This project seeks to use genetic, biochemical and physiological approaches to investigate the pathogenicity of oral bacteria. The three specific areas of investigation are: 1) molecular cloning and characterization of genes encoding surface structures that mediate attachment of oral microbes, 2) characterization of plasmid-coded and chromosomal metabolic genes from oral bacteria and 3) development of systems for genetic exchange in oral bacteria. A gene encoding the structural subunit of the Type 2 fimbriae of Actinomyces viscosus was cloned into Escherichia coli using the cosmid vector pHC79. This has was subcloned into p&C13 on a 10 Kbp HIND III fragment; the clone expresses the complete 59 Kdal fimbrial subunit. Subcloning for M13 dideoxy sequencing is now in progress. A gene encoding the synthesis of the Type 1 fimbriae was identified in a second genomic library; its sequence is under study. The gene for the Type 2 fimbriae of A. naeslundii was also cloned; it was found to share DNA-DNA homology with the gene isolated from A. viscosus. An unusual plasmid encoding the enzyme Beta-galactosidase was found in L. actobacillus casei; the structural gene was cloned into E. coli using the plasmid expression vector pKK223-3. Subclones are being prepared for sequencing and for analysis of L. casei promoter structure and function. For the first time Lactobacillus strains were transformed; using lactose plasmids encapsulated in liposomes prepared from lecithin, lactose fermenting transformants were derived from lactose negative strains of L. casei. Southern blot analysis confirmed the introduction of specific lactose metabolic genes into the L. casei chromosome. Current studies seek to optimize and improve the transformation system.
