Regulation of Metabolism in Mycobacteria and Cyanobacteria
Vickers, C. (2015). Regulation of Metabolism in Mycobacteria and Cyanobacteria (Thesis, Doctor of Philosophy (PhD)). University of Waikato. Retrieved from http://hdl.handle.net/10289/10620
Permanent Research Commons link: http://hdl.handle.net/10289/10620
Bacteria encounter changes in their environment and must adapt to these changes in order to survive. Their ability to adapt is determined by their capacity to efficiently regulate their cellular processes. The mechanisms of bacterial regulation at the transcriptional level have been investigated by structural and functional characterisation of the transcription factor AmtR from Mycobacterium smegmatis. M. smegmatis is a soil bacteria capable of utilising alternative nitrogen sources during nitrogen limitation. AmtR is an important transcription factor that regulates the cellular machinery involved in alternative nitrogen metabolism via a novel co-repressor induced mode of regulation. A second pathway that involves post-transcriptional regulation was investigated by preliminary characterisation of a previously unexplored VapBC family of Type II Toxin Antitoxin (TA) systems found in the cyanobacteria Microcystis aeruginosa and Synechocystis sp. PCC6803 (Synechocystis). Investigation of the biochemistry of M. aeruginosa VapBC systems was limited by poor expression, however a single TA system, VapBCMAE43230/20, was successfully purified by incorporating the SUMO fusion protein into the expression construct. VapBMAE43230 and VapCMAE43220 are coexpressed and form a tetrameric complex that appears to be copurified with DNA. Similar to M. aeruginosa, only one system, VapBCslr1209/1210, in Synechocystis was successfully purified and investigated in vitro. Preliminary EMSA assays indicate VapBCslr1209/1210 is autoregulatory and binds to the promoter region of its operon by recognising palindromic sequences. The RNA pentaprobe system demonstrates that VapCslr1210 has metal dependent endoribonuclease activity that can be inhibited by VapBslr1209. Markerless deletion strains of five Synechocystis vapBC operons were made and growth experiments of three of the deletion strains were conducted. Growth experiments identified a reduced growth rate in two of the deletion strains (when compared to the WT strains): ΔvapBCslr1209/1210 and ΔvapBCssl2138/sll1092. Transcriptomic analyses of both deletion strains were conducted. Preliminary data show that both VapBC systems appear to target genes involved in carbon assimilation and metabolism via both photosynthetic and heterotrophic processes. Interestingly, VapBCslr1209/1210 targets an operon involved in glucose transport, which is also the cellular process targeted by the only other well characterised VapBC system from M. smegmatis (VapBC1283/4). These preliminary results point toward a possible similarity in biological function for VapBC systems across the prokaryotic tree.
University of Waikato
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