................................... 1 σ-factors and Directed Transcription.......................................................................................... 1 The competition model of σ-selectivity.............................................................................. 3 Promoter elements and σ-Selectivity.................................................................................. 3 RpoS............................................................................................................................................ 5 Background......................................................................................................................... 5 Transcriptional regulation of rpoS...................................................................................... 9 Translational regulation of RpoS...................................................................................... 14 RpoS stability.................................................................................................................... 22 Fis.............................................................................................................................................. 22 Regulation of Fis............................................................................................................... 23 Functions of Fis ................................................................................................................ 25 Regulation of Translational initiation ....................................................................................... 27 Shine-Dalgarno sequence and translational regulation..................................................... 29 Role of the initiation codon .............................................................................................. 30 Structured Ribosome Binding Regions............................................................................. 31 Trans-acting factors and translational regulation ............................................................. 32 Literature Cited ......................................................................................................................... 33 Chapter 2 Role of ppGpp in rpoS Stationary Phase Regulation in Escherichia coli .............................. 53 Abstract..................................................................................................................................... 54 Introduction............................................................................................................................... 55 Materials and Methods.............................................................................................................. 57 Bacterial strains and construction. .................................................................................... 57 Media and growth conditions. .......................................................................................... 58 New mutations affecting putative trans-acting factors..................................................... 58 Other mutations affecting trans-acting factors. ................................................................ 60 rpoS-lac fusion.................................................................................................................. 61 Second method for making promoter fusions................................................................... 63 Assay of β-galactosidase................................................................................................... 64 Results......................................................................................................................................