The current and 7th pandemic of Vibrio cholerae caused by the El Tor biotype encodes two novel genetic islands called the Vibrio Seventh Pandemic Islands 1 and 2 (VSP-1 and VSP-2). Although acquisition of these islands is proposed to be key to initiation of the 7th pandemic, the function of these genes remains virtually unknown. The over-arching purpose of this proposal is to understand the function and regulation of a novel bacterial cytidine deaminase (CDA) regulatory system that we have discovered is encoded on VSP-1 and in many other Proteobacteria. This new CDA regulatory system consists of the multi-domain protein we named DcdV (deoxycytidine deaminase Vibrio) and its inhibitor named DifV (DcdV inhibitory factor Vibrio) encoded in a 222 NT region 5? of dcdV. These genes were first identified as our bioinformatic analysis indicated that they significantly cooccur in bacterial genomes with the VSP-1 encoded DncV/CapV cyclic GMP-AMP phage defense system that we previously discovered. Consistent with a potential role of DcdV-DifV to regulate phage defense, expressing DcdV in the absence of difV causes cell filamentation and disruption of dNTP pools in V. cholerae and Escherichia coli. Deoxycytidine deaminases (DCD) enzymes play critical roles in maintaining nucleotide homeostasis, hypermutation, and viral defense in both bacteria and eukaryotes, but in numerous respects, DcdV and its orthologs are quite different from any other previously studied DCDs. For example, all previously described DCDs are single domain proteins, while DcdV has an associated N-terminal nucleotide kinase (NK) domain that our genetic studies show is essential for DcdV activity. Furthermore, other DCDs are negatively regulated by allosteric binding of dTTP, while DcdV is instead regulated by DifV. For a litany of reasons based on preliminary studies described in the proposal, we hypothesize that activation of DcdV via inhibition of DifV skews the cellular nucleotide pool. More specifically, DcdV drives an increase in dUTP concentration and decrease in dCTP and dTTP concentrations as a two-fold phage defense mechanism, i.e., preventing accumulation of dNTP substrates for phage genome replication and promoting dUMP incorporation into phage genomes. Exactly how DcdV functions mechanistically, how this function is inhibited by DifV, and the contribution of this system to bacterial survival, for example, as part of a phage defense mechanism remains to be elucidated. We propose to study the mechanistic basis of DcdV function, its regulation by DifV, and the biological contribution of this newly discovered regulatory system to bacterial physiology in V. cholerae and other bacteria.
These aims will be pursued at the cellular and atomic level using the tools of cell biology, genetics, biochemistry, microscopy, and structural biology. By defining the mechanism and function of this novel CDA regulatory system we expect that our research will have a broad impact in multiple disciplines across both prokaryotic and eukaryotic fields.
Enzymes called cytidine deaminases are essential for living organisms to regulate nucleotide homeostasis, protect against viral infections, and drive mutations at specific parts of their genome. In this proposal, we will study the mechanism and biological function of a unique cytidine deaminase and its inhibitor that we have discovered in the bacterial pathogen Vibrio cholerae.