Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. Seasonal cholera outbreaks occur where the disease is endemic and can spread worldwide. V. cholerae's ability to cause epidemics is tied to its ability to survive in aquatic habitats. It has been proposed that V. cholerae's ability to form biofilms (i.e., matrix-enclosed, surface-associated communities) is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. The objective of this proposal is to improve our understanding of biofilm matrix components, the mechanisms and regulation of biofilm formation, the mechanism of cyclic dimeric guanosine monophosphate (c- diGMP) signaling, and their importance in the biology of V. cholerae. In Aim 1, we will focus on characterization of biofilm matrix components. We will determine Vibrio polysaccharide (VPS) binding capacities and localization patterns of the matrix proteins in biofilms, and test our hypothesis that these proteins bind to VPS in order to stabilize the matrix. We will determine which genes within the vps gene clusters are required for biofilm formation. We will also investigate the enzymatic properties of a putative VPS lyase. Finally, we will ascertain the contribution of known biofilm determinants in V. cholerae pathogenesis. In Aim 2, we will dissect regulation of biofilm formation. Biofilm regulatory network consists of two positive transcriptional regulators (VpsR and VpsT), a negative transcriptional regulator (HapR), and our recently-identified sensor histidine kinase (VpsS). We will determine whether VpsS phoshorylates VpsR and identify important signals that control VpsS. We will characterize the cis-acting features of the vps and matrix-protein genes and elucidate whether the transcriptional regulators VpsR, VpsT and HapR interact directly with these promoters. In Aim 3, we will elucidate the molecular mechanisms by which c-diGMP signaling controls biofilm formation. We will determine whether c-diGMP signaling proteins that modulate biofilm formation rely on protein-protein interactions. To identify the target proteins of the c-di-GMP signaling systems, we will search for proteins interacting with c-diGMP signaling proteins, as well as for c-diGMP receptor proteins within the rugose variants. We will then assess how these c-diGMP signaling proteins affect V. cholerae pathogenesis. Better understanding of the mechanism of biofilm formation, c-diGMP signaling, and the importance of both of these processes in V. cholerae biology will prove useful for the development of future strategies for predicting and controlling cholera epidemics, and will facilitate identification of novel drug targets for combating the pathogen during infection.