Speight, Richard Alan; (2000) The structure, function and regulation of mycobacterial porin-encoding genes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Two years ago Senaratne et al., (1998) published research into a gene, ompATb, which encodes an outer membrane protein in the genus Mycobacterium. It was shown that the protein, OmpATb, possesses a pore-forming ability in liposomes and can be incorporated into lipid bilayers in which it exhibits voltage-gated pore characteristics. This study examines the distribution of this gene, and the protein product OmpATb, amongst different mycobacterial species using a PCR based method, and demonstrates immunoblotting using an antibody previously raised against the protein. Regulation of the porin genes in Escherichia coli by a two-component sensor-regulator pair is discussed and parallels drawn to a putative method of regulation in the mycobacteria. Regulation of the mycobacterial porin is demonstrated by real-time RT-PCR of Mycobacterium bovis BCG RNA and the addition of osmotic stress to the bacterial culture is shown to have a dramatic effect on the levels of ompATb transcript. Two genes, Rv0902c and Rv0903c, are proposed to have a mode of action in controlling the expression of the porin gene in Mycobacterium tuberculosis. These were identified by homology to their Escherichia coli counterparts by searching the published genome of Mycobacterium tuberculosis (Cole et al., 1998). The regulatory component, Rv0903c, is cloned into an expression vector and the recombinant Rv0903 protein overexpressed in Escherichia coli. This protein product is purified using immobilised metal affinity chromatography and is used in gel retardation assays to show that two regions around the ompATb gene bind the protein. Phosphorylation of the protein in vitro is also shown to enhance the binding affinity. The porin-encoding gene is expressed in a non-native organism, Mycobacterium smegmatis, both from its native promoter and from the mycobacterial hsp60 promoter, the effect of overexpression of this gene and the protein product is investigated. The protein is also cloned into Mycobacterium smegmatis in the presence and the absence of the putative regulatory machinery. An attempt to characterise the promoter region of the ompATb gene is made using the construction of lacZ reporter vectors. These are used for assays of β-galactosidase activity in Mycobacterium smegmatis. It is shown that the ompATb promoter functions in this organism and that the length of upstream sequence included affects the promoter activity. A suicide vector for knockout of the ompATb gene is constructed and transformed into Mycobacterium tuberculosis H37Rv in an attempt to disrupt the function of the gene and a ΔompATb strain of Mycobacterium tuberculosis 1424 is characterised by Southern blot and PCR based methods. This strain is investigated by DNA and cDNA microarray analysis and by growth in media providing a variety of environmental stresses. Gene disruption by homologous recombination is also attempted on the regulatory gene Rv0903c, employing a different technique for suicide vector construction that uses 3 counterselectable markers. This technique has been previously applied with some success in the mycobacteria (Parish and Stoker, 2000; Parish and Stoker, 2000). A number of colonies exhibiting the correct phenotype for gene knockouts are examined using a PCR based method.

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