Previous results have indicated that the glucosyltransferase activities of mutans streptococci are required for sucrose-dependent colonization of tooth surfaces. We have constructed mutants of Streptococcus mutans GS5 that are altered in varying combinations of the three gtf genes present in this organism. A quantitative in vitro sucrose-dependent attachment system was used to demonstrate that the inactivation of the gtfC gene drastically reduced adherence to smooth surfaces. By contrast, inactivation of the gtfB gene resulted in a smaller, but significant, reduction in attachment while the gtfD mutant was only marginally affected. Furthermore, production of only the glucosyltransferase C enzyme allowed for attachment although at reduced levels compared to the wild-type organism. The results from reintroduction of single copies of each of the gtf genes into a mutant of strain GS5 lacking glucosyltransferase activity also demonstrated the crucial role of the glucosyltransferase C enzyme in colonization. These results suggest a unique role for the glucosyltransferase C enzyme in the sucrose-dependent colonization of tooth surfaces by S. mutans strains.

to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp 411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp 491 or His 561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.The important role of the Streptococcus mutans glucosyltransferases (GTFs) (EC 2.4.1.5) in the induction of human dental caries has been well documented (16). These enzymes catalyze the synthesis of both water-insoluble glucans (IG) and soluble glucans (SG) from dietary sucrose. Human strains of S. mutans normally express three distinct GTFs, GTF-I and GTF-SI, which synthesize primarily ␣-1,3-rich glucans, and GTF-S, which produces ␣-1,6-linked glucans exclusively (1,7,8). The combined action of the three enzymes is required for maximum dental caries in experimental animals (32). Despite intensive investigation of these enzymes over the past decade by both molecular genetic (13) and biochemical (18) approaches, structure-function analysis of the GTFs has been initiated only recently (19,21,25). This is due mostly to the relatively large sizes of the enzymes (approximately 1,450 amino acids), which have made structural investigations difficult. Nevertheless, the isolation of the genes coding for these enzymes (13), as well as for related enzymes from other mutans streptococci (23) in addition to Leuconostoc mesenteroides (31), has made it possible to deduce structural similarities with other well studied enzymes, including ␣-amylases (17). These later approaches have suggested that the GTFs are members of the (␤/␣) 8 barrel-containing protein family. Since certain amino acid positions appear to be highly conserved in such proteins, it appeared to be possible to predict which amino acid residues of the GTFs are essential for activity. Recent results by both biochemical (20) and molecular genetic (10) approaches have identified the catalytic GTF Asp residue involved in covalent attachment of the glucose residue of sucrose to these enzymes. However, documentation of the roles of other amino acids in GTF catalysis has not yet been provided. Nevertheless, a number of amino acid positions in the S. mutans GTFs have been demonstrated to influence the nature of the glucan product synthesized (25). Based upon a comparison of the enzymes shown to be related to the GTFs, the present investigation utilized site-directed mutagenesis in order to establish the essential role ...

In addition to the 1,3*-D-glucan synthetase (p1 4.9) and the highly-branched 1,6_cu-D-glucan synthetase (PI 3.9-4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (PI 5.5-7.0), which exhibited 87% 1,6_cu-bond-, 6% 1,3_cu-bond-and 7% 1,3,6-branchforming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween IO-supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.Streptococcus mutans Glucosyltransferase Glucan Tween 80

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Water-insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (a-1,3-glucanase) and dextranase (a-1,6-glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi-functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pESUMOstar Amp plasmid vector. The resultant his-tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non-adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol-sulfuric acid method, and reducing sugars were quantified by the Somogyi-Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose-dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.

A glucosyltransferase which synthesized insoluble glucan in polyacrylamide gel was isolated from the culture supernatant of Streptococcus mutans Ingbritt (serotype c) by ultrafiltration, ethanol fractionation, isoelectric focusing, and preparative gel electrophoresis. The enzyme preparation was electrophoretically homogeneous and immunologically distinct from the highly branched 1,6-alpha-D-glucan synthase and fructosyltransferase from the same strain and glucosyltransferases from serotypes a and g. The molecular weight was 99,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was 8.5. The enzyme had the optimum pH of 6.0 and Km value for sucrose of 9.4 mM. Besides the insoluble glucan with 96% 1,3-alpha linkage, this enzyme synthesized a considerable amount of diffusible glucan with 84% 1,6-alpha linkage, separately. This enzyme may be the one released from the enzyme aggregates by extracellular proteases, because the addition of extraneous trypsin to the crude enzyme preparation increased the amount of the enzyme species.

The formation of water-insoluble glucan by extracellular glucosyltransferase from Streptococcus mutans 6715 found to be greatly stimulated by various mono- or divalent cations. An enzyme preparation, obtained by ethanol fractionation, was able to catalyze the formation of water-insoluble glucan from sucrose in the presence of monovalent cations above 100mM or divalent cations above 20 mM at neutral pH. As the concentration of monovalent and divalent cations was reduced to below 10 mM and 1 mM, respectively, the formation of insoluble glucan decreased to a negligible amount. High concentrations of these cations were found to stimulate the formation of insoluble glucan in the following ways: (i) it increased the activity of total glucosyltransferase up to 1.6- and 2.7-fold in the absence and presence of a primer dextran, respectively, and (ii) it changed the formation of soluble glucan to insoluble. It was postulated that one of the essential factors for the formation of insoluble glucan would be to keep more than two water-soluble glucan chains close to enzyme aggregates and that such interaction could be enhanced by the presence of high cation concentrations.