The interaction between Streptococcus mutans dextransucrase (EC 2.4.1.5) and high-molecular-weight dextran was studied in both the presence and absence of substrate sucrose. Equivalent weight-percent solutions of primer dextrans that differed 200-fold in molecular weight were found to be equally efficient in priming new dextran synthesis. Sodium borohydride reduction of dextran had no effect on its priming ability. These results suggest that dextran synthesis proceeds by addition of glucosyl residues to nonreducing termini of primer dextrans and that several enzyme molecules simultaneously bind to single high-molecular-weight dextran molecules. Kinetic data suggested that dextransucrase contains only one dextran binding site per enzyme molecule. The nature of the commonly observed highly aggregated state of dextransucrase was also studied. Two types of enzyme aggregates were distinguished: (i) oligomeric enzyme aggregates that formed in the absence of dextran and were dissociated by 1 M KCl; and (ii) dextran-induced enzyme aggregates that were stable to 3 M salt. Oligomeric enzyme aggregates were obtained from supernatants of fructose-grown cultures, whereas dextran-induced enzyme aggregates appeared to be present in glucose-grown cultures. The molecular weight of the smallest species of dextran-free dextransucrase observed in solutions of 1 M KCl was estimated to be 40,000 by gel column chromatography. Addition of dextran to primer-dependent dextransucrase resulted in formation of complexes that were stable in CsCl density gradients and exhibited a buoyant density of 1.382 g/cm3 as compared with a buoyant density of 1.302 g/cm3 exhibited by dextransucrase. The enzyme-dextran complexes observed in CsCl density gradients contained about 25% dextran. This corresponded to 150 enzyme molecules (molecular weight, 40,000) per dextran molecule (molecular weight, 2 x 106). The implication of these results to the mechanism of sucrose-and dextran-induced aggregation of S. mutans is discussed.Streptococcus mutants elaborates a dextransucrase (EC 2.4.1.5) that exhibits a high affinity for dextran (4, 10) and is present in both an extracellular and cell surface-associated location (12,15,17,38). The cell-associated and extracellular enzymes do not differ significantly with respect to optimal pH, temperature, or kinetic parameters (25) and are probably alternate states of the same enzyme (25,29).The addition of either dextran or sucrose to broth cultures or washed-cell suspensions of S. mutans results in a rapid, dramatic cellular aggregation (14,17). Dextran-induced aggregation results from dextran cross-linking of cells (14). Sucrose-induced aggregation depends upon synthesis of dextran by dextransucrase (17) and subsequent cellular binding of the product. The ability of S. mutans to synthesize and bind dextran has been recognized to be of special significance for colonization of the smooth enamel surfaces of teeth (13-15). Since dextransucrase contributes to (i) the cellular aggregation phenomenon and (ii) adherence of the ...

The effect of dextranases (EC 3.2.1.11) from the oral isolates Actinomyces israelii and Bacteroides ochraceus on water-insoluble glucan production by the Streptococcus mutans dextransucrase (EC 2.4.1.5) and sucrose-dependent adherence to smooth glass surfaces by S. mutans was studied. Collection on membrane filters of water-insoluble polysaccharides synthesized from radioactive sucrose was used to demonstrate the marked sensitivity of insoluble glucan formation to the presence of dextranase. Concentrations of A. israelii dextranase as low as 0.002 U/ml inhibited insoluble glucan formation by 60%. Similar results were obtained with the B. ochraceus enzyme. An assay for sucrose-stimulated adherence of S. mutans to smooth surfaces involved attachment of radioactively labeled nongrowing cells to the bottom of glass scintillation vials. This facile and sensitive assay was utilized to demonstrate that sucrose-dependent adherence was affected by low levels of dextranase from either A. israelii or B. ochraceus. Enzyme at 0.005 U/ml reduced adherence of S. mutans by 80%. Treatment of S. mutans cells previously attached to glass with low concentrations of the dextranases resulted in removal of 50 to 60% of the bacteria. These results indicate that dextranase-producing oral bacteria may affect sucrose-dependent colonization of S. mutans on the tooth surface and offer a possible explanation for both the difficulties involved in implanting this bacterium into the human mouth and the limited intraoral transmission of S. mutans from one tooth surface to another. Recent studies in these laboratories (39) demonstrated that a significant proportion of the human dental plaque bacterial flora is capable of producing dextran-degrading enzymes. These bacteria were shown to be a heterogeneous mixture of cell types with varying morphological and biochemical properties. One representative isolate which produces an extracellular endohydrolytic dextranase activity has been identified as Actinomyces israelii (39; R. Staat and C.

The soluble dextransucrase (EC 2.4.1.5) activity produced by Streptococcus mutans strain 6715 during growth on a chemically defined synthetic medium (FMS) was compared to enzyme from glucose broth cultures (TSB). Growth on the two media was similar. The specific activity of ammonium sulfate-precipitated FMC enzyme was 17 times greater than similar TSB enzyme preparations. The FMC enzyme was stimulated 11-fold, whereas the TSB enzyme was stimulated 1.2-fold by the addition of exogenous primer dextran. In contrast to the TSB enzyme, the FMC activity could be disaggregated to a low-molecular-weight form by 1 M salt. Thus, low-molecular-weight S. mutans dextransucrase activity free of contaminating primer glucan may be readily obtained after growth of the bacterium in a chemically defined sucrose-free medium.

Ion-sensitive field-effect transistor (ISFET) pH electrodes were used to monitor changes in plaque pH at the base of artificial occlusal surface fissures and at interproximal sites. Bovine enamel was used to construct fissures (1.5 × 0.1 × 1.0 mm) containing a small ISFET electrode. The fissures were fixed to carrier appliances and worn by 4 human volunteers. After plaque accumulation for 4 days changes in pH were monitored by wire telemetry following 1-min rinses with 10% solutions of either sorbitol or sucrose. Results were compared to data obtained from interproximal sites in the same subjects. Responses to sorbitol in the fissure and on the proximal surfaces were minimal and showed no significant difference in minimum pH (5.9 ± 0.4 and 6.1 ± 0.3, respectively) and area under pH 7.0. The response to sucrose at the two sites was very different revealing unique pH profiles which were statistically significantly different with regard to minimum pH (5.0 ± 0.3, fissure and 4.3 ± 0.2, proximal) and area under pH 5.7. Thus, the acidogenic potential of fermentable carbohydrate at two caries-prone sites in the human dentition is significantly different and conclusions based on interproximal telemetry measurements may not be applicable to occlusal surface fissures.

Changes in sucrose-induced plaque pH profiles and the microbial composition of occlusal tooth surface fissures were analyzed using wire telemetry and bacterial culturing techniques. Four human volunteers wore appliances containing artificial fissures constructed with ion-sensitive field-effect transistor (ISFET) electrodes for 1 2 and 4 days; 1 subject kept the electrode for 3 weeks. After monitoring the plaque pH response at the base of the fissure to a 10% (w/v) sucrose rinse the plaque was removed and analyzed for total viable bacteria, total and specific streptococci, lactobacilli and Actinomyces spp. One-day-old plaque showed a rapid drop in plaque pH to a minimum of 4.8 ± 0.2, with 2-day-old plaque showing the most acidogenic pH profile (minimum pH 4.6 ± 0.2). The 4-day-old plaque response was less acidogenic (minimum pH 5.0 ± 0.3) than the results from days 1 and 2. Responses from 13- and 21-day-old fissure plaques showed greatly decreased acidogenic responses (day 21 minimum pH 5.7). Viable bacteria recovered from the fissure increased from approximately 4 × 10⁶ colony-forming units on day 1 to 1.2 × 10⁷ on days 2 and 4 and 1.7 × 10⁷ on day 21. Streptococci ( > 50%) and Actinomyces ( > 10%) dominated in the fissure plaques and their levels were related to minimum pH. Since fissure plaque of all ages tested contained high concentrations of acidogenic bacteria, the decreased acidogenic response at the base of fissures with increasing plaque age suggests that maturing fissure plaques provide an increasingly greater diffusion barrier to fermentable carbohydrates.