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 1 fimbriae of Actinomyces viscosus was cloned into Escherichia coli using pUC13 as an expression vector. The coding sequences were subcloned into pUC13 on a 1.9 Kbp Pst I-BamH I fragment; the clone expresses the complete 65 Kdal fimbrial subunit. The gene encoding the synthesis of the Type 2 fimbriae was subcloned on a 2.5 Kbp Sma I fragment. Other subclones of both genes expressed truncated proteins corresponding to the N and C termini of the fimbrial antigens. M13 dideoxy sequencing of both fimbrial genes is in progress. Purification schemes for both cloned proteins have been developed. The plasmid coded Beta-galactosidase of L. casei was subcloned into E. coli using the plasmid expression vector pKK223-3. The 5.2 Kbp Hind III fragment also encoded sequences homologous to ISL1, a putative transposon. The Beta-galactosidase was purified to homogeneity and shown to be composed of two 70 Kdal and two 42 Kdal subunits. Factor III of the lactose PEP:PTS was cloned from pLZ64 into coli. M13 sequencing of Beta-galactosidase and Factor III is in progress. High frequency transformation of L. casei protoplasts was obtained using the Beta-galactosidase plasmid, pLZ15, encapsulated in liposomes. Transformants were detected as blue colonies on X-gal plates; unaltered pLZ15 supercoiled DNA was isolated from transformants.
