Glucosyltransferase gene polymorphism among Streptococcus mutans strains.

Genetic polymorphisms in genes coding for the glucosyltransferases were detected among Streptococcus mutans serotype c strains by Southern blot analysis with DNA probes located within the gtfB gene (H. Aoki, T. Shiroza, M. Hayakawa, S. Sato, and H. K. Kuramitsu, Infect. Immun. 53:587-594, 1986). Restriction endonucleases were used to examine genomic DNAs isolated from serotype a to h strains. The variations were readily detected among 33 strains of serotype c by EcoRI and PstI restriction enzyme digestions. Serotypes e and f, which are genetically similar to serotype c, also had comparable polymorphism; however, serotypes a, b, d, g, and h did not hybridize to the same DNA probes in parallel experiments. Further analysis of enzymatic activities for glucan synthesis and sucrose-dependent adherence revealed no significant differences among the serotype c strains. Our results suggested that genetic polymorphisms existing in S. mutans serotype c strains may reflect a complexity in genes coding for the glucosyltransferases, which are produced ubiquitously in members of the S. mutans group.     

Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans.

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.     

Observation of beta-hemolysis among three strains of Streptococcus mutans.

Streptococcus mutans is normally alpha- or gamma-hemolytic on blood agar plates. However, three recently isolated S. mutans strains were observed to elicit beta-hemolysis. The production and nature of a hemolytic substance were studied.     

Variation in internal polysaccharide synthesis among Streptococcus mutans strains.

Five strains, representative of Streptococcus mutans genetic group III antigenic group d, synthesized and degraded less intracellular polysaccharide (IPS) then 17 strains representative of other S. mutans groups. The strains that synthesize IPS degraded it rapidly. The production of acid in titratable amounts from endogenous IPS was usually complete within 1 h. IPS synthesis in S. mutans increased abruptly at culture glucose concentrations between 0.2 and 0.5% and was quantitated as both iodine-and glucose oxidase-positive material in cell hyrolysates. IPS degradation was measured by acid production in a pH-stat maintained at 7. The existence within group III d of a strain recently shown to be cariogenic in experimental animals suggest that IPS may not be a prerequisite for virulence in these cariogenic bacteria.     

Inhibition of Streptococcus mutans strains by different mitis-salivarius agar preparations.

Several Streptococcus mutans strains were markedly inhibited by mitis-salivarius agar manufactured by Baltimore Biological Laboratories, but little, if any, inhibition was noted using Difco Laboratories' mitis-salivarius agar. Supplementation of the basic medium with sucrose and bacitracin for specific selection of S. mutans resulted in suppression of representative S. mutans type a strains regardless of manufacturer.     

Conservation of cell wall peptidoglycan by strains of Streptococcus mutans and Streptococcus sanguis

Turnover of the cell wall peptidoglycan fraction of six different strains of Streptococcus mutans and eight different strains of Streptococcus sanguis was examined. Cells were grown in the presence of [3H]lysine and [14C]leucine for at least eight generations and then chased in growth medium lacking the two labels. At intervals during the chase, samples of cultures were removed, and the amounts of the two labeled precursors remaining in the peptidoglycan and protein fractions were quantitated. Similar experiments were done in which the pulse-labeling technique was used. In addition, cells were labeled in the presence of tetracycline or penicillin, chased with growth medium containing no inhibitor, and assayed at intervals during the chase for the amount of [3H]lysine present in peptidoglycan fractions. Studies of cultures of S. mutans strains FA-1, OMZ-61, OMZ-176, 6715, GS-5, and Ingbritt and of S. sanguis strains 10558, M-5, Wicky, DL-101, DL-1, 71X26, and 71X48 maintained in the exponential phase of growth in a chemically defined medium failed to show evidence of loss of insoluble peptidoglycan via turnover. Similarly, for the strains of S. mutans, insoluble peptidoglycan assembled during 2 h of benzylpenicillin or tetracycline treatment was also conserved during recovery from growth inhibition.     

Biochemical and immunological differences between hydrophobic and hydrophilic strains of Streptococcus mutans.

Hydrophobic strains of Streptococcus mutans were compared with paired variants showing reduced hydrophobicity. Extracts of hydrophobic cells contained a number of high-molecular-weight proteins which were not present on cells with decreased hydrophobicity. The proteins were found in purified cell walls, suggesting that they are located on the bacterial surface. Trypsin treatment of whole cells destroyed the proteins and reduced the hydrophobicity. Chemical analysis did not reveal any marked differences in the proportion of cell wall constituents. The amino acid compositions and lipoteichoic acid contents of hydrophobic and hydrophilic cell walls were similar. Culture supernatants from the hydrophilic variants contained high-molecular-weight proteins similar to those extracted from the cell walls of the hydrophobic parent strains, indicating that the variants were impaired in their ability to incorporate the hydrophobicity-associated proteins into the cell wall. The dominant protein had a molecular weight of 190,000, similar to that of antigen I/II (B) of S. mutans.     

Serological characterization of Streptococcus mutans serotype polysaccharide g and its different molecular weight forms.

The serotype polysaccharide g from Streptococcus mutans 6715 was found to cross-react with serotype polysaccharide a from S. mutans HS6 and serotype polysaccharide d from S. mutans B13. Double immunodiffusion experiments indicated that the serotype polysaccharide g consisted of the following: (i) the type-specific g site; (ii) a cross-reactive site g-a that was in common with polysaccharide a; (iii) a cross-reactive site g-d that was in common with polysaccharide d; and (iv) a cross-reactive site g-(a-d) that was in common with both polysaccharides a and d. Moreover, by a procedure involving several column chromatography steps, six polysaccharide-containing fractions showing reactivity with anti-g serum were found. By gel filtration, the molecular weight estimates of fractions LI, LII, LIII, SI, SII, and SIII were 2 X 10(6), 5 X 10(5), 6 X 10(4), 3 X 10(4), 1.4 X 10(4), and 1 X 10(4), respectively. Double immunodiffusion analysis indicated that LI, LII, and LIII contained the four antigenic sites of the putative polysaccharide g. LII also contained another additional immunodominant region, designated site x. The analysis also suggested that fraction SI lacked the type-specific site g, fraction SII lacked sites g and g-a, and fraction SIII lacked sites g, g-a, and g-d.     

In vitro inhibition of adherence of Streptococcus mutans strains by nonadherent mutants of S. mutans 6715.

Four nonadherent mutants from Streptococcus mutans 6715 mutant UAB66 (serotype g) with similar phenotypes were shown to inhibit the adherence of adherence-proficient S. mutans serotypes c and g strains. One mutant, UAB108, was shown to inhibit adherence by wild-type strains representing serotypes a, d, and e as well. This inhibition of adherence was seen with pairs of strains grown in partially defined (PD) medium supplemented with 1% sucrose in both microtiter plates and glass tubes. The inhibiting factor was present in culture supernatant fluids of inhibiting strains grown in PD medium plus 1% sucrose and was heat stable. Ethanol precipitation of culture supernatant fluids of these strains yielded a water-soluble polymer which effectively inhibited the adherence of UAB66. This polymer, isolated from UAB108, was also shown to inhibit the adherence of UAB66 at lower concentrations than that needed to inhibit adherence with dextran T10. Partially purified glucosyltransferase, isolated from the culture supernatant fluids of glucose-grown UAB108, produced a water-soluble glucan which was shown to inhibit the adherence of UAB66 as well. The methods developed permit rapid screening for strains or mutants of strains or both that inhibit adherence or plaque formation or both by wild-type strains of S. mutans.     

Insoluble glucan synthesis by Streptococcus mutans serotype c strains

Both dextransucrase and mutansynthetase activities have been purified from the culture fluids of Streptococcus mutans GS-5 (serotype c). Although homogeneous dextransucrase preparations normally synthesize little insoluble glucan, essentially all of the glucan synthesized by this enzyme in the presence of 1.5 M (NH4)2SO4 was water insoluble. Linkage analysis of the insoluble glucans indicated that the presence of NH4+ increased the portion of alpha-1,3-glucose linkages relative to alpha-1,6-glucose units in the product. Chromatofocusing of aggregated glucosyltransferase fractions synthesizing predominantly insoluble glucan yielded primarily dextransucrase activity separable from relatively low levels of mutansynthetase activity. The latter enzyme was detected only in 18-h assays and synthesized primer-dependent insoluble glucan, which was decreased in the presence of NH4+. In the absence of primer dextran T10, the addition of dextransucrase also stimulated insoluble glucan synthesis by mutansynthetase. Dextransucrase and mutansynthetase appear to be distinct enzymes, since the latter possesses a higher molecular weight (155,000 compared to 140,000), a much lower isoelectric point, and did not cross-react with antibody directed against dextransucrase. These results are discussed relative to the mechanism of insoluble glucan synthesis by S. mutans serotype c strains.     

Impaired colonization of gnotobiotic and conventional rats by streptomycin-resistant strains of Streptococcus mutans.

Colonization of streptomycin-resistant mutants derived from Streptococcus mutans strain LB1, a human isolate, and strain FA-1, a rodent isolate, was studied in gnotobiotic and conventional rats. Mutants resistent to 2.0 mg of streptomycin per ml were isolated by using both stepwise (suffix "R"M) and one-step (suffix "R"1) selections. Rats were infected with mixtures of parental and streptomycin-resistant strains, and the proportions of each strain present in samples from the intestinal canal, tongue dorsum, teeth, and fissure plaque were determined. Combinations of strains investigated were LB1 and FA-1"R"M; FA-1 and LB1"R"M; LB1 and LB1"R"1; FA-1 and FA-1"R"1. In gnotobiotic rats, nonresistant strains predominated in every oral sample studied at 7 and 21 days after infection. Similarly, when conventional exgermfree rats were infected with FA-1 and FA-1"R"1, FA-1 dominated in all samples. Streptomycin-sensitive revertants were not detected in rats monoinfected with strains LB1"R"1 and FA-1"R"1 for 21 days. No antagonistic interactions were observed between the strains in in vitro experiments. Streptomycin-resistent mutants attached to hydroxyapatite treated with rat or human saliva in equal or higher numbers than did parental strains. However, parental strains appeared to grow faster in Trypticase soy broth then streptomycin-resistant mutants. These observations indicate that induction of streptomycin resistance frequently impairs the colonization properties of S. mutans strains, possibly by altering their rate of growth.     

Acetoin production by wild-type strains and a lactate dehydrogenase-deficient mutant of Streptococcus mutans.

Eleven different laboratory strains of Streptococcus mutans representing the various serogroups were found to produce an average of 6.0 +/- 4.8 mM acetoin when grown in glucose-containing medium under aerobic conditions. None of the strains produced detectable acetoin when grown anaerobically. A lactate dehydrogenase-deficient mutant produced acetoin both aerobically and anaerobically and in substantially greater amounts than the wild-type strains did. Substitution of mannitol for glucose resulted in decreased acetoin production by wild-type strains and the lactate dehydrogenase-deficient mutant, indicating a role for NADH2 in the regulation of the acetoin pathway. Pyruvate incorporated into the growth medium of a wild-type strain caused acetoin to be produced anaerobically and stimulated acetoin production aerobically. Cell extracts of a wild-type S. mutans strain were capable of producing acetoin from pyruvate and were (partly) dependent on thiamine PPi. Extracts prepared from aerobically grown cells had approximately twice the acetoin-producing activity as did extracts prepared from anaerobically grown cells. The results indicate that acetoin production by S. mutans may represent an auxiliary reaction of pyruvate dehydrogenase in this organism.     

变形链球菌gcp基因敲除菌株表达谱基因芯片

背景：前期研究中经证实变形链球菌内部存在单磷酸鸟苷环二聚体信号通路,构建了变形链球菌gcp基因敲除菌株。 目的：比较变形链球菌野生菌种和gcp基因突变菌株基因表达的差异情况,筛选与生物膜相关的基因,进入后续研究。 方法：提取两种细菌的总RNA,反转录后分别用cy3和cy5染色。与基因芯片杂交后,扫描结果,进行数据分析,获取差异基因信息,对筛选的基因进行Real-Time PCR验证。 结果与结论：差异基因主要与糖代谢、生物膜形成有关,选择了2个基因进行验证,PCR结果与芯片结果相符合。变形链球菌gcp基因敲除后,突变菌株ahpC基因表达上调,磷酸转移酶系统基因表达下调,说明这2个基因与c-di-GMP信号通路的下游途径相关。     

Oral implantation in humans of Streptococcus mutans strains with different degrees of hydrophobicity.

The more hydrophobic, rough-colony-forming, streptomycin-resistant Streptococcus mutans parent strains GW Smr and LK Smr and the less hydrophobic, smooth-colony-forming, streptomycin-resistant variant strains GW36 Smr and LK36 Smr were implanted in oral cavities. Strains GW Smr and LK Smr implanted significantly better than strains GW36 Smr and LK36 Smr. The hydrophobicity of and the colony morphology formed by the different S. mutans strains did not seem to be affected throughout the experiment.     

Plasmid-mediated transformation of Streptococcus mutans.

Streptococcus mutans GS-5 was transformed to erythromycin resistance with streptococcal plasmid pVA736. Transformation frequencies were higher with plasmids reisolated from transformed GS-5 cells relative to plasmid originally derived from S. sanguis Challis.     

Antigens of Streptococcus mutans I. Characterization of a Serotype-Specific Determinant from Streptococcus mutans

A membrane-associated glycerol teichoic acid antigen has been isolated from Streptococcus mutans AHT and a similar antigen has been demonstrated to be present in each of the other Bratthall serotype a organisms studied. Trichloroacetic acid-extracted material was resolved into two phosphorus-containing antigenic fractions (B and C) by agarose chromatography. Fraction B was preliminarily identified as a phospholipid moiety with a glycerol-to-phosphorus ratio of 2:1, and fraction C showed a ratio of 1:1 indicative of a glycerol teichoic acid. This latter fraction also was associated with glucose, galactose, alanine, and fatty acids. Diglycerol triphosphate, the compound characteristically released from 1-3 phosphodiester-linked glycerol teichoic acids by alkaline hydrolysis, was isolated and characterized. Alanine was identified as its alkaline-labile, ester-linked D-isomer. A glyceride was isolated containing a disaccharide of glucose and galactose attached to the 2-hydroxyl group of glycerol. Hapten inhibition analysis demonstrated that beta-galactosides were the greatest inhibitors of the precipitin reaction (>75%), whereas glucose and its derivatives inhibited to a much lesser extent (<30%). Comparative immunodiffusion and immuno-electrophoresis analyses demonstrated that all six Bratthall serotype a organisms tested contained this antigenic determinant and that it was absent in serotypes b, c, and d. It is suggested that the common antigenic determinant of this serotype within S. mutans may be a beta-galactoside associated with a glycerol teichoic acid and possibly other polymers.     

Inhibition of adsorption of Streptococcus mutans strains to saliva-treated hydroxyapatite by galactose and certain amines.

Adsorption of all of eight strains of Streptococcus mutans to saliva-treated hydroxyapatite (S-HA) surfaces was inhibited by galactose and melibiose, but not by other neutral sugars tested. This observation supports the hypothesis that lectin-like components participate in the attachment of these streptococci to salivary glycoproteins on saliva-treated hydroxyapatite surfaces. Adsorption of all strains was also inhibited by iodoacetate and spermine; other amines tested reduced adsorption of some strains, but not others.     

Serological purification of polysaccharide antigens from Streptococcus mutans serotypes a and d: characterization of multiple antigenic determinants.

The polysaccharide antigen preparations from serotype a and serotype d strains of Streptococcus mutans contained both a serotype-specific antigenic determinant and a common a-d antigenic determinant, as demonstrated by agar gel diffusion studies and a quantitative cross-precipitin assay. The chromatographically purified antigens were isolated by a method which depended on their serological specificity to determine if these two antigenic determinants were located on the same molecule. The a and d polysaccharides were recovered from specific antigen-antibody complexes and characterized with respect to their immunological specificity and chemical composition. Agar gel diffusion tests demonstrated that, in both the a and d preparations, the serotype-specific antigenic determinant and the common a-d antigenic determinant were present in one molecule.     

Expression of Streptococcus mutans gtf genes in Streptococcus milleri.

The Streptococcus mutans glucosyltransferase (GTF) genes gtfB and gtfC were ligated into Escherichia coli-streptococcus shuttle plasmids and introduced into Streptococcus milleri. gtfB transformant KSB8 formed an S. mutans-like rough colony on mitis salivarius agar and expressed an extracellular GTF-I, of 158 kDa, and two cell-bound GTF-Is, of 158 and 135 kDa. gtfC transformant KSC43 formed a semirough colony on mitis salivarius agar and expressed primarily an extracellular GTF-SI, of 146 kDa, and two cell-bound GTF-SIs, of 146 and 152 kDa. The extracellular GTFs from KSB8 and KSC43 were purified and characterized. The two types of GTF also reacted specifically with monoclonal antibodies directed against each enzyme. Both enzymes synthesized significant amounts of oligosaccharides, consisting primarily of alpha-1,6-glucosidic linkages, as well as water-insoluble glucans, containing alpha-1,3-glucosidic linkages. Insoluble-glucan-synthesizing activities of both enzymes were stimulated (three- to sixfold) by the addition of dextran T10 and were inhibited in the presence of 1.5 M ammonium sulfate. The Km(s) for sucrose and the optimal pHs were also similar for both enzymes. However, when the transformants were grown in Todd-Hewitt broth supplemented with sucrose, KSC43 cells, expressing GTF-SI activity, adhered to glass surfaces in vitro, while KSB8 cells, expressing GTF-I activity, did not. These results are discussed relative to the potential role of the gtfB and gftC genes in S. mutans cariogenicity.