Understanding the regulation of acid resistance in E. coli using whole genome techniques by Matthew David Johnson A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences The University of Birmingham September 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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. ABSTRACT The ability of bacteria to thrive in a variety of host environments depends on their capacity to sense and respond to a wide array of stressors. E. coli encounters many stresses during transit through the gastro-intestinal tract, including acid stress. Acid stress response in E. coli is regulated by a complex network called AR2. The AR2 network comprises several local regulators that collate signals from multiple two-component systems (TCS) including RcsBD, EvgAS and PhoPQ. We combined lab-based evolution and whole genome re-sequencing to generate and identify mutations that confer increased acid resistance in E. coli K-12. All of these mutations map in the gene encoding EvgS, the sensor kinase of the EvgAS TCS. Using a luciferase reporter system and phenotypic assays we characterised the nature of these evgS mutations and their contribution to acid resistance. We also used high-temporal resolution luciferase reporter assays to uncover novel aspects of this network and implicate PhoP in the repression of acid resistance. Finally, we used our evgS mutants to characterise novel interactions within the AR2 network between the two component systems RcsBD and EvgAS. These results are discussed in relation to the role of regulatory networks in bacteria. Dedicated to the loving memory of my nephew Harry J. Tree 2004 - 2010 ACKNOWLEDGEMENTS I would like to acknowledge my supervisor Pete Lund for his excellent supervision during my PhD. Pete’s support and advice for talks and the preparation of this thesis have been invaluable. I would also like to extend my gratitude to Chris Thomas, Steve Busby and Ian Henderson, for their advice and constructive criticism during this project. I would like to thank all those past and present who worked in T101 and T102 for making my experience in the lab thoroughly enjoyable, including, Neil and Olga, Tara, Tabish, Elsa, Andy, Martin, Rhiddi, Faye, Karina, Cathy, Tim and Doug. I acknowledge the Francesco Falciani and Robin May labs for their collaboration; in particular, I would like to thank Anna and Kerstin for all their help and patience. I would like to make a special thank you to Rebecca, Denisse, Sean, James, Chris, Amanda H and Dan for their friendship, which has been so supportive during my studies. Of course, I would also like to thank my parents, Dave and Barbara, for their support both financially and emotionally, and for keeping my feet on the ground; and my sister, Charmaine, whose courage and perseverance during times of extreme adversity has been inspirational. Finally, I would like thank Amanda, to whom I owe so much for her support as a scientist, a friend, and as a companion. TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ............................................................................................1 1.1 Introduction ............................................................................................................2 1.2. E. coli acid resistance..............................................................................................2 1.2.1. Acid resistance mechanisms of E. coli ...............................................................3 1.2.2. Components of amino acid dependent acid resistance mechanisms .............5 1.2.2.1. Characterisation of the acid dependent amino acid decarboxylases ..... 5 1.2.2.2. Activation of decarboxylase enzymes by low pH ..................................... 6 1.2.2.3. Characterisation of the membrane bound antiporters and the amino acid dependant acid resistance mechanism ................................................................ 7 1.2.3. Mechanism of amino acid dependent acid resistance systems ....................10 1.2.4. Amino acid independent acid resistance mechanisms – AR1 ......................13 1.2.5. Amino acid independent acid resistance mechanisms – The AFI ................13 1.2.6. Amino acid independent acid resistance mechanisms – YdeP......................17 1.3. Control and induction of acid resistance mechanisms ......................................18 1.3.1. Induction of acid resistance mechanisms ........................................................18 1.3.2. Promoter organisation of the GAD genes .......................................................18 1.3.3. Promoter organisation and local regulation of ADI ......................................22 1.3.4. Promoter organisation and local regulation of CAD .....................................25 1.4. Control of Acid Resistance mechanisms by the AR2 network .........................26 1.4.1. Repression of the AR2 network by CRP and H-NS.......................................26 1.4.1.1. Control of the AR2 network by cAMP receptor protein ...................... 30 1.4.1.2. Control of the AR2 network by H-NS .................................................... 31 1.4.2 Two-component systems involved in the AR2 network.................................32 1.4.2.1. The mechanism of TCSs........................................................................... 32 1.4.2.2. Activation of TCSs .................................................................................... 34 1.4.3. The EvgAS-YdeO-PhoPQ-GadE control network ........................................34 1.4.3.1. Characterisation of the EvgAS two-component system as an AR regulator …………………………………………………………………………….34 1.4.3.2. Characterisation of the EvgA-YdeO-GadE activation cascade ........... 35 1.4.3.3. Role of PhoPQ in the GAD Network ...................................................... 37 1.4.3.4. Role of RcsB in the GAD Network: An essential inactive activator .... 39 1.4.4. The RpoS-GadX-GadW-GadY regulatory circuit .........................................40 1.4.4.1. Characterisation of the components of the RpoS-GadY-GadX-GadW regulatory circuit ........................................................................................................ 40 1.4.4.2. Characterisation of GadX and GadW regulation ................................. 42 1.4.4.3. Characterisation of the RpoS-GadY-GadX-GadW regulatory circuit 42 1.4.5. Role of TrmE in the AR2 network ..................................................................43 1.5. Role of GadE in the AR2 network ......................................................................44 1.5.1. Characterisation of GadE ................................................................................44 1.5.2. Post-transcriptional control of GadE ..............................................................44 1.5.3. GadE is a central regulator of acid resistance in E. coli................................45 1.6. Regulatory reach of the AR2 network ................................................................46 1.6.1. AR2 network control of AR2 ...........................................................................46 1.6.2. AR2 network control of AFI ............................................................................47 1.6.3. Regulation of AR4 by the AR2 network .........................................................47 1.7. Experimental evolution experiments ..................................................................48 1.7.1 Evolving microbes .............................................................................................48 1.7.2 Applications of lab based evolution .................................................................49 1.7.2 Evolution of acid resistant E. coli K-12 ...........................................................50 1.8. Aims .......................................................................................................................51 CHAPTER 2: MATERIALS AND METHODS ..................................................................52 2.1. Bacterial strains and plasmids.............................................................................53 2.2 Growth conditions ................................................................................................61 2.3. Custom oligonucleotides.......................................................................................61 2.4. Molecular biology techniques ..............................................................................66 2.4.1. Preparation of genomic DNA ...........................................................................66 2.4.2. Preparation of plasmid DNA ...........................................................................66 2.4.3. Amplification of DNA by Polymerase Chain Reaction (PCR)......................66 2.4.4. PCR for cloning and sequencing procedures .................................................67 2.4.5. PCR purification ...............................................................................................68