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 early-colonizing microorganism in the oral cavity of humans. S. oralis is known to produce glucosyltransferase (GTase), which synthesizes glucans from sucrose. The enzyme was purified chromatographically from a culture supernatant 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-soluble glucans with no primer dependency. The addition of GTase markedly enhanced the sucrose-dependent resting cell adhesion of Streptococcus mutans at a level similar to that found in growing cells of S. mutans. The antibody against GTase-R inhibited the glucan-synthesizing activities of Streptococcus gordonii and Streptococcus sanguis, as well as S. oralis. The N-terminal amino acid sequence of GTase-R exhibited no similarities to known GTase sequences of oral streptococci. Using degenerate PCR primers, an 8.1-kb DNA fragment, carrying the gene (gtfR) coding for GTase-R and its regulator gene (rgg), was cloned and sequenced. Comparison of the deduced amino acid sequence revealed that the rgg genes of S. oralis and S. gordonii exhibited a close similarity. The gtfR gene was found to possess a species-specific nucleotide sequence corresponding to the N-terminal 130 amino acid residues. Insertion 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 transformants. These results indicate that S. oralis GTase may play an important role in the subsequent colonizing of mutans streptoccoci.
Fujiwara
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Department of Pedodontics, Osaka University Faculty of Dentistry, Suita-Osaka 565-0871, Japan. fujiwara@dent.osaka-u.ac.jp
Hoshino
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Ooshima
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Sobue
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Hamada
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D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019533:Streptococcus oralis / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D015534:Trans-Activators
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C074779:rgg protein, Streptococcus; D005964:Glucosyltransferases; C409029:GtfR protein, Streptococcus