A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non-wild-type colony morphology on mitis salivarius agar. These mutants were examined for (i) adherence and aggregation after overnight growth in sucrose-containing medium, (ii) aggregation of nongrowing cells in the presence of 200 jig of sucrose per ml or 20 yg of dextran per ml, and (iii) dextranase production on blue dextran agar plates. Although we isolated mutants which exhibited a variation from the parent strain in only one of the traits tested, the majority of mutants exhibited defects in two or more characteristics. Thirty-eight stable mutants of independent origin were categorized into 13 separate phenotypic groups.
Attachment of bacteria to the host tissue is regarded as a crucial step in the development of many types of infections. Recent
Previous studies have shown that adherence-defective mutants of Streptococcus mutans PS14, serotype c, can be grouped into several different phenotypic groups. In this study a method was developed to test for complementation between pairs of nonadhering mutants which possess different genotypic defects. Mutant strains UAB95 and a spectinomycin-resistant derivative of UAB95 (UAB516) were found to exhibit increased levels of adherence when grown together with UAB230 in media containing sucrose as compared to the adherence of each strain grown separately. An increase in caries was also observed in gnotobiotic rats mixedly infected with the two mutants as compared to either strain alone. Tests revealed that UAB95 produced more water-insoluble glucan than its parent strain but had a defect in glucan binding. UAB230 was found to produce levels of a defective glucan that could not be bound by mutant or wildtype cells. Our results suggest that UAB95 produces a water-insoluble glucan which is bound by UAB230, thus allowing complementation for adherence and caries production.
An inhibitor of Streptococcus sobrinus endodextranase was detected in the extracellular fractions of UAB66 mutants identified following ethyl methanesulfonate mutagenesis as either devoid of dextranase activity (Dex-) or overproducing water-soluble glucan. The two groups of mutants had the same phenotype and displayed no dextranase activity in assays of extracellular fractions (H. Murchison, S. Larrimore, and R Curtiss III, Infect.Immun. 34:1044-1055, 1981) and had been shown to be defective in adherence (Adh-) and capable of inhibiting adherence of wild-type strains during cocultivation in vitro (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 50:826-832, 1985) and in vivo in gnotobiotic rats (K. Takada, T. Shiota, R. Curtiss III, and S. M. Michalek, Infect. Immun. 50:833-843, 1985). By analysis of proteins in Western blots (immunoblots) and following blue dextran-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE), it was demonstrated that these Dex-mutants did synthesize enzymatically active dextranase. From the results of mixing experiments, it was determined that these Dex-Adh-mutants produced enhanced amounts of a cell surface-localized or a cell-associated dextranase inhibitor (Dei) (11,16,25). Two of these species, S. mutans and S. sobrinus, are prevalent in human plaques (5).The ability of these organisms to synthesize extracellular glucans and to concomitantly bind these glucans is thought to contribute to cell adherence and accumulation on the tooth surface (16). The enzymes involved in the synthesis of glucans have been characterized as extracellular, cell surface-associated glucosyltransferases (11,24). The discovery that the mutans group of streptococci produces a glucosyltransferase capable of producing ot-1,6-linked water-soluble glucan (WSG) sparked further interest in the possibility that the presence of an endodextranase (Dex) (9, 14) that releases oligosaccharides of the isomaltose series from dextran could play some role in adherence and aggregation in the sucrose-dependent step as originally suggested by Guggenheim and Burckhardt (14). Although the precise role of Dex is not yet fully understood, there are several possibilities: (i) Dex (a-1,6-glucan 6-glucanohydrolase; EC 3.2.1.11) can partially degrade water-insoluble glucan (WIG) (4, 13); (ii) Dex can inhibit the production of WIG (10,15,20,33,37,38); and (iii) Dex can inhibit the adherence of oral streptococci (10,15,20,37). Furthermore, the percentage of insoluble glucan synthesized has been correlated with the amount of Dex present (10, 38), suggesting a
A nitrosoguanidine-induced mutant, designated UAB90, of Streptococcus mutans PS14 (serotype c) strain UAB62, was identified on the basis of its unique colony morphology and isolated on brain heart infusion agar. Other mutants displaying similar colony morphologies on brain heart infusion agar were isolated after ethyl methane sulfonate mutagenesis of UAB62 and S. miltans 6715 (serotype g) strains UAB61 and UAB66, and these were found to exhibit abnormalities in cell morphology, chain length, or both. All mutants were examined further for (i) adherence and aggregation after overnight growth in medium containing sucrose, (ii) growth and aggregation in brain heart infusion broth and medium containing glucose, (iii) aggregation of nongrowing cells in the presence of 2 mg of sucrose per ml or 200 Vig of dextran per ml, (iv) dextranase activity, and (v) ease of cell lysis. Mutants isolated included several with long chains of enlarged cocci, and two of these strains, UAB261 and UAB433, along with UAB90, were more susceptible to cell lysis than were their parents. UAB261, isolated from UAB62, maintained other parental characteristics, whereas UAB433, isolated from UAB66, lost its ability to aggregate in the presence of either sucrose or dextran. The "fragile" mutant UAB90 was particularly useful in the isolation of high-molecular-weight DNA for early gene cloning experiments by our laboratory. Two other cell morphology mutants, UAB272 from UAB66 and UAB289 from UAB61, did not lyse better than their parents, but both lacked measurable dextranase activity. A final mutant, UAB276 from UAB66, displayed only increased chain length without apparent cell morphology variations. Chains produced by this mutant were up to 10 times longer than those produced by UAB66. UAB276 lysed slightly less well than its parent but retained all other wildtype characteristics examined.
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