The extension of alpha-D-1,3-branch linkages by 1,3-alpha-D-glucan synthase from Streptococcus sobrinus.

In the presence of an acceptor, 1,3-alpha-D-glucan synthase of Streptococcus sobrinus synthesizes water-insoluble glucans from sucrose. Under such conditions, 1,3-alpha-D-glucoside linkages were extended without any change in the glucose-residue number between the 1,3,6-branch points on the acceptor. From these results, the mechanisms of water-insoluble-glucan formation were proposed as follows: (i) the attachment of an acceptor to the glucan binding sites of 1,3-alpha-D-glucan synthase occurs during the initiation of the reaction, and concurrently determines the positions of the branched portions of 1,3,6 on the acceptor, and (ii) the 1,3-alpha-D-glucoside linkage extends from these positions.

Effect of calcium ions on cell surface electrostatics of Bacteroides gingivalis and other oral bacteria.

Surface electrostatics of Bacteroides gingivalis and other oral bacteria were examined. A polarization circuit was employed using platinum electrodes exposed in each bacterial suspension and the number of bacteria adsorbed to the anode and cathode were then estimated. In all bacteria (B. gingivalis, Streptococcus sobrinus, S. mutans, S. salivarius, S. sanguis and Actinomyces viscosus), the number of cells adsorbed to the anode were much greater than the number of cells adsorbed to the cathode. Treating these bacteria with calcium ions tended to decrease the ratio of the number of cells adsorbed to the anode to the number of cells adsorbed to the cathode in all bacteria examined. Moreover, in the case of B. gingivalis, the number of cells adsorbed to the anode and cathode was in an inverse relationship to the number counted before calcium ion treatment. These findings indicate that the cell surfaces of oral bacteria are generally negatively charged but only the cell surface electrostatics of B. gingivalis was dramatically affected by calcium ion treatment. Thus, divalent metal bridges such as calcium bridges contribute to the adherence of the periodontopathic bacterium, B. gingivalis rather than to that of other oral bacteria including cariogenic bacteria.

Mechanism of water-insoluble glucan synthesis in Streptococcus sobrinus.

Synthesis of water-insoluble glucan (IG) by 1,3-alpha-D-glucan synthase from Streptococcus sobrinus was examined using methylation analysis. The purified enzyme was incubated with sucrose and dextran T2000 (DT2000) for a given time and only IG was harvested by centrifugation. The remaining supernatant was incubated again, and IG was obtained. By repeating the above method using the residual supernatant, we obtained 5 varieties of IG precipitated in different periods. These IGs were methylated and examined using gas chromatograph mass spectrometry. The DT2000 water-insolubilized in the early reaction stage tended to have a highly ramified structure, with 1,3-alpha-D-glucan on a 1,6-alpha-D-glucan chain as the main chain. On the contrary, the DT2000 water-insolubilized in the late stage tended to have sparse side chains of 1,3-alpha-D-glucan which elongated with incubation. Specifically, the greater the number of side chains, the sooner the DT2000 was insolubilized. These results suggest that water-insolubilization of the water-soluble glucan not only depends on the increase of the ratio of 1,3-alpha-glucoside linkages to 1,6-alpha-glucoside linkages but also on the degree of branching of the 1,3,6-alpha-branched glucoside linkages.

Four different types of glucans synthesised by glucosyltransferases from Streptococcus sobrinus.

Four different kinds of glucosyltransferases (GTFs) were purified from the cariogenic bacterium Streptococcus sobrinus AHT. One of them (GTFP3) produced water-insoluble glucan with the alpha-1,3 linkage, exclusively. The others (GTFP1, P2 and P4) produced water-soluble glucans. GTFP2 produced oligosaccharides with linear 1,6-alpha-D-glucan structure. Since GTFP1 and P4 produce similar molecular weight glucans, the structural differences between these glucans remain unclear. To clarify the difference between GTFP1 and P4 products, the glucan structures were investigated by methylation analysis with gas liquid chromatography and gas liquid chromatography-mass spectrometry. The glucan synthesised by GTFP1 was 1,6-alpha-D-glucan with a high percentage (25.9 mol%) of 1,3-alpha-D-linked units. The other glucan synthesised by GTFP4 contained 1,6-alpha-D-glucan with 1,3,6-alpha-D-glucose (18.5 mol%).