Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular aggregation

Abstract

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 ...

Kinetic modeling of oligosaccharide synthesis catalyzed by Leuconostoc mesenteroides NRRL B-1299 dextransucrase

“…In most cases, dextran accelerates the reaction. This has been attributed to acceptor reactions, but also to the effect of dextran on the enzyme conformation, leading to modified responses to pH or to excess substrate inhibition (Côté and Robyt, 1983;Dols et al, 1997;Germaine and Schachtele, 1976;Robyt and Corrigan, 1977).…”

Cariogenicity in hamsters of Streptococcus mutans AHT mutants with varying in vitro adherence abilities

“…Second, glucose-grown M35 shows either no ability or a decreased ability to agglutinate upon addition of exogenous glucans such as mutan or Dextran T250 (Table 1). Some investigators have suggested that agglutination and adherence are mediated by different mechanisms (6,7,18,22,30,31,36), while others suggest that the ability of S. mutans cells to agglutinate reflects their capacity for adherence (8,15,23) and that a common mechanism governs both agglutination and adherence (26,27). Regardless of the relationship of agglutination to adherence, the strong agglutination of cells in plaque would provide tight cohesion of cells and matrix and prevent dissociation of plaque deposits by environmental factors in vitro or in vivo.…”

Glucosyltransferases of Streptococcus sobrinus C211 are both stimulated and inhibited by hydrogen peroxide

“…Fractions (4 ml) were eolleeted and assayed for GTF-S and GTF-I activity and absorbance at 280 nm. Fractions demonstrating GTF-S or GTF-I activity were pooled separately and eoneentrated (7,8,14). The preparations were free of fructosyltransfeiase, invertase, suerose phosphorylase and dextranase activities.…”

“…Assay for GTE activity. Production of glucan from sucrose by GTF-S was determined by a modification of the procedure of Germaine et al, (14). In a final volume of 450 /d, the reaction mixture contained I mM sucrose (2.5 //Ci [U-' ""C] suerose and unlabelled sucrose), 25 /Ug dextran, 70 /ig sodium fiuoride and GTF-S in a sodium acetate buffer, pH 5,5, Samples of 50 //I each were spotted onto Whatman 3 MM filter paper disks after 15 min incubation, and the disks were immediately immersed in absolute methanol.…”

“…Streptococcus mutans has the ability to adhere to saliva-coated smooth surfaces. Once the initial adhesion has taken place, a sucrose-dependent cellular accumulation process ensues in which bacteria are able to bind other baeteria (1,14,28), The ability of S, mutans to accumulate to hard, smooth surfaces may be promoted by insoluble glucans produced by glucosyltransferases (GTF) (29). There are 2 types of glucosyltransferases: one (GTF-S) produces a water-soluble glucan rich in a-1,6 linkages, whereas the other (GTF-I) produces a water-insoluble glucan composed primarily of a-1,3 linkages (2,10,13,19,20,23).…”