Regulation of gene transfer and motility in the bacterium Rhodobacter capsulatus by the dinucleotide cyclic dimeric guanosine monophosphate (c-di-GMP)

Gene transfer agents (GTAs) are bacteriophage-like particles produced by several bacterial and archaeal lineages that contain small pieces of the producing cells’ genomes that can be transferred to other cells in a process similar to transduction. One well-studied GTA is RcGTA, produced by the -proteobacterium Rhodobacter capsulatus. RcGTA gene expression is regulated by several cellular regulatory systems, including the CckA-ChpT-CtrA phosphorelay. A previous study on CtrA, a DNA-binding response regulator protein, showed that transcription of multiple other regulator-encoding genes was affected by its loss. These included genes encoding proteins predicted to be involved in the synthesis and hydrolysis of the second messenger bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP). In this thesis, I investigated these genes and found that disruption of four of them (rcc00620, rcc00645, rcc02629, rcc02857) affected RcGTA production and flagellar motility. I performed site-directed mutagenesis of key catalytic residues in the functional domains responsible for diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities involved in synthesizing and hydrolysing cyclic-di-GMP, respectively, and analysed the activities of the wild type and mutant proteins. Through this work, I demonstrated that c-di-GMP acts to inhibit RcGTA production and motility. I subsequently demonstrated that the enzymatic activity of one of the proteins, Rcc00620, is regulated through the phosphorylation status of its REC domain, which is controlled by a cognate histidine kinase protein, Rcc00621. In this system, the phosphorylated form of Rcc00620 is active as a PDE enzyme and stimulates gene transfer and motility. Interestingly, the rcc00620/rcc00621 genes were acquired via horizontal gene transfer from a distantly related α-proteobacterium. I also explored a potential mechanistic link between c-di-GMP and RcGTA regulation via the CckA-ChpT-CtrA phosphorelay and confirmed that c-di-GMP binds to CckA and this shifts its activity from kinase to phosphatase and thereby affects CtrA phosphorylation. All these results add c-di-GMP signaling to the collection of cellular regulatory systems controlling gene transfer in this bacterium and add gene transfer to the diverse list of activities affected by c-di-GMP in bacteria.