The regulation of production of the Rhodobacter capsulatus Gene Transfer Agent, RcGTA

Mercer, Ryan G. (2012) The regulation of production of the Rhodobacter capsulatus Gene Transfer Agent, RcGTA. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Horizontal gene exchange is the transfer of genetic material between cells and it plays an important role in the evolution of bacterial genomes. One mechanism contributing to high levels of transfer is virus-mediated transduction by bacteriophage (phage). Most bacterial species encode prophages within their genome and play host to actively transducing phage, which can lead to acquisition of new genes. An unusual form of genetic exchange is carried out by a phage-I ike particle called a gene transfer agent (GT A). The purple, non-sulfur, u-proteobacterium Rhodobacter capsulatus produces RcGT A that transfers - 4 kb of random genomic DNA in a process similar to generalized transduction. Expression of the - 15 kb gene cluster encoding the RcGTA particle is under the control of a cellular response regulator protein, CtrA, a homologue of the well-studied cell cycle regulator in Caulobacter crescentus. Despite the requirement for CtrA, the complete regulatory system controlling RcGT A has previously been poorly understood. The research in this thesis has shown that loss of CtrA has pleiotropic effects, as the protein is required for proper expression of - 6% of genes in the R. capsulatus genome. This includes all flagellar motility genes and genes encoding a number of other putative regulatory proteins. Mutations of the cckA and chpT genes in R. capsulatus revealed that the proteins encoded by these genes, along with CtrA- P, are required for motility and release of RcGTA from cells. It was also determined that both phosphorylated and unphosphorylated CtrA are capable of stimulating RcGTA gene expression. Proper expression of a set of genes encoding putative a factor regulatory proteins was also found to require CtrA and mutations in these genes affected RcGT A expression, motility, colony morphology and stationary phase cell viability. The results indicate that CtrA regulates RcGTA, at least in part, by controlling the activity of an important cr factor in R. capsulatus. The results of this thesis work have provided an excellent framework for further deciphering the regulation of this intriguing genetic exchange element.

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