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The lacE and lacG genes from Streptococcus mutans have been isolated and characterized, and their nucleotide sequence has been determined. The lacE gene encodes the lactose-specific Enzyme II component of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The lacG gene encodes the phospho-beta-galactosidase which cleaves the lactose phosphate that is formed by the lactose PTS. The S. mutans lacE and lacG genes are located in the same operon as the tagatose genes. S. mutans metabolizes lactose via the tagatose phosphate pathway. The deduced LacE and LacG proteins of S. mutans display high homology with the corresponding proteins from Lactococcus lactis, Staphylococcus aureus and Lactobacillus casei.

Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6.6 and exhibited optimal activity at pH 6.0. The H2O-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7.5. Both enzymes oxidized NADH (Km 0.05 and 0.025 mM for the H2O2- and H2O-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271, 383 and 449 nm for the H2O2-forming enzyme and 271, 375 and 447 nm for the H2O-forming enzyme. Antibodies raised against the H2O2-forming enzyme or the H2O-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences.

A gene encoding an ATP-dependent fructokinase from Streptococcus mutans GS-5 was identified within a 2 kb DNA fragment immediately downstream from the scrA gene. The gene cloned in Escherichia coli also expressed mannokinase activity. Insertional inactivation of this gene in S. mutans markedly decreased both fructokinase and mannokinase activities. Nucleotide sequence analysis of the 2 kb fragment revealed an ORF starting 199 bp downstream from the scrA gene, preceded by potential ribosome-binding (Shine-Dalgarno) and promoter-like sequences. This ORF specified a putative protein of 293 amino acids with a calculated M(r) of 31,681. The deduced amino acid sequence of the fructokinase gene, scrK, from S. mutans exhibited no significant similarity to fructokinase genes from Klebsiella pneumoniae, E. coli plasmid pUR400 or Vibrio alginolyticus, but was similar to a comparable gene from Zymomonas mobilis.

The aga gene coding for alpha-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage lambda. The gene was subcloned into plasmid vectors and shown to specify a novel protein of Mr 80,000. Characterization of alpha-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the alpha-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted Mr of 82,022 and revealed regions of homology to alpha-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all alpha-galactosidase activity and abolished the ability to ferment melibiose; alpha-glucosidase activity was also lost, due to an indirect effect on the dexB gene.

The nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.

Nucleotide sequencing of DNA in the region of the Streptococcus mutans chromosome adjacent to the previously characterized gtfA gene revealed the presence of two long open reading frames. One of these corresponds to the dexB gene and has been shown to encode an intracellular exodextranase (dextran glucosidase) which has short isomaltosaccharides as preferred substrates. Comparison with other published sequences showed that the dexB gene product shares regions of similarity with enzymes, from a variety of sources, which attack other glucose polymers.

Cell-free glucosyltransferase of Streptococcus mutans strain B13 (serotype d) exclusively synthesized water-insoluble glucan from sucrose. The insoluble glucan possessed strong glucan-associated glucosyltransferase activity even after extensive washing and lyophilization. Furthermore, cell-free glucosyltransferase became bound to heat-treated water-insoluble glucan or to heat-treated S. mutans B13 cells grown in Todd Hewitt broth, and the resulting glucan and cells adhered to a glass surface in the presence of exogenous sucrose. No other water-insoluble glucans bound significant quantities of glucosyltransferase. Glucan synthesis by free or glucan-bound glucosyltransferase was stimulated by low concentrations (1 to 5 mg ml-1) of isomaltose or water-soluble dextrans of various molecular weights, but higher concentrations (10 mg ml-1) inhibited glucan synthesis. The glucan synthesized in the presence of primer dextrans exhibited a reduced ability to adhere to a glass surface. Certain sugars such as maltose and fructose significantly lowered the yield of insoluble glucans. Preincubation of glucosyltransferase with the low molecular weight dextran T10 increased subsequent binding to S. mutans B13 insoluble glucan, whereas preincubation with higher molecular weight dextrans significantly inhibited the glucosyltransferase binding.

Dextranase activity was determined in cell extracts and cell-free filtrates of Streptococcus mutans strains which had been grown in batch culture. Exo-dextranase activity was located chiefly in cell extracts, whereas endo-dextranase was mainly extracellular. Release of endo-dextranase began early in the exponential phase of growth, and ended when the concentration of residual sugar was low. Thus, dextranase expression was associated with rapidly growing cells, the yield of dextranase was increased several fold when the initial concentration of D-glucose in the medium was changed from 0.5% to 2%. The endo-dextranase was not stable at pH 5, and control of the pH of the culture was essential to preserve active dextranase during overnight growth. Strain Ingbritt (serotype c) and serotype d strains were the best dextranase producers; other strains (serotypes a, b, c, e and f) displayed much lower activity. The ability to produce endo-dextranase, and to synthesize alpha-D-glucans with a high proportion of (1 leads to 3)-linked sequences, appeared to be related properties. The possibility is discussed that the release of two enzymes, namely endo-dextranase and the D-glucosyltransferase (GTF-I) that synthesizes (1 leads to 3)-alpha-D-glucan, are factors that contribute to the cariogenicity of S. mutans serotype d.

Extracellular glucosyltransferases (sucrose: 1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6.20 and 5.86 i.u. mg-1, respectively, exhibited slightly different isoelectric points (pI 4.5 and 4.2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5.5 and the same Km values of 1.3 mM for sucrose and of 83 microM-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-alpha-D-glucans with 20 and 24.5 mol% 1,3,6-branch points, respectively. Both are therefore bifunctional enzymes.

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of water-soluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1.55 M-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1.55 M-(MH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1.55 M-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranse, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1.55 M-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1 leads to 3)-alpha bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1 leads to 6)-alpha bond formation.

Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5.8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The Mr was estimated to be about 130,000 by SDS-PAGE and about 135,000 by ultracentrifugal analysis. The apparent Km value and pH optimum of the enzyme were 3.9 +/- 0.2 mM (mean +/- SD) and about 4.7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-alpha-D-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.

Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.

An extracellular glucosyltransferase synthesizing water-insoluble glucan (GTF-I) was purified from the culture supernatant of Streptococcus rattus strain BHT (mutans serotype b) by hydroxylapatite chromatography, DEAE-Toyopearl chromatography and preparative isoelectric focusing. The Mr of GTF-I was 155,000 by SDS-PAGE and the isoelectric point was pH 4.9. The specific activity, the optimum pH and the Km value for sucrose were 10.0 i.u. (mg protein)-1, 6.5 and 2.4 mM, respectively. The enzyme synthesized a water-insoluble glucan consisting of 69.4 mol% 1,3-alpha-linked glucose, 23.6 mol% 1,6-alpha-linked glucose, 2.6 mol% 1,3,6-alpha-branched glucose and 4.4 mol% non-reducing terminal glucose, and also a small amount (3% of the total glucan) of soluble glucan with 82.4 mol% 1,6-alpha-linked glucose. The Mr and pI values of purified GTF-I were identical with those of the enzyme in the culture supernatant.

A new serotype (h) of Streptococcus mutans was isolated from the dental plaque of monkeys (Macaca fascicularis). Serotype h strains fermented mannitol and melibiose but not sorbitol or raffinose, failed to hydrolyse aesculin and arginine, did not produce hydrogen peroxide and were unable to grow in the presence of bacitracin at 2 units ml-1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole-cell proteins showed serotype h strains to be closely related to strains of genetic group III (i.e. serotypes d and g). The serotype-specific antigen of serotype h contained glucose and galactose but was antigenically distinct from the polysaccharide antigens of serotypes a, d and g. Serotype h strains preferentially colonized developmental grooves of teeth and the proportion of serotype h in the plaque flora was greater in monkeys fed a sucrose-rich diet than in monkeys fed a starch-based diet. A serotype h strain was cariogenic for germ-free rats fed a high-sucrose diet, and serotype h strains appear to be implicated in the caries process in monkeys.

The effects of isomaltosaccharides of various molecular weights (isomaltose to dextran T2000) on glucan synthesis by a water-soluble glucan-synthesizing glucosyltransferase enzyme (GTase-S) and a water-insoluble glucan-synthesizing enzyme (GTase-I), both from Streptococcus mutans OMZ176, were examined. The activity of GTase-S was not affected by the addition of the isomaltosaccharides, but GTase-I was stimulated increasingly by isomaltosaccharides with degrees of polymerization more than 10. The GTase-I activity first increased and thereafter decreased slightly with increasing amounts of a soluble dextran. Maximal stimulation occurred at concentrations in the range 0.1 to 0.2 mg ml-1, when dextran T10 was used as a primer. The rate of glucan synthesis was highly enhanced by the combined action of GTase-S and GTase-I. The profile of the net activity of GTase-I in the presence of various amounts of GTase-S was similar to that of GTase-I in the presence of increasing amounts of an exogenous dextran. These results collectively suggest that soluble glucan produced by GTase-S from sucrose acts as an intrinsic primer for the glucan synthesis by GTase-I, indicating the contribution of autopriming in glucan synthesis by crude GTase of S. mutans.

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.

Streptococcus mutans strains Ingbritt, and its derivative B7 which had been passaged through monkeys, have been used to investigate how the synthesis of extracellular glucosyl- and fructosyltransferases is regulated. The most active enzyme from carbon-limited continuous cultures was a fructosyltransferase; enzymes catalysing the formation of water-insoluble glucans from sucrose were relatively inactive. Dextransucrase (EC 2.4.1.5), which catalyses soluble glucan synthesis, was most active in the supernatant fluid from cultures grown with excess glucose, fructose or sucrose, but full activity was detected only when the enzyme was incubated with both sucrose and dextran. Little dextransucrase activity was detected in carbon-limited cultures. It is concluded that glucosyl- and fructosyltransferases are constitutive enzymes in that they are synthesized at similar rates during growth with an excess of the substrate or of the products of the reactions which they catalyse. Although the Ingbritt strain was originally isolated from a carious lesion, it is now a poor source of glucosyltransferase activity. Glucosyltransferases were extremely active in cultures of a recent clinical isolate, strain 3209, and were apparently induced during growth with excess glucose.

An extracellular glucosyltransferase (GT-S) synthesizing water-soluble glucan was purified from the culture supernatant of Streptococcus mutans BHT (serotype b, subsp. rattus) by DEAE-Sepharose chromatography and preparative isoelectric focusing. The Mr of the enzyme was 155,000 and the pI was 4.5. The GT-S had a specific activity of 10.2 i.u. (mg protein)-1, an optimum pH of 6.0 and a Km value of 0.8 mM for sucrose, and was activated twofold by dextran T10. The GT-S was immunologically partially identical with the corresponding enzymes in crude preparations from serotypes c, e and f. The glucan synthesized de novo from sucrose by the GT-S was water-soluble and consisted of 29 mol% of non-reducing terminal, 49 mol% of 1,6-alpha-linked, 11 mol% of 1,3-alpha-linked and 11 mol% of 1,3,6-alpha-branched glucose residues.

Strains of Streptococcus mutans (serotypes c, e and f) were found to possess high levels of glucosyltransferase (GTase) activity, both cell-associated and in the culture medium, when grown in either sucrose-free or sucrose-containing broth media. The cell-associated GTase of S. mutans MT8148 (serotype c) was effectively extracted by treatment with 8 M-urea at 25 degrees C for 1 h. Approximately 95% of the GTase activity was solubilized by this treatment. The crude extract was purified by DEAE-Sephacel and hydroxylapatite column chromatography. For comparison, extracellular GTase was also purified from the culture supernatant of the same strain by ammonium sulphate precipitation, chromatofocusing and hydroxylapatite chromatography. The molecular masses of the cell-associated and extracellular GTase proteins were similar (156 kDa) as determined by SDS-PAGE. However, the pH optima for maximum GTase activity were different: pH 6.7 to 7.0 for the cell-associated enzyme and pH 5.5 to 6.5 for the extracellular enzyme. The product of cell-associated GTase from sucrose was almost exclusively water-insoluble glucan. On the other hand, extracellular GTase produced mainly water-soluble glucan from sucrose. This indicates that GTase synthesizing water-insoluble glucan is present primarily in a cell-associated form in serotype c S. mutans. Insoluble glucan synthesis by the cell-associated GTase from sucrose was not enhanced by addition of primer dextran T10 to the reaction mixture. The extracellular and cell-associated GTases were immunologically unrelated as determined by ELISA using monoclonal antibodies.

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.

Extracellular glycosyltransferases from 17 strains of Streptococcus mutans were examined by analytical isoelectric focusing. Three kinds of glucosyltransferase: highly-branched-1,6-alpha-D-glucan synthetase, 1,3-alpha-D-glucan synthetase and 1,6-alpha-D-glucan synthetase, were excreted from serotype a, d and g strains. The enzymes of serotype a strains were distinguishable from those of serotypes d and g by differences in their pI values. Serotype c, e and f strains excreted basic glucosyltransferase and acidic fructosyltransferase. Serotype b strains also excreted the glucosyl- and fructosyltransferases, but the pI values were different from those of the enzymes from the other serotypes. Thus, S. mutans strains could be divided into four groups by analytical isoelectric focusing of glycosyltransferases which corresponded well to the four genetic groups.

Streptococcus mutans serotype g secretes at least three kinds of glucosyltransferase with different enzymological and immunological properties. One of them is a primer-independent enzyme and seems to be the source of primer for the others, both of which are primer-dependent enzymes. Recently, we purified the primer-independent enzyme, the third glucosyltransferase in this group from S. mutans strain AHT-k serotype g. In the present study, we examined the specificity of the antiserum against the primer-independent glucosyltransferase using extracellular culture-conditioned fluids of many strains of the various serotypes of S. mutans. The antiserum cross-reacted with the extracellular culture fluids from strains of serotypes d and a, in addition to serotype g, but not with those of other serotypes, indicating that the primer-independent glucosyltransferase is secreted by the S. sobrinus and S. cricetus, but not by S. mutans and S. rattus. The antiserum did not completely inhibit the activity of the enzyme, even at more than twofold antibody excess, determined by indirect precipitation with immobilized staphylococcal protein A.

The effects of oxygen on growth and mannitol fermentation of eight strains of Streptococcus mutans were compared under aerobic and strictly anaerobic conditions. The growth of three strains was severely inhibited by oxygen, whereas the others were oxygen-tolerant. The growth of two of the oxygen-tolerant strains was significantly enhanced by oxygen. The activities of superoxide dismutase and NADH oxidase in extracts from aerobically grown bacteria showed a positive correlation with the growth rate under aerobic conditions. The activities of these enzymes in oxygen-sensitive strains grown aerobically were as small as those in anaerobically grown cultures. Moreover, the enzyme activities increased during aeration of anaerobically grown oxygen-tolerant strains, but not in oxygen-sensitive strains. In all strains, oxygen changed mannitol catabolism from heterolactic to homolactic fermentation. It was concluded that oxygen-tolerance of S. mutans is dependent on the ability of strains to induce NADH oxidase and superoxide dismutase.

Streptococcus mutans serotype c produces several extracellular proteins which bind to affinity columns of immobilized glucans. The proteins are three distinct glucosyltransferases and another glucan-binding protein (molecular weight 74000) which is now shown to be a fructosyltransferase. This enzyme is antigenically distinct and genetically independent of two other fructosyltransferases produced by the same organism. A mutant is described which lacks the glucan binding fructosyltransferase and has defective ability to form adherent colonies in the presence of sucrose. Although the production of glucans from sucrose results in the glucan binding protein becoming bound to the bacterial surface, and hence perhaps contributing to adherence, the fructans synthesized by the enzyme do not appear to contribute to this phenomenon.

Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.

Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S. mutans clinical variant, MT6801R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG14 and NG7183, were induced from strain MT6801R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to saliva-coated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801R and NG14, but not NG7183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de novo glucan synthesis is necessary for the accumulation of serotype c S. mutans cells on the tooth surface and the induction of dental caries.

Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67-1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67-25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.

A water-soluble glucan-synthesizing glucosyltransferase (GTase-S) and a water-insoluble glucan-synthesizing glucosyltransferase (GTase-I) were purified from culture supernatant of Streptococcus mutans 6715 (serotype g) by ammonium sulphate precipitation, chromatofocusing on a Polybuffer exchanger PBE 94 column, and subsequent phenyl-Sepharose CL-4B or hydroxyapatite column chromatography. The GTase-S and GTase-I activities were purified 4019- and 4714-fold, respectively, and the molecular weights were calculated to be 160000 and 165000, respectively. GTase-S had a pH optimum of 5.0, a Km of 8.8 mM for sucrose in the presence of 20 microM-dextran T10, and an isoelectric point of pH 4.3. GTase-I had two pH optima of 5.0 and 7.0, Km values of 4.9 mM (at pH 5.0) and 7.0 mM (at pH 7.0), mM (at pH 7.0), and an isoelectric point of pH 4.9. Methylation analysis indicated that the water-soluble glucan produced by GTase-S was a highly branched 1,6-alpha-linked D-glucan with 1,3-linked glucose residues, and that the water-insoluble glucan synthesized by GTase-I was composed of 1,3-alpha-linked glucose units.