File(s) under permanent embargo
Reason: Restricted by author. A copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library or by emailing email@example.com
Functional analysis of the conjugation system from Clostridium perfringens
thesisposted on 24.02.2017, 00:54 by Wisniewski, Jessica Ann
In Clostridium perfringens, toxins that cause histotoxic and gastrointestinal diseases have been identified on a family of large conjugative plasmids. The tcp transfer locus was initially identified on the prototype conjugative plasmid, pCW3. This locus has been shown to mediate conjugative plasmid transfer and studies on several of the Tcp proteins have identified their role in plasmid transfer. In general less is known about the mechanism of conjugative transfer in Gram positive bacteria, nonetheless some functional similarity between the protein families in the C. perfringens conjugation system and other Gram positive systems have been identified. To contribute to our understanding of the mechanism of conjugative transfer in C. perfringens this thesis examined the functional role of TcpM (formerly called IntP), TcpD, TcpE and TcpJ, all of which are encoded on the tcp transfer locus of pCW3 (Chapter 2 and 3), and identified components of the novel relaxosome of pCW3 (Chapter 3). To determine the role of the hypothetical proteins, TcpD, TcpE and TcpJ, independent homologous recombinant mutants were constructed. Conjugative mating studies with the pCW3∆tcpD and pCW3∆tcpE mutants and their complemented derivatives showed that TcpD and TcpE were essential for pCW3 transfer. The pCW3∆tcpJ mutant transferred at levels similar to wild-type, which showed TcpJ was not required for conjugative transfer. To further analyse the role of TcpD and TcpE, functional HA-tagged derivatives were constructed and shown to complement the respective mutants. Cell fractionation, western blot and immunofluorescence studies showed that TcpD and TcpE were localized to the cell envelope, primarily at the poles of C. perfringens cells, independently of proteins encoded on pCW3. These results provide evidence that the essential conjugation proteins, TcpD and TcpE, form part of the multi-protein transfer apparatus in C. perfringens donor cells. Although several components of the multi-protein transfer apparatus had been identified, nothing was known about the relaxosome complex that must be essential for C. perfringens plasmid transfer. Key components of this complex, the relaxase gene and the oriT site, had not been identified on pCW3. The first gene on the tcp locus, tcpM, was hypothesised to be the pCW3 relaxase due to its similarity to tyrosine recombinases, a family of DNA processing proteins. A tcpM insertion mutant was constructed and shown to have a significantly reduced level of plasmid transfer. Site-directed mutagenesis identified a tyrosine residue Y259 as the only conserved tyrosine recombinase catalytic residue involved in the in vivo activity of TcpM. The residue Y259 was also shown to be required for the ability of TcpM to relaxase plasmid DNA in an in vitro assay, which is consistent with the requirement for nucleophilic attack on the DNA substrate by a relaxase. A gene encoding a potential accessory protein, TcpK, of the pCW3 relaxosome was identified outside the tcp locus. Mutagenesis and complementation studies showed that TcpK was required for wild-type pCW3 transfer. Mobilization studies then were used to identify the pCW3 oriT site. A 150 bp fragment located within the intergenic region between tcpK and tcpM increased mobilization of a shuttle vector. Gel mobility shift studies showed that TcpM bound to the oriT site. The addition of TcpK resulted in the formation of a supershift, providing evidence that TcpK could bind to the TcpM-oriT complex. In conclusion, this thesis has completed the genetic analysis the tcp transfer locus on pCW3, identified a gene outside of the currently defined tcp locus as being involved in conjugative transfer and identified the DNA substrate, the pCW3 oriT site. Overall these results have lead to the identification of the novel pCW3 relaxosome. These findings have furthered our understanding of the mechanism of C. perfringens plasmid transfer and have contributed to the advancement of knowledge of Gram positive conjugation systems.