PROJECT SUMMARY/ABSTRACT Non-cholera Vibrio (NCV) species represent a notable and increasing threat to human health and food safety. The biofilms formed by Vibrio species are robust and highly relevant to host infection. Biofilms are tightly regulated communities of matrix-associated bacteria and are a major component of bacterial pathogenesis including drug resistance. Since an estimated 75% of bacterial infections involve biofilms, it is crucial to better understand how biofilms are formed. In this application, I propose to elucidate the molecular mechanism of an unusual regulatory protein necessary for controlling NCV biofilm formation. This protein is conserved in NCVs and controls the production of symbiosis polysaccharide (Syp), a component of the biofilm matrix involved in host infection. Based on preliminary findings, this protein has an atypical mode of action compared to well characterized homologs. My fundamental hypothesis is that this NCV biofilm regulator protein uses a novel mechanism to control biofilm formation and will challenge our current understanding of this family of proteins. My hypothesis will be addressed through two specific aims: 1) probing the protein’s function using biochemical and structural characterization studies, and 2) uncovering its position in the regulatory network by identifying binding partners. The proposed research is innovative because it focuses on a system that is crucial for biofilm formation, and to date, the system has only been investigated using cellular and genetic approaches. The structure-function approach proposed here will provide essential information needed to fill our knowledge gaps. The project is significant because it will provide a deeper understanding of the regulation of host-associated biofilms from the rising threat of NCV infection. Ultimately, the work proposed will answer key questions related to the molecular mechanisms of a protein within a critical and conserved pathway that regulates host-relevant biofilms. Support from this K22 award will facilitate my transition to an independent investigator by providing me with resources to generate data and publications that will strengthen my competitiveness for future funding.