UBC Theses and Dissertations
Studies of Rhodobacter capsulatus gene transfer agent recipient capability regulated by quorum-sensing and the CtrA response regulator Brimacombe, Cedric
Gene transfer agents (GTAs) are agents of genetic exchange that resemble small tailed DNA bacteriophages and transfer random segments of the producing cell's genome to recipient cells. The canonical GTA is produced by the α-proteobacterium Rhodobacter capsulatus, hereafter referred to as RcGTA. The RcGTA packages ~4 kb segments of genomic DNA, and is produced and released by the lysis of a sub-population of donor cells in the stationary phase of growth. The primary structural gene cluster is a ~ 15 kb genomic region. Production and release of RcGTA is regulated by several host systems, including the GtaI quorum-sensing system, and the CckA/ChpT/CtrA putative phosphorelay system. Prior to this work, studies on RcGTA focused primarily on aspects involved in the production of RcGTA particles, such as gene regulation, DNA packaging, and biological functionality. However essentially nothing was known about how RcGTA delivers DNA to recipient cells. Herein, several key aspects of the capability of a cell to receive an RcGTA carried genetic marker, defined as RcGTA recipient capability, are delineated. Initial studies on the GtaR/I quorum-sensing system showed that gtaR/I are co-transcribed, and indirectly regulate not only transcription of the RcGTA gene cluster, but also RcGTA recipient capability. Part of this quorum-sensing effect was attributed to regulation of capsular polysaccharide production, which was determined to be involved in RcGTA adsorption to cells. Additionally, it was found that CtrA is essential for, and a regulator of several genes required for RcGTA recipient capability. CtrA was found to regulate a set of natural competence genes involved in DNA entry into the cell and in RecA-mediated homologous recombination. These genes, DprA, ComM, ComEC, and ComF, are all essential for RcGTA recipient capability, and analyses of the encoded proteins were used to propose a pathway for acquisition of RcGTA-borne DNA. These findings indicate that the RcGTA horizontal gene transfer mechanism is a combination of two fundamentally different horizontal gene transfer (HGT) mechanisms, transduction and transformation, generating a very efficient mode of HGT.
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