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Characterization of a signal transduction phosphorelay controlling Rhodobacter capsulatus gene transfer agent (RcGTA) gene expression

University of British Columbia

The alphaproteobacterium Rhodobacter capsulatus mediates an unusual method of horizontal gene transfer within its population via the production of a small phage-like particle, a gene transfer agent (GTA). Although GTAs have been found to exist in a wide variety of microorganisms, the GTA of R. capsulatus (RcGTA) remains the best understood and has become a model for the study of GTAs. All known GTAs are under the control of bacterial regulators; specifically, in R. capsulatus the production of RcGTA is mediated by the CtrA response regulator. CtrA is mostly known as the master cell cycle regulator in Caulobacter crescentus, where it is the centerpiece of a regulatory phosphorelay that includes the CckA histidine kinase and the ChpT phosphotransferase. Homologues of CtrA, ChpT and CckA exist in R. capsulatus and their role in the regulation of RcGTA-mediated gene transfer has been demonstrated in different ways. CtrA is involved in both particle production and recipient capability, and CckA and ChpT are essential for particle maturation and release. With these roles in mind and the sequence similarity between the R. capsulatus and C. crescentus CckA, ChpT and CtrA proteins, it was thought that these proteins form a similar phosphorelay in R. capsulatus. However, although a growing body of evidence demonstrates the regulatory roles of these proteins in RcGTA-mediated gene transfer, no conclusive evidence existed to show that they form such a phosphorelay in R. capsulatus. Here, through a set of in vitro phosphorylation assays and PhosTag polyacrylamide gel electrophoresis analysis, I present evidence that the R. capsulatus CckA, ChpT and CtrA proteins form a phosphorelay. CckA autophosphorylates in the presence of ATP and transfers this phosphate to the ChpT phosphotransferase, which in turn transfers it to CtrA. I also show evidence that the CckA kinase responds to cyclic-di-GMP, switching its activity from a kinase to a phosphatase and inverting the phosphate flow in the phosphorelay. These data, coupled with the analysis of the effect of three different site-directed CckA mutants on the phosphorelay, support a model of how RcGTA gene expression is regulated differentially by CtrA and CtrA~P, the phosphorylated form of CtrA.

Text

2020-04-27

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/74186

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/