Production of the gene transfer agent of Rhodobacter capsulatus, RcGTA, is dependent upon several cellular regulatory systems, including a putative phosphorelay involving the CtrA and CckA proteins. These proteins are also involved in flagellar motility in R. capsulatus. The interactions of proteins in this system are best understood in Caulobacter crescentus where CtrA is activated by phosphorylation by the CckA‐ChpT phosphorelay. CtrA~P activity is further controlled by SciP, which represses ctrA transcription and CtrA activation of transcription. We show that R. capsulatus chpT and cckA mutants both have greatly reduced motility and RcGTA activity. Unlike the ctrA mutant where RcGTA gene transcription is absent, the decrease in RcGTA activity is because of reduced release of RcGTA from the cells. The sciP mutant is not affected for RcGTA production but our results support the C. crescentus model of SciP repression of flagellar motility genes. We show that both unphosphorylated and phosphorylated CtrA can activate RcGTA gene expression, while CtrA~P seems to be required for release of the particle and expression of motility genes. This has led us to a new model of how this regulatory system controls motility and production of RcGTA in R. capsulatus.

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.