Regulation of Metabolism in Mycobacteria and Cyanobacteria

Vickers, Chelsea

Regulation of Metabolism in Mycobacteria and Cyanobacteria

dc.contributor.advisor	Arcus, Vickery L.
dc.contributor.advisor	McDonald, Ian R.
dc.contributor.author	Vickers, Chelsea
dc.date.accessioned	2016-09-01T23:59:50Z
dc.date.available	2016-09-01T23:59:50Z
dc.date.issued	2015
dc.date.updated	2016-09-01T03:58:56Z
dc.description.abstract	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.
dc.format.mimetype	application/pdf
dc.identifier.citation	Vickers, C. (2015). Regulation of Metabolism in Mycobacteria and Cyanobacteria (Thesis, Doctor of Philosophy (PhD)). University of Waikato. Retrieved from https://hdl.handle.net/10289/10620	en
dc.identifier.uri	https://hdl.handle.net/10289/10620
dc.language.iso	en
dc.publisher	University of Waikato
dc.rights	All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subject	AmtR
dc.subject	VapBC
dc.title	Regulation of Metabolism in Mycobacteria and Cyanobacteria
dc.type	Thesis
thesis.degree.grantor	University of Waikato
thesis.degree.level	Doctoral
thesis.degree.name	Doctor of Philosophy (PhD)