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Growth Phase-Dependent Rpos Levels in Escherichia Coli

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Growth Phase-Dependent Rpos Levels in Escherichia Coli GROWTH PHASE-DEPENDENT RPOS LEVELS IN ESCHERICHIA COLI LABORATORY AND ENVIRONMENTAL STRAINS GROWTH PHASE-DEPENDENT RPOS LEVELS IN ESCHERICHIA COLI LABORATORY AND ENVIRONMENTAL STRAINS BY PARDIS HAGHIGHI, 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 Pardis Haghighi, February 2016 M.Sc. thesis – P. Haghighi; McMaster University – Biology MASTER OF SCIENCE (2016) McMaster University (Biology) Hamilton, Ontario TITLE: Growth phase-dependent RpoS levels in Escherichia coli laboratory and environmental strains AUTHOR: Pardis Haghighi, B.Sc. (Ferdowsi University of Mashhad, Mashhad, IRAN) SUPERVISOR: Dr. Herb E. Schellhorn NUMBER OF PAGES: x, 58 ii M.Sc. thesis – P. Haghighi; McMaster University – Biology ABSTRACT RNA polymerase and the sigma factors in Escherichia coli play role in maintaining the bacterial transcriptional profile in dynamic environmental conditions during growth cycle. Previously, transcriptom analysis between E. coli laboratory strain MG1655 and pathogenic strain EDL933 showed that a lot of genes are highly RpoS-dependent in EDL933 without RpoD to compete against RpoS. In the first project, the RpoD level was examined in these two strains. The same RpoD level between the two E. coli strains was shown suggesting the independent expression of RpoS regulon. In prototypical E. coli K12 MG1655 laboratory strain, RpoS levels, the stress- response sigma factor, are not detectable during exponential phase while several genes are controlled by RpoS in this phase. Although, RpoS levels in E. coli pathogenic strain O157:H7 was higher during exponential phase, the RpoS expression pattern in other isolates of E. coli was not known. In second project, the E. coli environmental isolates were examined as we have access to a large number of isolates, collected from a variety of gut to aquatic sources. To examine if RpoS in environmental isolates has exponential phase expression similar to that of laboratory strains, immunoblots were performed on forty isolates at exponential phase (OD600 ~ 0.3). Twelve isolates showed higher levels of RpoS protein compared to laboratory strain during exponential phase. Also, the immunoblot analysis on some of the isolates at early stationary phase (OD600 ~ 1.5) and 24 h after subculture demonstrate the decrease in expression of RpoS protein at stationary phase to iii M.Sc. thesis – P. Haghighi; McMaster University – Biology overnight culture. This finding was different from the RpoS expression in E. coli K12 laboratory strain. In conclusion, the relatively lower levels of RpoS in the laboratory strains might occur due to selection for attenuation in RpoS levels in the laboratory strains after being cultivated for many years in laboratory conditions. iv M.Sc. thesis – P. Haghighi; McMaster University – Biology ACKNOWLEDGMENTS Pursuing graduate studies as an international student in a foreign country was a big challenge for me at the beginning and I could not handle it without the tremendous support from the amazing people in the Schellhorn lab. First and foremost, I would like to sincerely thank my supervisor, Dr. Herb Schellhorn, for his consistent support and constructive feedback on my work. I appreciate his guidance and patience during my time here, and I am very honored and thankful for being given the opportunity to learn and grow academically and as a person. I will always value our conversations and his advice on how to be a good grad student. I also thank my co-supervisor, Dr. Radhey Gupta for his guidance and helpful feedback on my project during my committee meetings. I would like to thank current and past members of Schellhorn lab (in no particular order): Mahi, Sakis, Shirley, Deepinder (especially for being good company while running the Western blots and correcting my English), Rachelle, Steve, Rachel, Paul, Cathy, Vivian, Ashish, Kristine and William. Thank you guys for making my academic life more fun and memorable. The “Lab’s mom” will never forget any of those moments. Lastly, I have to say a big thank you to my family for their endless love and for supporting me even from miles away. And special thanks to my dearest friend and partner, Hooman, for being in my life, listening to all my worries during stressful times and his invaluable support. v M.Sc. thesis – P. Haghighi; McMaster University – Biology TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii ACKNOWLEDGMENTS .................................................................................................. v TABLE OF CONTENTS ................................................................................................... vi LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x Project 1: RpoD expression level in E. coli K12 laboratory strain and E. coli O157:H7 pathogen strain in exponential and stationary phases ......................................... 1 Chapter 1.1 Project Rationale and Overview ...................................................................2 1.1.1 Quantitative Immunoblot ................................................................................. 4 Chapter 1.2 Methods ........................................................................................................5 1.2.1 Sample collection ............................................................................................ 5 1.2.2 SDS-PAGE ...................................................................................................... 5 1.2.3 Immunoblot ..................................................................................................... 6 Chapter 1.3 Results ..........................................................................................................7 Chapter 1.4 Discussion ....................................................................................................7 vi M.Sc. thesis – P. Haghighi; McMaster University – Biology Project 2: Exponential expression levels of RpoS sigma factor in E. coli environmental strains ................................................................................................................ 15 Chapter 2.1 Introduction ................................................................................................16 2.1.1 Escherichia coli RNA polymerase core enzyme ........................................... 16 2.1.2 Sigma factors in E. coli.................................................................................. 17 2.1.3 Sigma factors competition ............................................................................. 18 2.1.4 RpoS; a global regulator ................................................................................ 19 2.1.5 RpoS expression in exponential phase .......................................................... 19 2.1.6 RpoS regulation and other transcriptional regulators .................................... 20 2.1.7 Project rationale and objectives ..................................................................... 22 Chapter 2.2 Materials and Methods ...............................................................................23 2.2.1 Bacterial strains ............................................................................................. 23 2.2.2 Growth conditions and sample collection ..................................................... 23 2.2.3 Immunoblot analysis...................................................................................... 24 2.2.4 Quantify the blots using ImageJ 1.47 ............................................................ 25 Chapter 2.3 Results ........................................................................................................26 vii M.Sc. thesis – P. Haghighi; McMaster University – Biology 2.3.1 Growth curve ................................................................................................. 26 2.3.2 RpoS expression level in exponential phase .................................................. 26 2.3.3 Quantitation of RpoS protein expression using ImageJ 1.47 ........................ 27 Chapter 2.4 Discussion ..................................................................................................28 2.4.1 Possible factors involved in RpoS expression level in exponential phase .... 29 Chapter 2.5 Conclusion and Future work ......................................................................30 References ......................................................................................................................... 39 Appendix ........................................................................................................................... 45 Standard Operating Procedures ......................................................................................45 Thesis Defence Slides............... .....................................................................................53 viii M.Sc. thesis – P. Haghighi; McMaster University – Biology LIST OF TABLES Table 2.1. Escherichia coli sigma factors. ...................................................................... 32 Table 2.2. Summary of source types for the 40 environmental E. coli isolates used in this study .......................................................................................................
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