The function and evolution of the response regulator CtrA in Rhodobacter capsulatus and Alphaproteobacteria

Kollipara, Roshni S. (2020) The function and evolution of the response regulator CtrA in Rhodobacter capsulatus and Alphaproteobacteria. Masters thesis, Memorial University of Newfoundland.

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Abstract

Rhodobacter capsulatus is a model organism for studying gene transfer agents (GTAs). GTAs are a unique facilitator of gene transfer in prokaryotes. The DNA binding response regulator CtrA plays a key role in modulating GTA activity in R. capsulatus, as well as flagellar biosynthesis and cell motility. CtrA is an OmpR/PhoB response regulator with an N-terminal receiver domain and a C-terminal transcriptional regulator domain. One unusual aspect of CtrA function in R. capsulatus is that it regulates gene expression in both the phosphorylated and nonphosphorylated forms. Using overlap extension PCR, the constructs for expression of three of different versions of ctrA in R. capsulatus were prepared: wild type, phosphomimetic, nonphosphorylatable. These constructs place the genes under the control of the R. capsulatus puf promoter for high level of expression and the encoded proteins have 6×-histidine tags for purification in studies aimed at determination of the DNA binding sites of the different versions of CtrA. Horizontal gene transfer is an interesting way that bacteria can increase their genetic diversity. In this work, the distribution of ctrA in the Alphaproteobacteria was examined and evidence of horizontal gene transfer of this gene was found. Using phylogenetic analyses, several instances of apparent misclassification of alphaproteobacteria to the wrong orders were found and one candidate ctrA horizontal gene transfer event that may have occurred in an ancestral bacterium that subsequently evolved into one lineage within the order Sphingomonadales was found.

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