Roles of fructosyltransferase and levanase-sucrase of Actinomyces naeslundii in fructan and sucrose metabolism

The ability of Actinomyces naeslundii to convert sucrose to extracellular h omopolymers of fructose and to catabolize these types of polymers is suspec ted to be a virulence trait that contributes to the initiation and progress ion of dental caries and periodontal diseases. Previously, we reported on t he isolation and characterization of the gene, ftf, encoding the fructosylt ransferase (FTF) of A. naeslundii WVU45. Allelic exchange mutagenesis was u sed to inactivate ftf, revealing that FTF-deficient stains were completely devoid of the capacity to produce levan-type (beta2,6-linked) polysaccharid es. A polyclonal antibody was raised to a histidine-tagged, purified A. nae slundii FTF, and the antibody was used to localize the enzyme in the supern atant fluid. A sensitive technique was developed to detect levan formation by proteins that had been separated by sodium dodecyl sulfate-polyacrylamid e gel electrophoresis, and the method was used to confirm that the levan-sy nthesizing activity of A. naeslundii existed predominantly in a cell-free f orm, that a small amount of the activity was cell associated, and that the ftf mutant was unable to produce levans. By using the nucleotide sequence o f the levanase gene of a genospecies 2 A. naeslundii, formerly Actinomyces viscosus, a portion of a homologue of this gene (levJ) was amplified by PCR and inserted into a suicide vector, and the resulting construct was used t o inactivate the levJ gene in the genospecies 1 strain WVU45. A variety of physiologic and biochemical studies were performed on the wild-type and Lev J-deficient strains to demonstrate that (i) this enzyme was the dominant le vanase and sucrase of A. naeslundii; (ii) that LevJ was inducible by growth in sucrose; (iii) that the LevJ activity was found predominantly (>90%) in a cell-associated form; and (iv) that there was a second, fructose-inducib le fructan hydrolase activity produced by these strains. The data provide t he first detailed molecular analysis of fructan production and catabolism i n this abundant and important oral bacterium.