Characterisation of gene regulation in mycobacteria

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.