Actinobacillus actinomycetemcomitans (Aa) has been implicated in the pathogenesis of juvenile and adult periodontitis. This microorganism also is capable of causing more serious human infections such as endocarditis and soft tissue abscesses, and the likely source of these infections is Aa derived from oral sources. The ability of Aa to cause these disease states clearly depends on its ability to colonize and/or invade oral, cardiac, and soft tissues, and to elaborate various products that contribute to its survival in the host, or to cause damage to these tissues. The identification of Aa virulence traits, and tests for their actual roles in pathogenesis, will depend on the availability of modern genetic and molecular tools, and suitable animal models. With regard to the former, genetic and molecular analyses of Aa and its virulence have been lacking, due in large measure to the paucity of genetic transfer systems for this microorganism, and the total absence of appropriate host-vector systems for the application for recombinant DNA technology, including the delivery of manipulated genetic information for mutagenesis and studies of gene regulation. The applicant has described the isolation of plasmid DNA from two strains of Aa, and the construction of E. coli/Aa shuttle plasmids from one of the Aa replicons (pVT736-1), and the use of one of the shuttles to establish an electroporation-based transformation system for this species. More recently, the applicant has examined the properties of pVT736-1 in greater detail, and obtained preliminary data on the replication in Aa of three additional plasmids, two of Gram-negative and one of Gram- positive bacterial origin. The goal of this proposal is to assess the potential of all four plasmids as molecular and genetic tools for the analysis of Aa. Two specific aims are: 1) to evaluate the properties of each plasmid relative to its potential as an E. coli/Aa shuttle vector; and 2) to construct a vector with properties that enhance recombinational events between Aa genomic DNA and plasmid DNA.