The role of the ECF sigma factor SigG in Mycobacterium tuberculosis Alison Emma Gaudion March 2011 A thesis submitted in fulfilment of the requirements of University College London for the degree of Doctor of Philosophy Division of Mycobacterial Research MRC National Institute for Medical Research The Ridgeway Mill Hill London Declaration I Alison Emma Gaudion confirm that the work presented in this thesis is my own. When information has been derived from other sources, I confirm that this has been indicated in the thesis. A.E. Gaudion 2 Abstract Mycobacterium tuberculosis is the causative agent of Tuberculosis (TB). Two billion people are currently infected with M. tuberculosis bacilli, one in ten of whom will develop active TB in their lifetime. M. tuberculosis is able to survive within macrophages but the exact mechanisms used for intracellular survival are poorly understood. DNA-dependent RNA polymerase is the enzyme responsible for transcription in all living organisms. In bacteria this enzyme recognises different promoters by binding to sigma factors that recognise those promoters. This study focuses on the role and regulation of the M. tuberculosis extracytoplasmic function (ECF) sigma factor, SigG. ECF sigma factors are responsible for upregulating genes necessary for bacterial stress responses. SigG has previously been shown to be upregulated in response to DNA damage and during macrophage infection. It has been demonstrated that sigG is expressed from at least 2 promoters and that only promoter P1 is DNA-damage inducible. sigG is co-transcribed with the two downstream genes Rv0181c and Rv0180c, which were hypothesised to be anti- and anti-anti-sigma factors to SigG. Protein-protein interaction studies showed that SigG and Rv0181c do not interact. Potential anti-sigma factors to SigG were identified, the most promising of which was the thioredoxin family protein Rv1084. Two potential SigG-dependent genes had previously been identified, Rv0887c and Rv0911. It has been demonstrated that SigG is able to bind to the promoter regions of these genes but this interaction was not specific. 3 An M. tuberculosis sigG-Rv0180c deletion strain was constructed and complemented with the whole operon as well as with sigG alone. The phenotype of the mutant strain was examined in vitro as well as in vivo to test the hypothesis that SigG has a role during infection. Use of a phenotype microarray revealed an enhanced susceptibility of the mutant strain to oleic acid and its ester, Tween 80, leading to investigation of the sensitivity of the strains to a range of fatty acids. 4 Acknowledgments I would like to thank the MRC for providing the funding for this project. I would like to express my thanks and utmost gratitude towards my supervisors Elaine Davis and Ian Taylor for their guidance and unlimited advice. I would also especially like to thank Katherine Smollett for creating and analysing the SigG overexpression strain, optimising expression conditions for His-tagged SigG, assisting with macrophage and animal experiments and for her help, advice and endless patience throughout this project. I would like to thank Alan Williams for all his help with protein expression and purification. Kathryn Lougheed for her assistance with the alamar blue and Biolog assays. Thanks to Joanna Dillury for all her help with the macrophage and animal experiments, to Debbie Hunt for providing the yeast-2-hybrid positive control plasmids and the anti-CRP antibody, David Goldstein for the large scale purification of Rv0181c protein and to Steven Howell for performing the MALDI-MS analysis. Thanks also go to Melanie Stapleton for discussion regarding protein-DNA binding assays and Mark Buttner for providing the positive control and advice for the in vitro transcription assays. Finally I would like to thank all past and present members of the Division of Mycobacterial Research for their advice and discussion. 5 Table of Contents DECLARATION ........................................................................................................ 2 ABSTRACT ................................................................................................................ 3 ACKNOWLEDGMENTS ......................................................................................... 5 TABLE OF CONTENTS ........................................................................................... 6 LIST OF FIGURES ................................................................................................. 13 LIST OF TABLES ................................................................................................... 16 LIST OF ABBREVIATIONS ................................................................................. 17 WORK TIMELINE ................................................................................................. 21 1. INTRODUCTION ....................................................................................... 23 1.1 Mycobacteria and the Mycobacterium tuberculosis complex ................... 23 1.2 Tuberculosis the Disease ............................................................................. 24 1.2.1 Incidence and therapy ............................................................................ 24 1.2.2 Pathogenesis and the host response ....................................................... 25 1.3 The role of fatty acids during M. tuberculosis infection of macrophages ....................................................................................................................... 29 1.3.1 Macrophages produce mycobactericidal fatty acids .............................. 29 1.3.2 Fatty acids as a carbon source for M. tuberculosis ................................ 30 1.4 Transcription and sigma factors in bacteria ............................................. 31 1.5 Response to stress – two-component systems and extracytoplasmic function sigma factors ................................................................................. 36 1.6 The sigma factors of M. tuberculosis .......................................................... 39 1.6.1 Structure and conserved regions ............................................................ 39 1.6.2 Sigma factor regulation in M. tuberculosis ............................................ 41 1.7 M. tuberculosis ECF sigma factor, SigG .................................................... 45 1.7.1 Expression of sigG ................................................................................. 45 1.7.2 The sigG operon ..................................................................................... 48 1.8 Identification of a possible SigG-dependent promoter ............................ 50 1.8.1 The probable dioxygenase, Rv0654 ....................................................... 50 6 1.8.2 Expression of Rv0887c and Rv0911 increased in a SigG overexpression strain ....................................................................................................... 50 1.9 The toxic electrophile methylglyoxal ......................................................... 52 1.9.1 Methylglyoxal and its detoxification ..................................................... 52 1.9.2 Methylglyoxal and DNA damage .......................................................... 54 1.9.3 Methylglyoxal in E. coli – a fine line between survival and cell death . 55 1.10 Hypotheses & Aims ..................................................................................... 56 2. MATERIALS AND METHODS ................................................................ 58 2.1 Bacterial and Yeast Strains & Growth Media .......................................... 58 2.2 Plasmids ........................................................................................................ 58 2.3 Recombinant DNA techniques ................................................................... 65 2.3.1 Polymerase chain reaction (PCR) .......................................................... 65 2.3.2 Agarose gel electrophoresis ................................................................... 66 2.3.3 Plasmid DNA extraction ........................................................................ 66 2.3.4 Restriction enzyme digestion of DNA ................................................... 67 2.3.5 PCR product & digested DNA purification ........................................... 67 2.3.6 Dephosphorylation and ligation of DNA ............................................... 67 2.3.7 RNA extraction ...................................................................................... 68 2.3.8 Ethanol precipitation of nucleic acids .................................................... 69 2.3.9 Site-directed Mutagenesis (SDM) .......................................................... 69 2.3.10 Transformation of competent E. coli ..................................................... 70 2.3.11 E. coli frozen stocks ............................................................................... 70 2.3.12 Sequencing of DNA ............................................................................... 70 2.4 Mycobacteria-specific Techniques ............................................................. 70 2.4.1 DNA Extraction ..................................................................................... 70 2.4.2 Preparation of competent Mycobacteria ................................................ 71 2.4.3 Electroporation of Mycobacteria ........................................................... 72 2.4.4 Preparation