For many bacteria, humans are their only habitat, and evolution in these bacteria is in response to selective pressure from the host environment. As each human host represents a unique ecological niche, bacterial virulence factors that contribute to colonization and pathogenicity may need to be tailored to the specific host, in order to maximize fitness of the microorganism. Although all bacteria are able to diversify their genomes by random mutagenesis, species that also transfer DNA between strains have an added advantage in their ability to re-assort useful mutations into the same bacterial cell. Porphyromonas gingivalis is a Gram-negative anaerobe that colonizes plaque biofilms in the human subgingival crevice, and is a causative agent in the development of chronic and severe periodontal disease. P. gingivalis is a genetically diverse species, but little is known about how this diversity is generated, or how it contributes to the fitness of these important pathogens during chronic infections. The long-term goal of this project is to understand how genetic diversity contributes to fitness in chronic periodontal infections. As a first step towards this goal, the objective of this application is to determine the molecular mechanisms underlying genetic variability in P. gingivalis, and to measure the effect of DNA changes on bacterial fitness, using multi-species biofilm formation as a model system. These goals have been formulated based on a fundamental new behavior discovered in P. gingivalis: chromosomal DNA transfer between strains, which we hypothesize, is a major diversity generating mechanism for these oral pathogens. We will pursue the following three specific aims to address this hypothesis. Aim One: Identify the genes that control horizontal transfer of chromosomal DNA in P. gingivalis. Aim Two: Determine the prevalence of chromosomal DNA transfer within the species. Aim Three: Identify genotypic changes that contribute to fitness improvements during P. gingivalis biofilm formation. The experiments described in this proposal will determine the effect of horizontal DNA transfer on complex bacterial phenotypes. The identification of bacterial genes that are transferred and favorably selected in response to environmental pressure will provide a novel framework for the identification of therapeutic targets for treatment of periodontal disease.