Properties of Streptococcus sanguinis glucans formed under various conditions.

The aim of our study was to determine whether the structure of glucans formed by glucosyltransferase from Streptococcus sanguinis (GtfSs) on a surface differ from those formed in solution and to explore the effects of antiserum to Gtfs, control normal rabbit serum, starch hydrolysates (STH) and dextran on S. sanguinis (GtfSs) glucan. Linkage analyses showed that solution-formed glucans are predominantly alpha-1,6-linked and have a small amount of alpha-1,3-linked glucose. Surface-formed glucans have enhanced susceptibility to mutanase. Solution- and surface-formed glucans made in the presence or absence of sera, STH, and dextran contain linkages which differ in both amount and type from control glucans. The GtfSs enzyme in solution exposed to antiserum behaves as if it is adsorbed to a surface. Binding of Streptococcus mutans GS-5 and Actinomyces viscosus OMZ105E (Ny1) to S. sanguinis glucan differs if the glucan is formed in the presence of antiserum. The information could help to define the role of glucans in the formation of pellicle, colonization of tooth surfaces and the accumulation of dental plaque.

Structural aspects of glucans formed in solution and on the surface of hydroxyapatite.

Streptococcus mutans glucosyltransferases (GtfB, -C, and -D) and their products formed from sucrose, glucans, play an essential role in the pathogenesis of dental caries. Enzymatically active Gtf is found in whole human saliva (solution), and incorporated into the salivary pellicle that is formed on teeth in vivo (surface). GtfB glucans are predominantly 1,3-linked; however, surface-formed glucans from GtfB contain greater amounts of 3-linked glucose than glucans formed in solution. In contrast, the major linkage of glucans formed on the surface by GtfB in the presence of sucrose and starch hydrolysates in 4-linked glucose. GtfC-derived glucans in solution have a major linkage of 6-linked glucose, while surface-formed glucans from the same enzyme have 3-linked glucose as the major linkage. GtfD glucans formed either in solution or on the surface are predominantly 1,6-linked; however, surface-formed glucans contain more 6-linked glucose than solution-formed glucans. Digestion with the glucanohydrolases mutanase and dextranase shows differences in susceptibility among glucans formed either in solution or on the surface by each of the Gtf enzymes, and differences are also seen in the soluble end products from these digestions. Our results show that the same Gtf enzyme can form structurally distinct glucans in solution and on a surface. These observations are important in the study of naturally occurring microbial films.

Characterization of glucosyltransferaseB, GtfC, and GtfD in solution and on the surface of hydroxyapatite.

GlucosyltransferaseB, GtfC, and GtfD were purified by hydroxyapatite column chromatography, followed by ultrafiltration from the culture supernatant fluids of three Streptococcus milleri constructs (gift from Dr. H.K. Kuramitsu) which harbored individual gtf genes of Streptococcus mutans GS5. GtfB, GtfC, and GtfD were enzymatically active both in solution and in an experimental pellicle (HA-CWS-Gtf) formed by adsorbing Gtf onto the surface of clarified human whole saliva (CWS)-coated hydroxyapatite (HA). The Km values for sucrose for all three enzymes were lower when the enzyme was adsorbed to a surface, compared with when it was in solution. In solution phase assays, and in the absence of primer dextran, glucan production was enhanced 75% when both GtfB and GtfD were present in the reaction mixture, compared with the sum of the individual enzyme activities (p < 0.005). This enhancement did not occur when GtfC was additionally present, or when the GtfB+GtfD enzyme pair was adsorbed onto HA-CWS. In additional experiments, glucan formed by GtfB or GtfC, but not by GtfD, on a HA-CWS-Gtf surface increased adherence of Streptococcus mutans GS5 and Streptococcus sobrinus 6715 by seven- to nine-fold compared with adherence when no glucan was present on the pellicle surface (p < 0.001). Further, treatment of the HA-CWS-GtfB-glucan or HA-CWS-GtfC-glucan pellicle with alpha-1,6 dextranase significantly reduced adherence of both streptococcal strains (p < 0.001). These results show that GtfB, GtfC, and GtfD are enzymatically active in an adsorbed state and that the nature of their product glucan can influence the adherence of cariogenic oral streptococci to an experimental pellicle.

Adherence of microorganisms to rat salivary pellicles.

Numerous studies, using models to mimic the formation of the acquired pellicle, have concentrated on human saliva-coated hydroxyapatite; in contrast, although the rat is frequently used as an animal model, the information concerning the formation of pellicles from rat saliva is sparse. We compared the ability of Streptococcus and Actinomyces species to adhere to hydroxyapatite coated by rat saliva (rsHA) and human saliva (sHA). We also explored the influence of glucan synthesized in situ on the adherence of organisms. We show that each strain of organism has a distinct binding pattern, which was essentially the same with surfaces coated with either rat or human saliva. Dissolved rsHA beads revealed a major protein band (35-49 kD), identified as glutamine/glutamic-acid-rich protein; acidic proline-rich protein (31-45 kD and alpha-amylase (66 kD) were also identified. Overall, these in vitro data strongly suggest that the principles of bacterial adhesion to rsHA are similar to those observed with sHA.

Adhesion of actinomyces isolates to experimental pellicles.

The ability of oral bacteria to adhere to surfaces is associated with their pathogenicity. Actinomyces can adhere to pellicle and cells through extracellular fimbriae. Research on adhesion of actinomyces has been conducted with use of hydroxyapatite (HA) coated with mammalian-derived salivary constituents, whereas the bacterial-derived components of the acquired pellicle have been largely ignored. The influence of the cell-free bacterial enzyme, glucosyltransferase (GTF), on adhesion of human and rodent isolates of Actinomyces viscosus was examined. Cell-free GTF was adsorbed onto parotid saliva-coated hydroxyapatite (sHA). Next, A. viscosus was exposed to the pellicle following the synthesis of glucan formed in situ by GTF. Glucans formed on the pellicle served as binding sites for adhesion of a rodent strain of A. viscosus. Conversely, the presence of in situ glucans on sHA reduced the adhesion of human isolates of A. viscosus compared with their adhesion to sHA. Adhesion of the rodent strains may be facilitated through a dextran-binding protein, since the rodent strains aggregated in the presence of dextrans and mutan. The human isolates were not aggregated by dextran or mutan. Pellicle harboring A. viscosus rodent strains interfered with the subsequent adhesion of Streptococcus mutans to the bacterial-coated pellicle. In contrast, the adhesion of S. mutans to pellicle was not decreased when the pellicle was pre-exposed to a human isolate of A. viscosus. The experimental data suggest that human and the rodent isolates of A. viscosus have distinct glucan adhesion properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus.

A single copy of the Streptococcus faecalis transposon Tn917, located in the Bacillus subtilis chromosome, was able to transpose onto the SPO2 cos plasmid pPL1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. Selection for pPL1017::Tn917 chimeras was performed by SPO2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr). The transposition of Tn917 onto plasmid pPL1017 occurred with a frequency of 10(-5) and was dependent on the presence of a subinhibitory dose of erythromycin. Twelve chimeras were subjected to genetic and physical analyses. Two Cams transductants harbored plasmids whose chloramphenicol acetyltransferase genes had been insertionally inactivated by Tn917. Several transpositions in the vicinity of the phi 105 immunity region were detected. However, all of the 300 MLSr, Camr transductants screened were immune to phi 105 infectious activity. One pPL1017::Tn917 chimera, pLK200, was transferred by SPO2 plasmid transduction into the Bacillus amyloliquefaciens prototrophic strain DSM7. Plasmid pLK200 was effective in the mutagenesis of the DSM7 chromosome and yielded auxotrophs at a frequency of 0.5 to 5.3%. Generation of auxotrophs was also dependent on the presence of a subinhibitory dose of erythromycin. Forty-four auxotrophs representing at least nine amino acid requirements were recovered.

Characterization of Bacillus subtilis DSM704 and its production of 1-deoxynojirimycin.

A Bacillus subtilis strain, DSM704, was characterized by genetic means, and its production of a human intestinal sucrase inhibitor, 1-deoxynojirimycin, was described. Synthesis of this compound is detected concomitant with the detection of heat-resistant spores. The amount of 1-deoxynojirimycin produced is highly dependent on the carbon source, with growth on substrates metabolized via glycolysis giving the greatest amount of production (up to 1 mg/ml). 1-Deoxynojirimycin appears to be nonmetabolizable by the producing strain in that it cannot serve as a sole carbon or nitrogen source.