Rao, Tara (2010)
Ph.D. thesis, University of Birmingham.
Although most bacteria contain a single gene for the essential oligomeric chaperonin Cpn60, many contain two or more cpn60 genes. The non-pathogenic Mycobacterium smegmatis has three cpn60 homologues, while the pathogenic Mycobacterium tuberculosis has two. This study is a functional characterisation of the chaperonins of M. smegmatis. Expression of cpn60.1, cpn60.2 and cpn10, but not cpn60.3, was found to be induced under stress conditions, particularly heat shock. Studies of the cpn10-cpn60.1 operon concluded that transcription is from a single promoter, with a subsequent post-transcriptional cleavage of the mRNA between the two genes. Cpn60.1 in M. smegmatis is required for biofilm maturation. Using this assay, we intended to test various Cpn60 homologues for their ability to function in M. smegmatis. Preliminary results obtained revealed that only the M. tuberculosis Cpn60.1 can fully complement for loss of the M. smegmatis Cpn60.1. While E. coli GroEL and Cpn60.3 appear to only partially complement, Cpn60.2 shows no complementing ability. Cpn60.2 expresses well and complements for loss of the E. coli Cpn60 homologue GroEL even at higher temperatures (42°C). Using native gels and analytical ultracentrifugation, purified Cpn60.2 did not oligomerise under normal conditions, however in the presence of nucleotide and high concentrations of salt, the formation of large oligomers was observed. Neither Cpn60.1 nor Cpn60.3 complemented for loss of GroEL.
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