UBC Theses and Dissertations
Evolution and regulation of the gene transfer agent (GTA) of Rhodobacter capsulatus Taylor, Terumi Anne
The gene transfer agent (GTA) of Rhodobacter capsulatus is an unusual entity that resembles a bacteriophage (phage), but does not appear to cause cell lysis and does not form plaques. GTA packages random 4.5 kb fragments of double-stranded bacterial genomic DNA and is capable of transducing recipient R. capsulatus cells. GTA has mainly been used as a mechanism to create genetic mutants, however little about the evolutionary history and regulation has been investigated, considering its relevance as a model of unusual viral-bacterial interactions. This work describes a phylogenetic analysis of GTA genes that indicates predominantly vertical descendence and limited horizontal transfer between related viruses. Investigation into the regulation of GTA revealed a link to quorum sensing through the discovery of the gtal gene. The Gtal protein is a Luxl homologue that acts as an acyl-homoserine lactone synthase. When gtal is knocked out, GTA activity is reduced significantly (5-7 times less activity). Also described is the creation of a tool to measure GTA activity that is independent of the packaging, transfer and incorporation of genetic markers that is typically used in the investigation of GTA activity, also known as the bioassay. Instead, an immunoassay that requires less time to perform (two days instead of four) was created using a tagged GTA capsid protein as an antigen to obtain antisera. This GTA immunoassay was tested and a standard protocol is described for optimal use in R. capsulatus strains. The immunoassay was employed for preliminary investigation of environmental factors affecting GTA production. The results show that under phosphate limitation in a minimal medium, GTA production increases to levels similar to the rich medium YPS. It was also found that GTA production increased in response to carbon limitation in a minimal medium, but this appeared to affect only intracellular amounts of GTA, with little release into the culture medium.
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