Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which there have been seven pandemics. The first six pandemics (1817-1923) have been attributed to strains of the classical biotype while the seventh pandemic (1961- current) is being perpetuated by circulating strains of the El Tor biotype. It is hypothesized that El Tor's acquisition of two unique genomic islands of unknown origins, Vibrio Seventh Pandemic Island 1 & 2 (VSP-1 & 2), potentiated its displacement of classical strains in both environmental and clinical reservoirs prior to the seventh pandemic. Despite their connection to the pandemic evolution of the El Tor biotype and the likelihood they encode a wealth of novel biological functions the majority of the 303036 ORFs in the VSP islands have remained uncharacterized. The works presented in the thesis represent examples of our collective efforts to understand the unique traits afforded to the El Tor biotype by the VSP islands. .In 2012, ten years after the initial discovery of the VSP islands, the cyclic di-nucleotide synthase DncV was identified in VSP-1 and shown to synthesize the novel second messenger cyclic-GMP-AMP (cGAMP). Despite this significant discovery, cGAMP remained an orphan second messenger in bacteria for six years. In Chapter 2, I present our identification of the first cGAMP signaling network in bacteria by connecting the synthesis of cGAMP to the allosteric activation of the VSP-1 encoded patatin-like phospholipase vc0178, now named CapV. This finding helped catalyze the revelation that homologous cyclic-dinucleotide signaling networks contribute to bacteriophage-immunity and are distributed widely across the bacterial phyla.dncV and capV represent just two of the 103031 ORFs encoded in VSP-1, therefore we hypothesized that cGAMP signaling may extend to include additional genes within the island. Bioinformatic analysis of VSP island genes performed in collaboration with the laboratory of Eva Top at the University of Idaho predicted that dncV was likely to share a biological pathway with the VSP-1 encoded putative deoxycytidylate deaminase vc0175, we have now named DcdV. In Chapter 3, I describe our ongoing efforts to understand the biological function of DcdV and the discovery of its novel negative regulator DifV.The work presented in this thesis has made fundamental contribution to understanding the pandemic evolution of the El Tor biotype and the utility homologous genes afford to their bacterial host while providing a roadmap for further exploration of the biological role of the VSP-1 and VSP-2 islands in pandemic V. cholerae.
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