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Regulation of Expression of the Gabdtp Operon and Utilization of GABA As a Sole Nitrogen Source in Escherichia Coli

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Regulation of Expression of the Gabdtp Operon and Utilization of GABA As a Sole Nitrogen Source in Escherichia Coli REGULATION OF THE GABDTP OPERON AND GABA UTILIZATION IN E. COLI REGULATION OF EXPRESSION OF THE GABDTP OPERON AND UTILIZATION OF GABA AS A SOLE NITROGEN SOURCE IN ESCHERICHIA COLI By MICHAEL SCHERTZBERG, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Master of Science McMaster University © Copyright by Michael Schertzberg, December 2004 MASTER OF SCIENCE (2004) McMaster University (Biology) Hamilton, Ontario TITLE: Regulation of Expression of the gabDTP Operon and Utilization of GABA as a Sole Nitrogen Source in Escherichia coli AUTHOR: Michael Schertzberg, B.Sc. (McMaster University) SUPERVISOR: Professor H.E. Schellhorn NUMBER OF PAGES: xi, 181 ii Abstract The gabDTP operon is regulated by a number of different signals and stress conditions including general stress via as, carbon limitation, cell-to-cell signaling, alkaline pH and nitrogen limitation. The mechanism of regulation by many of these conditions is unclear. Operon lacZ-fusions to the genes of the gabDTP operon, isolated in a previous search for rpoS-dependent genes in E. coli, showed a 10-fold rpoS-dependence in rich LB media. Here we provide evidence, utilizing Northern analysis of wildtype E. coli strains without any insertions in the gabDTP operon, that expression of the gabDTP operon was highly RpoS- and stationary phase-dependent. The corresponding enzymes GabD (SSDH) and GabT CGSST) showed a 20 fold higher activity in stationary phase cultures of wildtype cells compared to isogenic rpoS-mutant strains. However, contrary to expected results, growth on GABA as a sole nitrogen source is better in rpoS- mutants relative to wildtype strains. Similar growth patterns are observed when the same strains are grown on L-glutamate as the sole nitrogen source, suggesting that the limitation for growth with GABA as the nitrogen source may be due to an inability to utilize glutamate efficiently as a sole nitrogen source. These results confirm that the gabDTP operon is part of the RpoS regulon, and suggest the metabolism of GABA plays a role in stationary phase stress resistance in E. coli. 111 Acknowledgements First of all, I would like to thank my supervisor, Dr. H.E. Schellhorn, for giving me the opportunity of pursuing a M.Sc. degree under his supervision. His guidance and discussions on my experiment were both insightful and helpful. I would also like to thank him for giving me the opportunity to present my research at the ASM General Meeting in 2003; the experience was incredible and helped me to discuss my work with other researchers from my field. I would also like to thank him for giving me some leeway to conduct experiments and pursue research that interested me personally. I would also like to thank Dr. T.M. Finan for offering his helpful comments and for being a part of my thesis committee and Dr. Robin Cameron for being on my thesis examination committee. I would like to thank my parents for their support throughout my entire life, especially while attending McMaster University. Their support was vital to me succeeding during my undergraduate work and completing my post-graduate work at Mac. They have always encouraged me and believed in me and I never would have gotten this far without them. I have met numerous people since I started working in the lab, many of whom deserve thanks. First I would like to thank Pramuk Weerasinghe for his friendship over the last 4 years. His diligence in the lab, and his success after completing his M.Sc. inspired me to complete this work. I would also like to thank Sarosh Yousuf for showing me how to prepare and run Northern blots (even though they never fully worked at the time) and for passing on this project to me. I would like to thank the "London Crew", lV Mark, Vijay and Dirk, for being good friends, providing some entertainment in the lab, and for providing valuable discussions and feedback on experiments and ideas. I hope you guys keep in touch. I would also like to extend thanks to some of the past members of the Schellhorn lab who were valuable co-workers: Cheryl Patten, Sabine Maxwell, Galen Chen, Kelly Seto, lana Gilles, Rovena Tey, Michael Ha, and Mathew Trudeau. Working in the lab wouldn't have been the same without you. Most importantly, I would like to thank Saima Tariq, for all her help and support throughout the entire time I've worked in the lab. Some of our growth experiments would have been impossible to complete without working together. During the course of my M.Sc. work she has become my best friend, and without her encouragement I never would have finished this thesis. v Table of Contents Abstract .......................................................................................................................... iii Acknowledgements ........................................................................................................ iv Table of Contents ........................................................................................................... vi List of Figures ................................................................................................................ ix List of Tables .................................................................................................................. x List of Abbreviations ..................................................................................................... xi CHAPTER 1 INTRODUCTION ................................................................................. 1 1.0 Nitrogen Source Utilization in Escherichia coli .......................................... 1 1.1 Utilization of Ammonia as a Nitrogen Source ...•........................................ 1 1.11 Transport and Assimilation of Ammonia as a Nitrogen Source ................. 1 Assimilation at High Ammonia Concentrations ................................................. 2 Assimilation at Low Ammonia Concentrations .................................................. 3 1.12 Regulation of Ammonia and Alternative Nitrogen Source Assimilation ...... 4 The Regulators of the Ntr Operons ..................................................................... 4 Control of the Ntr Response ............................................................................... 6 Regulation of GOGAT, GS and GDH ................................................................ 8 1.2 Transport and Utilization of Amino Acids as Nitrogen Sources ............ 11 Transport and Utilization of Glutamine ................................................................ 12 Transport and Degradation of Aspartate and Glutamate ...................................... 13 Transport and Degradation of Arginine, Agmatine, Ornithine and Putrescine .... 14 1.3 G ABA as a Nitrogen and Carbon Source .................................................. 17 1.31 Sources of GABA ..................................................................................... 17 1.32 Metabolism of GABA: The gabDTP Operon and Adjacent csiD and ygaF Genes ......................................................................................................... 18 1.4 Regulation of the gabDTP Operon ............................................................ 19 1.41 Regulation by "GabC" .............................................................................. 19 1.42 Regulation by Stationary Phase and the RpoS Sigma Factor ................... 21 1.43 Regulation by Cell Density Signals .......................................................... 21 1.44 Regulation by pH ...................................................................................... 22 1.45 Regulation by Carbon and Nitrogen Source Limitation ........................... 22 1.46 Regulation by CsiD and CsiR ................................................................... 23 5.7 Goals of This Study ..................................................................................... 24 CHAPTER 2 MATERIALS AND METHODS ....................................................... 33 2.1 Media and Growth Conditions .................................................................. 33 2.2 RNA Isolation Methods for Northern Analysis and RT-PCR ................ 33 2.21 Isolation of Total RNA from Strains Harbouring the gab Operon lacZ- Fusions for Northern Analysis of gabD and gabP Expression ............................. 33 2.22 Isolation of RNA from MG 1655 and GC4468 Cultures for Northern Analysis of ygaF and gabDTP Expression and RT -PCR Analysis of the gab vi Operon ................................................................................................................... 34 2.23 Purification of mRNA for Northern Analysis of gabD Expression in Wildtype, rpoS- and gabC Mutants ...................................................................... 35 2.3 Probe Preparation and Northern Analysis ...............•..........•..•........•........ 35 2.4 RT- PCR of the gab Operon and Upstream Genes ................................... 36 2.5 Enzyme Assays ............................................................................................ 37 2.51 ~-Galactosidase Assays ............................................................................ 37 2.52 Isolation of Crude Protein Extracts ........................................................... 38 2.53 Determination of Succinic Semi aldehyde Dehydrogenase (SSDH) and GABA Transaminase (GSST) Activity .................................................... 38 2.6 Deletion of the ygaE, nac, and cyaA Genes ..............................................
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