Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis

Streptococcus oralis is a member of the oral streptococcal family and an ea rly-colonizing microorganism in the oral cavity of humans. S. oralis is kno wn to produce glucosyltransferase (GTase), which synthesizes glucans from s ucrose, The enzyme was purified chromatographically from a culture supernat ant of S, oralis ATCC 10557. The purified enzyme, GTase-R, had a molecular mass of 173 kDa and a pI of 6.3. This enzyme mainly synthesized water-solub le glucans with no primer dependency. The addition of GTase markedly enhanc ed the sucrose-dependent resting cell adhesion of Streptococcus mutans at a level similar to that found in growing cells of S. mutans. The antibody ag ainst GTase-R inhibited the glucan-synthesizing activities of Streptococcus gordonii and Streptococcus sanguis, as well as S. oralis. The N-terminal a mino acid sequence of GTase-R exhibited no similarities to known GTase sequ ences of oral streptococci. Using degenerate PCR primers, an 8.1-kb DNA fra gment, carrying the gene (gtfR) coding for GTase-R and its regulator gene ( rgg), was cloned and sequenced. Comparison of the deduced amino acid sequen ce revealed that the rgg genes of S. oralis and S, gordonii exhibited a clo se similarity. The gtfR gene was found to possess a species-specific nucleo tide sequence corresponding to the N-terminal 130 amino acid residues. Inse rtion of erm or aphA into the rgg or gtfR gene resulted in decreased GTase activity by the organism and changed the colony morphology of these transfo rmants. These results indicate that S. oralis GTase may play an important r ole in the subsequent colonizing of mutans streptoccoci.