The proposed project will decipher the molecular mechanisms that control adhesin production and biofilm formation in alpha-proteobacteria, a physiologically heterogeneous group that includes a number of pathogens that cause serious human diseases. Biofilm formation plays a crucial role in the survival of bacteria in diverse environments and is of great relevance to public health. Cells in biofilms attach recalcitrantly to biotic and abiotic surfaces, develop increased resistance to antimicrobial agents, and contribute to persistent infections. Although limiting biofilm formation has the potential to prevent and restrict bacterial infections, little is known about biofilm formation by alpha-proteobacteria and how it facilitates host colonization. Recently, we discovered a mutation in the conserved cell polarity factor PodJ in domesticated, laboratory strains of Sinorhizobium meliloti -- a model alpha-proteobacterium that establishes mutualistic symbioses with compatible legumes by colonizing their root tissues and fixing molecular nitrogen in exchange for nutrients from host plants. Correcting the mutation and restoring the PodJ polarity factor to the version found in environmental isolates of S. meliloti enabled the Rm1021 laboratory strain to produce a polar adhesin, called holdfast, and to develop robust biofilms. Furthermore, the corrected allele (podJ+) conferred a competitive advantage during symbiosis. These novel phenotypes have never been characterized because commonly used S. meliloti strains all possess the mutation. The goal of this proposal is to elucidate the genetic circuitry that links the cell cycle-dependent polarity factor PodJ to the regulated production of adhesive holdfast, which promotes biofilm formation and enhances fitness, particularly during host colonization. Achieving this goal involves accomplishing the following four specific aims: (1) determine the subcellular locations of PodJ and holdfast as a function of the cell cycle; (2) evaluate environmental conditions for their effects on holdfast synthesis and biofilm formation; (3) assess if holdfast increases attachment to host tissue; and (4) identify and define the roles of cellular factors that participae in holdfast production. Results from the investigation will provide a better model of how conserved and species-specific pathways can contribute to adhesion and biofilm formation in alpha-proteobacteria. Such knowledge will provide the foundation for developing new methods to combat biofilms and bacterial infections. In addition to accomplishing the scientific objectives described above, funding of this proposal will enhance the research productivity and grant competitiveness of the investigator and allow students, particularly those from underrepresented backgrounds, to gain exposure to an exciting area of research, thus nurturing their pursuits of biomedical careers.