Characterisation of VapBC Toxin-Antitoxins from Mycobacterium tuberculosis
Sharrock, A. V. (2013). Characterisation of VapBC Toxin-Antitoxins from Mycobacterium tuberculosis (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/7935
Permanent Research Commons link: http://hdl.handle.net/10289/7935
Toxin-antitoxin (TA) systems were identified more than 20 years ago on the mini F plasmid of Escherichia coli as plasmid stability elements; components responsible for purging bacterial cells that lack the plasmid from the population. More recent discovery of TA systems spanning a wide diversity of prokaryotic chromosomes, including that of Mycobacterium tuberculosis (M. tb), suggests a broader biochemical role. TA systems can be classified into a number of families, with the vapBC systems being by far the largest. The biochemical role of vapBC systems in M. tb remains unclear despite their abundance within the genome. This thesis describes the biochemical and functional characterisation of two vapBC systems encoded by operons Rv0065a/c and Rv0617a/c in the M. tb genome. VapCRv0617 overexpression had a bacteriostatic effect on the growth of Mycobacterium smegmatis cultures. Therefore, the biochemistry underlying this phenotype was investigated, along with that of the Rv0065a/c system. VapCRv0065 and VapCRv0617 are Mg2+-dependent, sequence-specific ribonucleases targeting GC rich 4-mers. Ribonuclease assays with in vitro synthesised RNA transcripts suggested an additional layer of target recognition resides in RNA secondary structure, and revealed that both VapC proteins exhibit high activity against isolated M. smegmatis 16S and 23S rRNA. Electrophoretic mobility shift assays with purified VapBC protein and labelled DNA demonstrated an autoregulatory function for the VapBCRv0617a/c complex and not the VapBCRv0065a/c complex. VapBCRv0617a/c bound specifically to a near-perfect inverted repeat in the Rv0617a/c promoter region that overlaps an annotated -10 M. tb promoter element. In contrast, the VapBCRv0065a/c complex exhibited no DNA binding activity against a putative Rv0065a/c promoter region. Individualised vapBC transcriptional regulation mechanisms may help explain the persistence of such an expanded number of these systems in the genome. Knowledge of the physiological role of vapBC systems in M. tb will enable a better understanding of how they contribute to the pathogenicity of this bacterium. This would serve as the basis for the design of drugs which interfere with vapBC system functioning and in turn the ability of M. tb cells to enter the persistent state.
University of Waikato
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