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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