Actinobacillus actinomycetemcomitans (Aa) is a Gram-negative, facultative, capnophylic, coccobacillus found in the oral cavities of healthy and periodontally affected individuals. Aa has been implicated as a cause of localized juvenile periodontitis and some forms of adult periodontitis, as well as other types of human infections, such as endocarditis and soft tissue abscesses. A number of Aa phenotypes have been proposed as candidate virulence traits, and two, the ability to invade epithelial cells, and the production of a potent leukotoxin, currently are under intense investigation. However, a complete and thorough investigation of the roles of these traits, as well as others, must await the availability of sophisticated molecular biological and genetic tools for the construction of well-characterized isogenic mutants, and for the trans and cis complementation of such mutations, in order to confirm their functions, and to analyze their regulation. The construction of molecular and genetic tools will require an understanding of the replication and genetic transfer mechanisms of extrachromosomal genetic elements, such as plasmids, that are either indigenous to Aa, or that are derived from other bacterial species, but are able to replicate in, and/or transfer to and from Aa. It also will be necessary to assess the genetic behavior of Aa strains relative to their potential as hosts for the development of host/vector systems. It is the goal of the proposed studies to obtain such information by 1) the continued assessment of potential candidate Aa/E. coli shuttle vectors, as well as vectors for direct cloning in Aa, 2) initiating studies of Aa properties that may impact on their suitability for genetic and molecular biological analyses, such as their transformability, the presence of restriction/modification systems, and the functions of recA gene homologs, 3) an examination of the vegetative (oriV) and transfer (oriT) origins of an indigenous, conjugative Aa plasmid, and 4) a characterization of the conjugation system of this plasmid. The latter studies will be pursued with an eye toward the development of a plasmid mobilization system for the transfer of recombinant molecules to strains of Aa that either are poorly transformable, or not transformable at all.
