UBC Theses and Dissertations

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UBC Theses and Dissertations

A site-directed mutagenesis approach to study the functions of the histidine kinase CckA in Rhodobacter capsulatus gene transfer agent production and recipient capability Wiesmann, Christina


The Rhodobacer capsulatus gene transfer agent (RcGTA) is a phage-like particle capable of packaging, and transferring, ~4 kbp fragments of DNA between different strains of R. capsulatus. This genetic transfer is dependent upon a variety of factors, including the regulation of transcription of the main structural gene cluster, particle maturation, release, and uptake (RcGTA recipient capability) by other, ‘RcGTA competent’ cells. These processes have been previously demonstrated to be regulated by the CckA-ChpT-CtrA phosphorelay pathway. I have created three CckA site-directed mutants thought to be involved in mediating kinase, phosphatase, or cyclic-di-GMP binding activities of the CckA protein. My results provide strong evidence that these three activities play roles in regulating the transcription, maturation, and RcGTA ‘competency’ of these cells. My thesis also provides evidence that the ChpT, and DivL regulatory proteins play a role in the regulation of RcGTA recipient capability. I further demonstrate that cell growth and morphology are not noticeably affected by mutations in CckA activity, and that differences in RcGTA recipient capability are not due to differences in the ability of RcGTA particles to bind, or adsorb, to cells. Overall, my thesis provides novel insights in to how RcGTA production and recipient capability are regulated, furthering our understanding of how this novel horizontal gene transfer mechanism is regulated.

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