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STUDIES ON GENE EXPRESSION IN A PATHOGENIC BACTERIUM BY MD. SHAHIDUL ISLAM A THESIS SUBMITTED TO THE UNIVERISTY OF BIRMINGHAM FOR THE DEGREE OF DOCTOR OF PHILOSOPHY SCHOOL OF BIOSCIENCES THE UNIVERSITY OF BIRMINGHAM EDGBASTON BIRMINGHAM B15 2TT UK JUNE 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Synopsis Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 Sakai is an emerging human pathogen. The genes responsible for EHEC virulence are contained in a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE consists of five major polycistronic operons (LEE1-5), which co-ordinately encode a type three secretion apparatus and effector molecules that are associated with the attaching and effacing lesions in the intestine. Expression of the genes in LEE is primarily coordinated by expression of the LEE1 operon. GrlA is a LEE-encoded transcription regulator that has been proposed to be involved in the regulation of expression of the LEE1 operon. To study GrlA-dependent effects at the LEE1 operon regulatory region further, a simple plasmid-based system is described in this work. The work reveals that GrlA can activate transcription initiation at the LEE1 P1 promoter by binding to a target located within the 18 base pair spacer between the promoter -10 and -35 elements, which were defined by mutational analysis. Shortening this spacer to 17 base pairs increases P1 promoter activity and short-circuits GrlA-dependent activation. Hence, at the P1 promoter, the action of GrlA resembles that of many MerR family transcription activators at their target promoters. A cryptic promoter, designated P1A, was found that can initiate transcription within the LEE1 operon regulatory region. Mutational- and biochemical analyses revealed that the P1A promoter overlaps the principal P1 promoter and transcription from the P1A starts at a site located 10 base pairs upstream of the P1 promoter. P1A is likely to compete with the P1 since it can only be active when the P1 promoter is mutated. A single base substitution in the P1 consensus -35 element unmasks P1A promoter activity. In contrast, P1A activity is much less when P1 is inactivated by a mutation in its -10 hexamer element. This suggests that, even when P1 is inactive, a consensus -35 element can sequester RNA polymerase and prevent its access to the P1A promoter. The LEE1 operon regulatory region has ~170 bases-long leader sequence. Deletion and mutational analyses revealed that the LEE1 operon leader sequence contains a short translated ii open reading frame, which has a strong ribosome binding site in front of two adjacent alternative translation start sites, followed by a lysine codon and a nonsense codon. Inactivation of this mini-gene significantly reduced the expression of the downstream ler gene, suggesting that optimal expression of the ler gene needs prior translation of the upstream mini-gene. Deletion and subsequent reporter gene assays revealed that overexpressed Ler represses expression from the LEE1 promoter regulatory region i.e., autoregulates its own transcription. In contrast, it activates expression from the LEE2 promoter by negating H-NS-mediated repression. This suggests that both the GrlA and the mini-gene play positive roles in the expression of the ler gene whilst overexpressed Ler exhibits a negative role on its own expression and activates the expression from the LEE2 promoter. iii To my parents for their blessings and support throughout my life iv Acknowledgements This thesis is the result of three years of work whereby I have been accompanied and supported by many people. I have performed all the works except where mentioned. It is a pleasant aspect that I have now the opportunity to express my gratitude to all who supported me. First and foremost, I offer my deepest gratitude to my supervisor, Professor Steve Busby, whose encouragement, guidance and support from the initial to the final level enabled me to develop an understanding of the project. It would have been next to impossible to write this thesis without his support. His overly enthusiastic and integral view on research has made a deep impression on me. I am really glad to have been a student with him. I also owe my sincerest gratitude to Professor Mark Pallen for his help and advice during the period of this study. I am grateful to Professor Jeff Cole and Professor Ian Henderson for reviewing the introduction of the thesis. I am indebted to Dr Lewis Bingle, Dr David Lee, Dr Kerry Hollands, Dr Mohamed Samir Elrobh and Dr Robert Shaw for their help and guidance with their scientific knowledge. I would like to thank Dr Jennie Mitchell for her patience in reading the thesis and making grammatical corrections in the text. I would like to show my gratitude to Rita, Chris and Lesley for their help and humor. In my daily lab work, I have been blessed with a friendly and cheerful environment shared by a group of fellow students from both Busby and Cole Groups. I would like to thank all of them, especially Jack, Ian, Chismon, Claire and Amanda. I offer my regards and blessings to all of the others who supported me in any respect during the completion of the project. I am grateful to the authority of the Commonwealth Scholarship Commission, UK for providing scholastic support. Lastly, I would like to show my gratitude to my wife, Bentul Mawa for her great patience, encouragement and understanding me throughout the period of this study. v Contents Content Page Title page…………………………………………………………………………... i Synopsis…………………………………………………..………………………... ii Acknowledgements…………………………………………………….………….. v Contents……………………………………………………………………………. vi List of Figures………………………………………………………………..……. xii List of Tables…………………………………………………………………….… xvi List of Abbreviations……………………………………………………………… xvii Chapter 1: Introduction…………………………………………………………... 1-55 1.1 Escherichia coli…………….........................................………………………... 2 1.2 Pathogenic E. coli................................................................................................. 4 1.3 E. coli pathovars EPEC and EHEC...................................................................... 6 1.3.1 Epidemiology and outbreak of E. coli O157:H7 serotype............................ 7 1.3.2 Clinical features of E. coli O157:H7 pathogenesis....................................... 8 1.3.3 Major pathogenic determinants of E. coli O157:H7 serotype: toxins.......... 8 1.3.4 Major pathogenic determinants of E. coli O157:H7 serotype: T3SS........... 10 1.3.5 Coordination of LEE gene activity............................................................... 19 1.4 Regulation of bacterial transcription: an overview...…………………………... 20 1.4.1 The importance of regulation..................................................................... 20 1.4.2 RNA polymerase…………………………………………………………. 20 1.4.3 Promoters………………………………………………………………… 23 1.4.4 Transcription initiation…………………………………………………… 25 1.4.5 Small ligands……………………………………………………………... 27 1.4.6 Transcription factors…………………………………………………....... 28 vi 1.4.7 Nucleoid-associated proteins…………………………………………….. 32 1.5 Post-transcriptional regulation............................................................................. 35 1.5.1 Regulation of translation initiation................................................................ 35 1.5.2 Messenger RNA turnover.............................................................................. 38 1.5.3 Regulation by small RNAs............................................................................ 39 1.6 An overview of regulation of the LEE genes.....….......................…………..… 40 1.6.1 Activities of GrlR/GrlA as transcription regulators….…………….……… 45 1.6.2 Ler and its regulation........................................................………………… 50 1.7 Aims and an outline of the project……………………....……………………... 54 Chapter 2: Materials and Methods…………………………………….………… 56-131 2.1 Suppliers............................................................................................................... 57 2.2 Buffers, solutions and reagents………………………………………………… 57 2.2.1 Gel electrophoresis of DNA and proteins.......……….…………………… 57 2.2.2 Extraction and purification of DNA fragments……...…………………… 58 2.2.3 DNA transformation in E. coli……………………………………………. 58 2.2.4 DNA sampling……………………………………………………………. 59 2.2.5 Pull down assays………………………………………………………….. 59 2.2.6 KMnO4 footprinting………………………………………………………. 59 2.2.7 In vitro transcription assays………………………………………………. 60 2.2.8 β-galactosidase assays……………………………………………………. 60 2.3 Bacterial growth media………………………………………………………… 60 2.3.1 Liquid media……………………………………………..……………….. 60 2.3.2 Solid media…………………………..…………………………………… 61 2.3.3 Antibiotics………………………...………………………………………. 61 2.4 Bacterial strains and plasmids..……………………………………………...…. 62 2.4.1 Bacterial strains and growth conditions………………………………….. 62 vii 2.4.2 Plasmids………………………………………………………………….. 62 2.5 Gel electrophoresis …………………………………………………………….. 62 2.5.1 Agarose gel electrophoresis