The phages and phage-like elements of Rhodobacter capsulatus

Hynes, Alexander P. (2014) The phages and phage-like elements of Rhodobacter capsulatus. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Rhodobacter capsulatus is a purple non-sulfur bacterium that produces the gene transfer agent RcGTA. This phage-like particle is capable of transferring ~4-kb of host DNA to other R. capsulatus cells in a process analogous to transduction. The genes known to encode this particle are located in a ~15-kb region called the RcGTA structural gene cluster. As this cluster is larger than the packaging capacity of the particles, RcGTA is non-replicative. In addition to lacking key functions required by a phage, the RcGTA structural gene cluster lacks genes likely to encode functions necessary for its gene transfer activity, such as a cellular release mechanism. The costs and benefits of RcGTA production for R. capsulatus are unclear, making it difficult to explain its persistence in the genome. I investigated R. capsulatus SB1003 prophages to search for other genes involved in RcGTA production and gene transfer activity. I identified two functional prophages, RcapNL and RcapMu, which I characterized and found to be linked to RcGTA production. I also compared the genome-wide transcriptional profiles in a variety of R. capsulatus strains and growth conditions affecting RcGTA production to identify genes consistently co-regulated with the RcGTA structural gene cluster. I found nine such genes at six separate loci. In characterizing several of these genes, I identified a gene required for release of RcGTA by cell lysis, a pair of putative tail fibre-encoding genes, and an additional gene whose product is required for binding to the recipient cell. Finally, I characterized features of RcGTA production by quantitatively determining the packaging frequency of all loci on the R. capsulatus chromosome. Remarkably, while any gene can be transferred, the RcGTA structural gene cluster region was under-represented iii in the particles relative to the genome average. The search for a mechanistic explanation of this anomaly led to the discovery that RcGTA gene expression is elevated in ~3% of the cells in a population, and that these cells undergo lysis to release RcGTA particles. This provided us with the first quantification of the cost of RcGTA production.

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