Characterisation of gene regulation in mycobacteria. Laura Dixon A thesis submitted for the degree of: Doctor of Philosophy September 2017 Department of Molecular Biology and Biotechnology The University of Sheffield 1 Abstract This study focuses on the characterisation of gene regulation in mycobacteria in response to various signals within the cell. The regulatory mechanisms explored include two types of protein regulators (cAMP-receptor proteins (CRPs) and CsoR) and an RNA-based method of regulation (a member of the ydaO-type riboswitch). The signals concerned are copper and the small nucleotide molecules cyclic AMP and cyclic di-AMP. The study focuses primarily on the regulation of rpfA, which encodes a resuscitation promoting factor that is involved in resuscitation of Mycobacterium tuberculosis from dormancy. The main findings of the study are as follows. A copper-sensitive repressor protein from M. tuberculosis, CsoR, was purified and shown to bind to the rpfA gene. The presence of copper caused the release of the DNA:protein complex and kinetic parameters of the interactions were determined. Two mycobacterial CRP proteins, Rv3676 from Mycobacterium tuberculosis and Msmeg_6189 from Mycobacterium smegmatis, were purified and their interaction with the rpfA or sdh1 promoters were characterised. The effect of cyclic AMP binding on the interaction with DNA was assessed and thermodynamic and kinetic parameters of the interaction between the CRP proteins and cyclic AMP was studied. Comparisons were made with the well characterised E. coli CRP protein and the two mycobacterial proteins. Finally, a putative ydaO-type riboswitch present in the 5’ untranslated region of rpfA messenger RNA was shown to play a role in regulation of the gene in a Mycobacterium marinum salt stress model. In vitro evidence was gathered to suggest the riboswitch is able to bind cyclic di-AMP and pApA, which favours a shift to a specific structural confirmation. Mutation of G168C/G169C residues in the riboswitch abolished this effect. 2 Acknowledgements First and foremost, I would like to thank my supervisor, Professor Jeff Green, for giving me the opportunity to carry out this PhD. Thank you for all the help and guidance you have given me throughout the project and especially for your support during difficult times. I am truly grateful to have had such an understanding supervisor. I would also like to thank all members of the Green lab, past and present, for your help with methods, techniques and suggestions for my research. A special thanks to Dr Laura Smith for her help with all things mycobacterial! I would also like to thank Katie, Ben and Peter for all the laughs and good times! I would like to thank Jenni, whose contributions to my CsoR chapter have been invaluable. I really enjoyed supervising you during your Masters project (during which no Gilson pipettes were harmed..!). Who would have thought we would end up being such close friends after the project. I would like to also thank my best friends Constantinos and Tom for all the good times, weekend trips and laughs over the years. I would like to thank my parents for their love and support and my brother, Sam. I have the fondest memories of the years that you lived with me in Sheffield during the PhD. I would like to thank Nam. You have always believed in me. I honestly don’t think I could have got this far without you there by my side cheering me on. Thank you. Finally, I would like to dedicate this thesis to Tanner. You always brought a smile to my face. 3 Publications Aung, H.L. in, Dixon, L.L., Smith, L.J., Sweeney, N.P., Robson, J.R., Berney, M., Buxton, R.S., Green, J., and Cook, G.M. (2015). Novel regulatory roles of cAMP receptor proteins in fast- growing environmental mycobacteria. Microbiology 161, 648–661. 4 List of Abbreviations AIDS Acquired immune deficiency syndrome ASP Ammonium sulphate precipitation ATP Adenosine triphosphate BLItz Biolayer interferometry βME β-mercaptoethanol bp Base pair(s) CCCP Carbonyl cyanide m-chlorophenyl-hydrazone CDS Coding sequence CFE Cell-free extract CHISAM Chloroform- isoamyl alcohol 24:1 Ci Curie (unit) CIAP Calf intestinal alkaline phosphatase Cyclic di-AMPSS Phosphorothioate-modified analog of cyclic di-AMP Cso Copper sensitive operon DNA Deoxyribonucleic acid DosR Dormancy survival regulon DSF Differential scanning fluorimetry DTT Dithiothreitol ELISA Enzyme-linked immunosorbent assay EMSA Electrophoretic mobility shift assay EMP Ethambutol F-cyclic di-AMP 2'-O-(6-[Fluoresceinyl]aminohexylcarbamoyl)-cyclic di-AMP gDNA Genomic DNA HIV Human immunodeficiency virus HPLC High-performance liquid chromatography ICP-MS Inductively coupled plasma mass spectrometry IF-γ Interferon-gamma ILP In-line probing INH Isoniazid iNOS Inducible nitric oxide synthase IPTG Isopropyl β-D-1-thiogalactopyranoside ITC Isothermal titration calorimetry IVEGI in vivo expressed genomic island IVT in vitro transcription LC Liquid chromatography LTBI Latent tuberculosis infection MctB Mycobacterial copper transport protein B MDR-TB Multi-drug resistant TB M. tuberculosis Mycobacterium tuberculosis mRNA messenger RNA MS Mass spectrometry MST Microscale thermophoresis MTBC Mycobacterium tuberculosis complex MWCO Molecular weight cut off NADH Reduced nicotinamide adenine dinucleotide NAG-NAM N-acetyl glucosamine-N-acetyl muramic acid nm Nanometers NRAMP1 Natural resistance associated membrane protein PAGE Polyacrylamide gel electrophoresis PE protein Proline-glutamic acid protein 5 PEI Polyethyleneimine PG Peptidoglycan PPE protein Proline-proline-glutamic acid protein PreQ1 7-aminomethyl-7-deazuguanine PrpfA Region of rpfA promoter containing CRP site PZA Pyrazinamide RicR Regulated in copper repressor RIF Rifampcin RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species rpfAL, 185 bp region of rpfA gene containing CsoR site rpfAS, 48 bp region of rpfA gene containing CsoR site rpm Revolutions per minute STB Smooth tubercle bacilli T4 PNK T4 polynucleotide kinase T7 RNAP T7 RNA polymerase TB Tuberculosis TLC Thin layer chromatography Tm Melting temperature or transition midpoint TNF Tumour necrosis factor UTR Untranslated region UV Ultraviolet WHO World Health Organisation WT Wild-type XDR-TB Extensively-drug resistant TB 6 List of Figures Chapter 1 Figure 1.1: Composition of the mycobacterial cell wall. Following page 18. Figure 1.2: Overview of the tuberculosis disease. Following page 19. Figure 1.3: Pathology of TB and granuloma structure. Following page 20. Figure 1.4: Copper in M. tuberculosis. Following page 24. Figure 1.5: Overview of cAMP signalling in M. tuberculosis. Following page 27 Figure 1.6: Overview of cyclic di-AMP synthesis and degradation in M. tuberculosis. Following page 29 Chapter 2 Figure 2.1: Calibration curve of protein standards for gel filtration. Following page 47. Chapter 3 Figure 3.1: Alignment of CsoR protein sequences from difference bacterial species. Following page 51. Figure 3.2: Crystal structures of M. tuberculosis CsoR with Cu(I) associated. Following page 52. Figure 3.3: Two CsoR tetramers bind per target DNA site. Following page 52. Figure 3.4: Map of binding sites and regulatory elements of the M. tuberculosis rpfA gene. Following page 53. Figure 3.5: Map of pGS2528 plasmid used for the overproduction of CsoR. Following page 54 Figure 3.6: Initial purification of CsoR. Following page 54 Figure 3.7: Degradation of the C-terminal tail of CsoR. Following page 56 Figure 3.8: Optimised purification of CsoR. Following page 58 Figure 3.9: Region of the rpfA sequence used for CsoR binding experiments. Following page 59 Figure 3.10: Agarose gel electrophoresis analysis of the products of PCR amplification of rpfAL. Following page 59 Figure 3.11: EMSA analysis of the interaction between CsoR and rpfAL in the presence or absence of copper. Following page 60 Figure 3.12: EMSA analysis of the interaction between CsoR and rpfAS. Following page 60 Figure 3.13: EMSA analysis of the interaction between CsoR and rpfAL with different copper concentrations. Following page 60 Figure 3.14 CsoR does not bind to the PRv1675c. Following page 61 Figure 3.15: Overview of a BLItz kinetic experiment. Following page 62 Figure 3.16: BLItz analysis of the interaction between CsoR and rpfAL. Following page 62 Figure 3.17: BLItz analysis of the interaction between CsoR and Pcso. Following page 62 7 Figure 3.18: Differences in the dissociation CsoR and rpfAL and the effect of copper during dissocation. Following page 63 Figure 3.19: Generation of constructs for overexpression of phosphomimetic CsoR proteins. Following page 65 Figure 3.20: Purification of full-length CsoR T93D and CsoR T93E. Following page 65 Figure 3.21: EMSA analysis of the interaction between phosphomimetic CsoR mutants (T93D and T93E) and rpfAL. Following page 66 Figure 3.22: BLItz analysis of the interaction between the phosphomimetic CsoR mutants and rpfAL. Following page 66 Chapter 4 Figure 4.1: X-ray crystal structures of apo- and holo- E. coli and M. tuberculosis CRP proteins. Following page 70 Figure 4.2: Sequence alignment of Rv3676 from M. tuberculosis and CRP from E. coli. Following page 71 Figure 4.3: The Rv3676 binding site. Following page 72 Figure 4.4: Alignment of M. tuberculosis Rv3676 and M. smegmatis Msmeg_6189. Following page 74 Figure 4.5: Gene expression ratio in response to deletion of Msmeg_0539 or overexpression of Msmeg_6189. Following page 76 Figure 4.6: Agarose gel electrophoresis of pGS2132 treated with EcoRI and XhoI. Following page 78 Figure 4.7: SDS-PAGE analysis of trial overproduction of His-Rv3676 under different conditions. Following page 79 Figure 4.8: Nickel affinity chromatography purification of His-Rv3676. Following page 79 Figure 4.9: SDS-PAGE analysis of fractions after nickel affinity chromatography purification of His-Msmeg_6189. Following page 80 Figure 4.10: The structure of PrpfA region of the rpfA gene promoter that contains the CRP binding site. Following page 80 Figure 4.11: The effect of cyclic AMP on the interaction between Rv3676 and PrpfA.