Graduate Theses, Dissertations, and Problem Reports 1999 Regulation of the synthesis and protein stability of the alternative sigma factor RpoS in Salmonella typhimurium Christofer Lee Cunning West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Cunning, Christofer Lee, "Regulation of the synthesis and protein stability of the alternative sigma factor RpoS in Salmonella typhimurium" (1999). Graduate Theses, Dissertations, and Problem Reports. 3125. https://researchrepository.wvu.edu/etd/3125 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. 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Christofer Lee Cunning DISSERTATION Submitted to the School of Medicine at West Virginia University in Partial Fulfillment of the Requirement for the degree of Doctor of Philosophy in Microbiology Tom Elliott, Ph.D., Chair Nyles Charon, Ph.D. Meenal Elliott, Ph.D. Marilyn Evans, Ph.D. David Yelton, Ph.D. Department of Microbiology/Immunology Morgantown, West Virginia 1999 Keywords: RpoS, stationary phase, Host Factor, DsrA, MviA Copyright 1999 Christofer L. Cunning Abstract RpoS is an alternative sigma factor which is required for the expression of genes that help bacteria cope with environmental stress. The regulation of RpoS is multifactorial and occurs at the levels of synthesis and protein turnover. Our lab has previously identified a rpoS translation enhancing factor in Salmonella typhimurium, the RNA-binding protein HF-I. An antisense element consisting of rpoS mRNA secondary structure is predicted to sequester the rpoS ribosome binding site and inhibit translation. HF-I may interact with this element to increase translation. Using rpoS genetic fusions, we have shown that native rpoS promoters can be substituted with tac or lacUV5 promoters without altering the normal HF-I mediated rpoS regulation. A long deletion from the 5' end of the rpoS transcript, which still contains the antisense element, was no longer regulated by HF-I. This suggests that HF-I may not simply interact with the secondary structure to enhance rpoS translation. DsrA is a small untranslated RNA which has been shown to stimulate rpoS translation. We have shown that DsrA RNA interacts directly with rpoS mRNA. Mutational analysis has identified which RNA bases are important for the pairing between DsrA and rpoS mRNA that enhances rpoS translation. The protein stability of RpoS is controlled by a putative two-component regulatory system. This system contains a protease, ClpXP, and a putative response regulator, MviA in S. typhimurium. We have tested several models which may explain MviA action and have confirmed that RpoS protein stability in S. typhimurium is dependent on the same genes as in Escherichia coli. We have shown that phosphorylation of MviA is important for its activity and that the source of this phosphate is probably not acetyl phosphate or the PTS system. Additionally, we observed an increase of RpoS during exponential growth on acetate as the ii sole carbon and energy source. Surprisingly, this RpoS increase during growth on acetate is independent of HF-I action. iii Acknowledgments I would like to thank my advisor Dr. Thomas Elliott. His patience, effort, and support have been immeasurable. In him I saw a perfect example of how a dedicated scientist challenges himself to the fullest every day. I would also like to thank the members of my committee Dr. Meenal Elliott, Dr. Marilyn Evans, and Dr. David Yelton for their time, advice, and encouragement. I would especially like to thank Dr. Nyles Charon who gave me my first job in science and showed me how to link creativity to research. I would also like to thank my wife Wendy and my mother who have supported me throughout my time as a graduate student. I am also grateful for my faith in God which has given me the strength to persevere. 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 General properties of Sigma Factors........................................ 1 The Sigma Factors............................................................. 4 The Housekeeping Sigma............................................ 5 The Nitrogen Metabolism Sigma.................................... 5 The Heat-Shock Sigmas............................................. 6 The Flagella Biosynthesis Sigma................................... 11 The Iron Citrate Transport Sigma................................... 12 The Stationary Phase/Stress Sigma................................. 13 Transcriptional Regulation of s38......................... 18 Translational Regulation of s38............................ 20 Control of s38 Stability..................................... 25 Chapter 2: Promoter substitution and deletion analysis of upstream region required for rpoS translational regulation........................................................... 27 Abstract......................................................................... 28 Introduction..................................................................... 29 Materials and Methods........................................................ 31 Bacterial strains and constructions.................................. 31 Media and growth conditions........................................ 31 Construction of lac fusions.......................................... 32 b-galactosidase assays.............................................. 33 Results.......................................................................... 34 Deletion analysis of sequences required for translational control of RpoS.................................... 34 Secondary structure in rpoS transcripts from deletion constructs.............................................. 36 Osmotic challenge..................................................... 39 v Continuous growth at high osmolarity............................. 42 Discussion...................................................................... 44 Literature Cited................................................................. 48 Chapter 3: DsrA RNA regulates translation of RpoS message by an anti antisense mechanism, independent of its action as an antisilencer of transcription......... 55 Abstract......................................................................... 56 Introduction.................................................................... 56 Materials and Methods........................................................ 57 Bacterial strains....................................................... 57 Plasmid constructions and DNA manipulations......................................................... 58 b-galactosidase assays............................................... 59 Northern blot analysis................................................ 59 RNA half-life determination......................................... 60 Results.......................................................................... 60 Regulated expression of dsrA....................................... 60 Mapping the active domains within DsrA.......................... 63 A model for translational regulation of rpoS by sequences in S-L1 of dsrA................................ 66 Mutational analysis of loop 1 of DsrA.............................. 68 Analysis of the unpaired region at the 5' end of the RpoS leader structure................................. 72 Discussion...................................................................... 72 Chapter 4: RpoS synthesis is growth-rate regulated in Salmonella typhimurium but its turnover is not dependent on acetyl phosphate synthesis or PTS function............................................................................ 79 Abstract......................................................................... 80 Introduction.................................................................... 81 Materials and Methods........................................................ 82 Bacterial strains and constructions.................................. 82 Media and growth conditions........................................ 84 vi Screen for mutants with a defect in RpoS turnover................................................................ 84 Screen for point mutants affecting RpoS turnover...............................................................