UBC Theses and Dissertations
Regulation of production, maturation and lytic release of the Rhodobacter capsulatus gene transfer agent Westbye, Alexander Bauer
The Rhodobacter capsulatus gene transfer agent (RcGTA) is a phage-like particle that mediates horizontal gene transfer between R. capsulatus strains, and its production is regulated by several bacterial systems, including quorum sensing and the CckA-ChpT-CtrA phosphorelay. This thesis presents evidence that RcGTA is released from cells by cell lysis, that lysis is modulated by the concentration of inorganic phosphate in the growth medium and that lysis requires an endolysin and holin gene. The expression of the lysis genes is regulated by the histidine kinase CckA, the phosphotransferase ChpT and the response regulator CtrA, and requires phosphorylation of CtrA. The endolysin and holin were characterized by expression in E. coli. High resolution electron microscopy images of affinity-purified RcGTA confirmed that RcGTA contains tail fibers and head spikes, and newly revealed the presence of a baseplate-like structure. RcGTA was found to undergo a maturation process similar to that of phages, and this maturation was regulated by the CckA-ChpT-CtrA phosphorelay. Cells lacking CckA produced tail-less particles containing DNA and polytube structures. During particle assembly spikes are attached to the head of RcGTA, and spike formation required ghsA and ghsB, which appear to be co-transcribed and regulated by CckA-ChpT-CtrA and quorum sensing. Spikes were required for efficient binding of RcGTA to the R. capsulatus capsular polysaccharide. Two new regulators of RcGTA, ClpX and DivL, were identified. ClpX was required for transduction, and capsid formation in cells lacking ClpX appeared to halt RcGTA production prior to DNA packaging. DivL appeared to be involved in regulating the CckA kinase activity; however a loss of DivL resulted in opposite phenotypes for an RcGTA overproducer and a wild type strain. Additionally, RcGTA production was found to be stimulated by temporary depletion of amino acids, but did not require the (p)ppGpp-mediated stringent response or a homologue of the general stress response sigma factor EcfG.
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