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The establishment of the subgingival microbiota is dependent on successive colonization of the implant surface by bacterial species. Different implant surface topographies could influence the bacterial adsorption and therefore jeopardize the implant survival. This study evaluated the biofilm formation capacity of five oral streptococci species on two titanium surface topographies.In vitrobiofilm formation was induced on 30 titanium discs divided in two groups: sandblasted acid-etched (SAE-n=15) and as-machined (M-n=15) surface. The specimens were immersed in sterilized whole human unstimulated saliva and then in fresh bacterial culture with five oral streptococci species:Streptococcus sanguinis,Streptococcus salivarius,Streptococcus mutans,Streptococcus sobrinus, andStreptococcus cricetus. The specimens were fixed and stained and the adsorbed dye was measured. Surface characterization was performed by atomic force and scanning electron microscopy. Surface and microbiologic data were analyzed by Student’st-test and two-way ANOVA, respectively (P<0.05).S. cricetus,S. mutans,andS. sobrinusexhibited higher biofilm formation and no differences were observed between surfaces analyzed within each species (P>0.05).S. sanguinisexhibited similar behavior to form biofilm on both implant surface topographies, whileS. salivariusshowed the lowest ability to form biofilm. It was concluded that biofilm formation on titanium surfaces depends on surface topography and species involved.

Antigen I/II protein is a cell surface protein antigen of Streptococcus mutans and is implicated in the adhesion mechanism to tooth surfaces. We have identified the antigen I/II homologous gene (paaA) from Streptococcus cricetus, a member of mutans streptococci. To clarify the mechanism of paaA expression, we sequenced the flanking regions of the paaA gene utilizing the gene-walking method in this study. In the upstream region of paaA, three homologous genes found in the corresponding region of Streptococcus sobrinus were identified. One of the corresponding genes in S. sobrinus was par, which has been characterized as a transcription represser of the antigen I/II (spaA) gene. Therefore, the homologous gene in S. cricetus could have a role in transcription of paaA. In the downstream region of paaA, the paaA homologous gene was found and designated as paaB. The paaB gene was interrupted by a novel insertion sequence element (ISScr1), a member of the IS982 family. Southern hybridization analysis of S. cricetus par indicated that no gene homologous to S. cricetus par was found in the oral streptococci used in this study. Furthermore, the downstream region of antigen I/II (spaA) in S. sobrinus was sequenced using the gene-walking method. No similar gene to antigen I/II gene was found in the region. It was suggested that the paaA might be regulated by the par in S. cricetus.

Thirty eight strains of oral streptococci were divided into six types by two-dimensional gel electrophoresis (2-DE) followed by glycosyltransferase (GLT) activity stain: Type 1, Streptococcus mutans; Type 2, Streptococcus rattus; Type 3, Streptococcus sobrinus and Streptococcus downei; Type 4, Streptococcus cricetus; Type 5, Streptococcus salivarius; and Type 6, Streptococcus sanguis, Streptococcus oralis and Streptococcus gordonii. In Types 1, 2 and 5, two glucosyltransferases synthesizing water-insoluble (GTF-I) and water-soluble glucans (GTF-S) and a fructosyltransferase (FTF) were detected. In Types 3 and 4, GTF-I and two GTF-Ss were detected. Only one GTF-S was detected in Type 6. The 2-DE patterns for these six types were clearly distinguished from each other based on the kind, number and location of GLTs in gel.

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.

Oral bacteria, such as Streptococcus cricetus and S. sobrinus, are aggregated by high molecular weight fractions of the linear α-(1→6) dextran produced by Leuconostoc mesenteroides. The linkage specificity of this interaction with a cell surface lectin of the streptococci is high. As a further probe of factors affecting recognition, we prepared partially methylated samples of dextran of average MW of 2 × 106 and 104. 13C NMR spectroscopy proved to be a convenient method for determining both site and degree of monomethylation and dimethylation of the polymer: relative reactivity of the OH groups was established as 02: 04: 03 = 4.3: 1.7: 1.0. A methylation DS of only 0.17 caused almost complete loss of recognition by the lectin. A structural feature of dextran oligomers is suggested to explain this result.

Streptococcus cricetus HS-6 grown with or without added manganous ions in their growth medium, i.e. with or without GBLs respectively, show distinct differences in the pH dependence of their zeta potentials and their adhesion to hexadecane confirming that non-specific surface properties can be a corollary of the presence of specific adhesins. It was suggested that at certain pH values, the GBLs are shielded by other cell surface components. The delay in glucan induced aggregation at pH 4 but not at pH 7 and 9 was taken as support for this suggestion. Furthermore, the pH dependence of the glucan induced aggregation of S. cricetus HS-6 is suggested to demonstrate clearly the electrostatic nature of the highly specific interaction between glucans and lectins and therewith provides support for our suggestion that the same fundamental forces are responsible for specific and non-specific interactions

The cariogenic bacteria Streptococcus sobrinus and S. cricetus were shown to have an absolute requirement for manganous ion in order to bind glucans or to adhere to glass in the presence of sucrose. The bacteria possessed a reasonably high affinity transport system for 54Mn2+, yielding a Km of about 12 microM. The Vmax for uptake of 54Mn2+ in S. sobrinus was increased when the bacteria were grown in Mn-depleted medium, but the Km remained the same. There was no evidence for two Mn2+ uptake systems, commonly observed for many bacteria. Ions such as Ca2+, Co2+, Co3+, Cu2+, Fe2+, Fe3+, Hg2+, Mg2+, Ni2+, and Zn2+ did not inhibit the uptake of 54Mn2+ by the bacteria, although Cd2+ was a potent inhibitor. Fractionation experiments showed that manganese was distributed in protoplasts (67%) and in the cell wall (33%). Approximately 80% of the 54Mn2+ in S. sobrinus was rapidly exchangeable with nonradioactive Mn2+. Electron spin resonance experiments showed that all of the manganese was bound or restricted in mobility. Proton motive force-dissipating agents increased the acquisition of 54Mn2+ by the streptococci, probably because the wall became more negatively charged when the cell could no longer produce protons.

Three different alpha-D-glucosyltransferases (GTFs) were separated from culture filtrates of Streptococcus cricetus strain AHT grown in a complex, standard medium in batch culture or under defined conditions of growth in the chemostat. Two of the enzymes (GTF-S1 and GTF-S2) converted sucrose into branched, soluble dextrans, and the third (GTF-I) produced a relatively linear, water-insoluble, predominantly (1----3)-linked alpha-D-glucan. When the organism was grown in complex medium modified by the removal of the fraction of high molecular weight, only GTF-S1 and GTF-S2 were released, and no GTF-I was detected. The water-insoluble glucan fraction obtained by incubating the cell-free filtrate with sucrose contained from 17 to 25% of (1----3)-glucosidic linkages, and accounted for up to 78 and 4% of the total glucans derived from growth in standard and modified medium, respectively. The soluble glucans produced in the same reaction were fractionated with ethanol to give, from both media, two distinct dextrans comprising (1) a highly branched dextran similar to the S1-dextran product of GTF-S1 and (2) a dextran containing fewer branch linkages and up to 86% of (1----6)-alpha-D-glucosidic linkages. A GTF responsible for the synthesis of the latter dextran was not separated. The structures of the glucan fractions and the products of the separated GTF were examined by enzymic degradation and methylation analysis.

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.

Mutans streptococci comprise a group of seven closely related, yet distinct species. The distinction between the four species used in this study, namely Streptococcus sobrinus, Streptococcus cricetus, Streptococcus rattus, and Streptococcus mutans, has been made only recently on the basis of DNA homologies. In order to determine if there is a difference in the physicochemical surface properties of these species, strains were characterized by contact angles, zeta potentials and isoelectric points (IEP), elemental surface compositions by X-ray photoelectron spectroscopy, and molecular moieties by infrared spectroscopy. Contact angles, particularly when measured with water, can be considered a measure of cell surface hydrophobicity; zeta potentials reflect the charge of the outermost cell surface; X-ray photoelectron spectroscopy yields the relative abundance of carbon, oxygen, nitrogen, and phosphorus over the outer 5 nm of the bacterial cell surface; infrared spectroscopy enables a molecular characterization in terms of proteins, phosphates, and polysaccharides. All four species were homogeneous with regard to their physicochemical surface properties. However, the S. rattus species were clearly different from the others on the basis of the low water contact angle (21 +/- 2 vs. 26-31 degrees), highly negative zeta potential and lack of IEP, and high oxygen/carbon (0.50 +/- 0.02 vs. 0.41-0.43) and phosphorus/carbon (0.016 +/- 0.001 vs. 0.006-0.008) surface concentration ratios. Amongst the other differences observed, each species had a characteristic pH dependence of their zeta potential measured in phosphate buffer, yielding an IEP of 1.7, 2.1, and 2.5 for S. cricetus, S. sobrinus, and S. mutans, respectively. However, a cluster analysis on the zeta potential data showed only an isolated cluster for the S. rattus species. Thus it is likely that the higher cariogenicity of S. sobrinus with respect to S. cricetus and S. mutans is, in addition to a higher acidogenicity, due to the smaller negative surface charge, giving an increased adhesion to negatively charged tooth surfaces. S. rattus is expected to be the least adherent strain in the oral cavity as it carries a relatively high negative surface charge.

Summary: A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.

Strains of Streptococcus cricetus and Streptococcus rattus exhibited striking differences in their ability to bind to different types of collagen. For example, S. cricetus AHT bound in highest numbers to hydroxyapatite (HA) treated with human type V collagen, while rat type I collagen was ineffective. In contrast, human type V collagen was least effective in promoting attachment of S. rattus LB-1, while treatment with rat or human type I collagen was effective. Adsorption of both species to human type I collagen-treated HA showed a high correlation with a Langmuir model. Estimates of adsorption parameters indicated there were greater numbers of binding sites with higher affinity for S. rattus LB-1 than for S. cricetus AHT. Treatment of HA with either the alpha 1 (1) or alpha 2 (1) polypeptide chains of collagen was effective in promoting adhesion of S. rattus LB-1 cells. In contrast, the alpha 2 (1) chain was more effective than the alpha 1 (1), chain for S. cricetus AHT. These observations indicate that S. cricetus AHT and S. rattus LB-1 cells bind to different segments of collagen molecules. Adhesion of both species was also promoted by collagen-rich fractions of human dentin.

As compared to the previous precipitin inhibition tests differences were found in thereactivities of monoclonal antibodies (MAbs), a-4 and a-84 with Streptococcus cricetus (serotype a) in an enzyme immunoassay using whole cells, purified cell wall antigen and haptenic sugars coated onto microtitre wells. Investigation into the differences led to the finding that the purified antigen from S. cricetus cells consisted mainly of three forms with different molecular weights and sugar contents. MAb a-4 reacted with a high molecular weight form (AgI, molecular weight of 46 000) and low molecular weight forms (AgII and AgIII, molecular weights of 9 800 and 20 000, respectively) whereas MAb a-84 reacted only with the high-molecular form. Gas chromatographic analysis revealed that all antigens contained rhamnose, galactose and glucose but in different ratios of the sugars. Although the binding site of AgII/AgIII with MAb a-4 seemed to be slightly different from that of AgI with MAb a-84, the predominant immunodeterminant of the antigens was considered to be the same. On the basis of these results, the chemical structures of the antigenic determinants are suggested. The nature of the antigen-antibody reactions is discussed.

The flanking region of the antigen I/II gene, paaA, in Streptococcus cricetus was examined using the gene-walking technique. In the region downstream of the paaA gene, another antigen I/II gene designated as paaB was found. The paaB gene was disrupted at the alanine-rich region (A region) by a novel insertion sequence element, ISScr1. ISScr1 is a member of the IS982 family and is composed of a 962-bp sequence and duplicated target DNA (the sequence 5'-TAGCTAAAT-3') resulting from its insertion. To clarify the structural divergence of the two antigen I/II proteins (PAaA and PAaB), computational analysis of the paaB gene was performed and the two structures were compared. The amino acid sequence homology indicated that PAaB resembled PAaA, but the middle region showed little similarity to that of PAaA. Phylogenetic analysis showed that PAaB was better classified in a major group with S. mutans PAc and S. gordonii SspA and SspB than with PAaA. The transcriptional expression of paaA and paaB was demonstrated by reverse transcription (RT)-PCR. In the region upstream of the paaA gene, three genes homologous to the genes located in the region upstream of the S. sobrinus antigen I/II gene (pag) were found. Of the three genes, ORF3 showed homology to the par gene encoding a transcriptional repressor for the pag gene in S. sobrinus. Therefore, ORF3 was designated the par gene of S. cricetus. Southern hybridization revealed that the par gene of S. cricetus was not found in other oral streptococci examined in this study.

With reference to the first Principle of the International Code of Nomenclature of Bacteria, which emphasizes stability of names, it is proposed that the original names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, Erwinia ananas, Eubacterium tarantellus, Lactobacillus sake, Nitrosococcus oceanus, Pseudomonas betle, Rickettsia canada and Streptomyces rangoon, all included in the Approved Lists of Bacterial Names, be conserved. Request for an Opinion.

Two tandem genes encoding glucosyltransferase synthesizing water-soluble glucan (GTF-S) were cloned from the lambda gene library of Streptococcus cricetus HS-6 (serotype a) using anti-GTF-S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C-terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF-S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.

The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.

The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.

In this study, native cells of Streptococcus mutans VA-29R and Streptococcus rattus FA-1 displayed significantly higher aminopeptidase activity than did cells of Streptococcus cricetus AHT or Streptococcus sobrinus 6715 toward the nitroanilide derivatives of leucine, alanine, methionine, arginine, and lysine. These differences in cellular aminopeptidase activity led us to investigate the subcellular localization of the aminopeptidase in these mutans group streptococci. Following conversion of native cells to protoplasts by treatment with lysozyme, most of the aminopeptidase activity detected in the native-cell preparations remained associated with the intact protoplasts. After lysis of protoplasts and differential centrifugation, most of the total cellular aminopeptidase activity was recovered with the cytoplasmic fraction. Membrane-associated aminopeptidases represented only minor activities in these mutans group streptococci. Although the four strains showed no differences with respect to a predominant cytoplasmic localization for the aminopeptidase activities, the levels of activity in the cytoplasmic fractions from S. cricetus AHT and S. sobrinus 6715 were significantly lower than those measurable in the corresponding fractions from S. mutans VA-29R and S. rattus FA-1. These results support the conclusion that the differences in aminopeptidase activity expressed by these streptococci reflect quantitative differences rather than differences in enzyme subcellular localization.

The growth inhibition of the Streptococcus mutans group, including Streptococcus mutans, Streptococcus cricetus, Streptococcus rattus and Streptococcus sobrinus, on glucose by oxygen and the properties of reduced nicotinamide adenine dinucleotide (NADH) oxidase induced by oxygen, using a representative oxygen-tolerant strain, were examined. The growth response to oxygen varied among strains and correlated with the level of NADH oxidase activity in the cell extract. The induced synthesis of NADH oxidase as well as superoxide dismutase was affected by oxygen tension and energy sources. The induced NADH oxidase involved at least two types, major H2O-forming NADH oxidase and minor H2O2-forming NADH oxidase activity. In the presence of a scavenger of H2O2, pyruvate, the growth inhibition by oxygen in an oxygen-sensitive strain (GS5) was protected but not in another oxygen-sensitive strain (MT8148). The high level of induced H2O-forming NADH oxidase activity protected against oxygen toxicity.

Propolis, the resinous hive product collected by bees, is important in the defense of the hive. The effects of propolis on growth and glucosyltransferase activity of Streptococcus sobrinus 6715, Streptococcus mutans PS14 and Streptococcus cricetus OMZ61 in vitro, and on dental caries in rats infected with S. sobrinus 6715 were investigated. Propolis had antimicrobial activity against S. sobrinus, S. mutans and S. cricetus, and inhibited both water-insoluble glucan synthesis and glucosyltransferase activity. In rats inoculated with S. sobrinus, about half of their fissures were carious, while dental caries was significantly less (p = 0.01) in rats given propolis. Dental caries was markedly decreased by the multiple actions of propolis which had antimicrobial activity, inhibited water-insoluble glucan synthesis, and inhibited glucosyltransferase activity. No toxic effects of propolis on the growth of rats were observed under experimental conditions in this study. These results suggest that propolis can control dental caries in the rat model system.

Summary The complete nucleotide sequence of the gene for a cell-surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N-terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.

Three monoclonal antibodies (MAb) were prepared against a cell surface antigen which cross-react between Streptococcus mutans (serotypes c, e and f) and Streptococcus sobrinus (serotypes d and g). Two of the MAb also recognise a determinant on the surface of Streptococcus cricetus (serotype a). The common antigen shared between S. mutans and S. sobrinus was demonstrated by Western blotting to be about 200 kD in size. This antigen is shared not only by the cell surfaces of serotypes a, c, d, e, f and g, but also by the major cell surface antigen of S. mutans of 185 kD and another of 150 kD. These MAb identify all but one mutans type of streptococci and can be utilised as analytical reagents.

A DNA fragment encoding the sucrose-6-phosphate hydrolase component of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system has been recovered from a plasmid-based genomic library of strain GS5. The locus, designated scrB, was found to reside within a 2.9-kilobase-pair restriction fragment present on the chimeric molecule pVA1343 (7.3 kilobase pairs). Minicell analysis of pVA1343-directed translation products revealed that the scrB product synthesized in Escherichia coli V1343 was a single peptide of Mr 57,000. This polypeptide was reactive with antiserum prepared against S. mutans intracellular invertase, which has been previously shown to have an Mr of 43,000 to 48,000. The basis of this difference in Mr was not established but may represent a posttranslational proteolytic event which occurred in S. mutans but not in recombinant V1343. Sucrose-6-phosphate hydrolase purified to homogeneity from V1343 exhibited Michaelis constants of 180 mM for sucrose and 0.08 mM for sucrose-6-phosphate. Deletion analysis of pVA1343 facilitated the assignment of a coding region for the hydrolase within the insert, as well as an orientation for the transcription of scrB. scrB-defective strains of S. mutans constructed by additive integration of an insertionally inactivated scrB locus exhibited the sucrose sensitivity characteristic of this mutant class. Similar loci were detected by DNA-DNA hybridization in additional strains of S. mutans and two strains of Streptococcus cricetus, but not in single strain representatives of S. rattus, S. sobrinus, S. sanguis I and II, S. salivarius, or S. mitis.

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.

The four species, each represented by two strains, all produced high levels of fissure caries in the upper and lower jaws of gnotobiotic Fisher rats. Streptococcus rattus strains and Streptococcus mutans NCTC 10832 gave low levels of approximal caries, whereas Streptococcus cricetus, Streptococcus sobrinus and Strep. mutans NCTC 10449 gave high levels. The only strains to produce buccal surface lesions were Strep. sobrinus and Strep. cricetus. All strains were able to form dense plaque in the central fissure of the second molar. Strep. cricetus, Strep. rattus and Strep. sobrinus strains were capable of producing smooth-surface plaque. This difference in plaque-forming ability did not correlate precisely with differences in cariogenicity, and such differences occurred between strains of the same species.

The specificity of the glucan-binding lectin (GBL) of Streptococcus cricetus AHT was determined. Examination of the kinetics of aggregation of cell suspensions with glucans containing various percentages of alpha-1,6, alpha-1,4, alpha-1,3, and alpha-1,2 anomeric linkages revealed that only glucans with at least 80% alpha-1,6 linkages promoted strong aggregation. Moreover, only linear glucans with molecular weights greater than 5 X 10(5) were capable of causing rapid aggregation of the bacteria. The lectin was observed to be present on S. cricetus strains, on Streptococcus sobrinus, and on several Streptococcus mutants strains. Preincubation of suspensions of S. cricetus AHT with glucan T10 (molecular weight of 10,000) before the addition of high-molecular-weight glucan resulted in competitive inhibition in a concentration-dependent manner. Inhibition was achieved also with isomaltopentaose, isomaltohexaose, and isomaltooctaose, but at higher concentrations than glucan T10. In contrast, no inhibition was observed with maltoheptaose, providing additional evidence for the specificity of GBL. Treatment of suspensions of S. cricetus AHT with trypsin before and after aggregation with high-molecular-weight glucan revealed a substantial level of protection of GBL when in a bound state. Collectively, these results indicated that GBL has an absolute affinity for glucans rich in alpha-1,6 linkages and possesses an active site which recognizes internal sequences and accommodates isomaltosaccharides of at least nine residues. This unusual specificity may contribute to the colonization of S. cricetus, S. sobrinus, and S. mutans in glucan-containing plaque in the oral cavity.

We identified and characterized a recombinant Escherichia coli containing the entire gene for surface protein antigen A (spaA) of Streptococcus sobrinus 6715. The recombinant E. coli was isolated from a cosmid gene bank of size-fractionated S. sobrinus DNA fragments, and recombinants expressing the SpaA protein were detected immunologically. Subcloning experiments showed that the DNA sequences encoding the SpaA protein could be isolated on two contiguous EcoRI fragments, 3.7 and 3.3 kilobases (kb) in size, both contained on a 16.2-kb BglII fragment. Southern blot hybridization experiments using the EcoRI fragments to probe genomic DNAs from various serotypes of the mutans group of streptococci revealed DNA sequence homology not only to S. sobrinus 6715 (serotype g) chromosomal DNA but also to S. sobrinus serotype d DNA. Weak hybridization signals to Streptococcus mutans serotypes c, e, and f and to Streptococcus cricetus serotype a were observed with the 3.3-kb EcoRI fragment. These results suggest that the coding sequence for the spaA gene is probably conserved in S. sobrinus strains. Plasmid-encoded polypeptides made in E. coli minicells revealed that transcription of the spaA gene was initiated on the 3.7-kb EcoRI fragment and that its product size was about 210 kilodaltons. The cloned SpaA protein was purified from the periplasmic protein of E. coli, and monospecific antiserum against the cloned product was prepared in rabbits. The data obtained from various physiochemical and immunological procedures allowed us to conclude that the coding sequence for the entire spaA gene of S. sobrinus 6715 had been successfully cloned in E. coli and that faithful expression of the cloned product could be obtained.

Strains of streptococci originally isolated from the dental plaque of monkeys (Macaca fascicularis) and designated as Streptococcus mutans serotype h were compared with the other species of the mutans streptococcus group. Despite the close resemblance noted previously between these strains and Streptococcus sobrinus, closer examination revealed several important differences. Strains of serotype h ferment mannitol but not sorbitol, melibiose, inulin, or raffinose, do not produce hydrogen peroxide, and are unable to grow in the presence of bacitracin at 2 units per ml. They exhibit a distinct polypeptide pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and possess several antigens absent from S. sobrinus as revealed by Western blotting (immunoblotting). Virtually identical polar lipid patterns were observed by two-dimensional thin-layer chromatography for both serotype h strains and S. sobrinus, although on the basis of long-chain fatty acid analysis by capillary gas-liquid chromatography, the former could be distinguished by the presence of a peak tentatively identified as cyclopropane acid (cis-9, 10-methyleneoctadecanoate (ΔC 19:0). Deoxyribonucleic acid (DNA)-DNA hybridization studies by both the S1 nuclease and renaturation rate methods showed that serotype h strains differ from S. mutans, S. sobrinus, Streptococcus cricetus, Streptococcus rattus, Streptococcus ferus, and Streptococcus macacae. On the basis of these data, we believe that S. mutans serotype h strains represent a distinct species for which the name Streptococcus downei is proposed. The DNA base composition is 41 to 42 moles percent guanine plus cytosine. The type strain is strain MFe28 (NCTC 11391T), which is cariogenic in monoassociated germfree rats.

Streptococcus cricetus AHT exhibited a requirement for manganese for growth and expression of the glucan-binding lectin. While low concentrations of manganese (0.1 to 10 microM) were able to support growth, higher concentrations (greater than 100 microM) were required for full expression of the glucan-binding lectin. The manganous-aquo ion may be important in cellular adhesion and accumulation processes in dental plaque.

Treatment of 10 strains representing 5 species of related oral streptococci with penicillin G resulted in measurable levels of cellular lysis. This lysis was dependent on penicillin concentration and cell density. At similar penicillin concentrations and cell densities, the 10 strains demonstrated appreciable differences in their lytic responses. Lysis values for Streptococcus mutans (GS-5 and Ingbritt) and Streptococcus rattus (BHT and FA-1) were greater than those for Streptococcus sobrinus (01 and B13), Streptococcus cricetus (HS6 and AHT), and Streptococcus ferus(8S1 and HD3).

Treatment of Streptococcus mutans GS-5 cells with concentrations of penicillin G within a relatively narrow range resulted in substantial lysis. This penicillin-induced lysis was dependent upon cell density and pH of the lysis medium. Other oral streptococci (Streptococcus sobrinus, Streptococcus rattus, and Streptococcus cricetus) also demonstrated substantial levels of penicillin-induced lysis under appropriate conditions. Lesser degrees of lysis were seen in a related organism, Streptococcus ferus.

Rat saliva agglutinated Streptococcus mutans Ingbritt and NCTC 10449 and Streptococcus sanguis NCTC 7864 but not S. mutans NCTC 10921, GS 5, or LM 7, Streptococcus sobrinus 6715-13 or OMZ 65, or Streptococcus cricetus HS 6, as measured turbidometrically. The specificity of agglutination by rat saliva was the same as that by human saliva. Agglutination was associated with a mucin complex (rat salivary agglutinin complex [rS-A]) of sulfated sialoglycoproteins, with a trace of associated lipid and an apparent Mr of 1.6 X 10(6), isolated by gel-filtration Fast Protein Liquid Chromatography. The complex was dissociated in a high-ionic-strength buffer containing 6 M urea and then fractionated by gel filtration and anion-exchange Fast Protein Liquid Chromatography into four sulfated sialoglycoprotein components, designated rS-A-1Q1, rS-A-1Q2, rS-A-1Q3, and rS-A-2, with rS-A-1Q2 being polydisperse through differential glycosylation of the polypeptide backbone. The dissociation destroyed agglutination activity. The polypeptide backbones contained up to 42% serine plus threonine and up to 40% glycine plus alanine plus proline plus valine. The carbohydrate moiety of the rS-A sialoglycoproteins consisted of N-acetylgalactosamine, sialate, galactose, fucose, N-acetylglucosamine, and small amounts of mannose, with the predominant sugar being N-acetylgalactosamine. Agglutination was inhibited by 1 mM EDTA but was restored by 1.5 mM CaCl2. Agglutination was also inhibited by 5 mM CaCl2; nonimmune sera; cationic polymers; and wheat germ, lentil, soybean, and peanut lectins. However, agglutination was not affected by lipoteichoic acid, various anionic proteins, or various sugars. Neuraminidase treatment of rS-A did not affect activity, but tryptic digestion of S. mutans did prevent agglutination. The results are consistent with calcium bridging the negative groups within the rS-A complex and allowing the approach of rS-A to the bacterial cell surface to effect a specific conformational attachment.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.

High-molecular-weight polymers of alpha-1,6-linked D-glucans are insoluble in alcohol solutions. Whole, but not parotid, saliva prevented the precipitation of D-glucans by 80% (vol/vol) ethanol, showing that the whole saliva contained a factor which complexed with the glucan to render it alcohol soluble. The glucan-binding factor was retained on a column of Sephacryl S-200 which had been preequilibrated with 80% ethanol. The factor was then eluted with water. Passive hemagglutination assays revealed that the glucan-binding factor could sensitize erythrocytes to agglutination with anti-poly(glycerolphosphate), suggesting that the active glucan-binding component with lipoteichoic acid. The glucan-solubilizing factor was resistant to heat (100 degrees C), proteases, sialidase, lysozyme, lactoperoxidase, trichloroacetic acid, and Triton X-100. When sucrose was added to saliva, a suspension of Streptococcus cricetus AHT, or a suspension of Streptococcus sanguis 10556, relatively large amounts of glucan-binding factor were released in a soluble form. In addition, penicillin G caused the release of the glucan-solubilizing component from a suspension of S. cricetus AHT. It is suggested that whole saliva contains a component, tentatively identified as lipoteichoic acid, which can complex with glucans in a relatively hydrophobic solvent. This type of complex formation may be important in the adhesion of oral streptococci to saliva-coated surfaces.

The prevalence of mutans streptococci (Streptococcus mutans, Streptococcus cricetus, Streptococcus sobrinus, and Streptococcus rattus) was determined in the salivas of 169 elderly individuals ranging in age from 60 to 87 years. Approximately 40% of these individuals were edentulous and wore full upper and lower dentures. With the exception of a higher proportion of saliva counts below 1,000 CFU/ml in the full-denture wearers, the prevalence and the serotype and species distributions of the mutans streptococci were similar in the denture wearers and individuals with natural teeth only. The species and serotype distributions of mutans streptococci in this elderly population were also consistent with reported observations of other workers on younger, more caries-prone populations. A total of 87 representative isolates of the mutans streptococci were tested for cariogenic potential in a hamster model system. A considerable degree of variation in virulence between different strains was observed. However, these differences were not relatable to individual species or serotypes or to whether the organisms were isolated from denture wearers or naturally dentate subjects. The results of our studies indicate that elderly individuals with either natural or artificial dentitions may be a hitherto unrecognized reservoir of mutans streptococci having varying degrees of potential cariogenicity. Hence, in close family situations they could serve, along with parents and siblings, as vectors in the initial transmission of cariogenic microorganisms to young children.

Streptococcus sanguis I gave a significantly greater percentage coverage (cell-pellicle attachment) of saliva-coated glass in the presence of sucrose than did Strep. sanguis II (p less than 0.025), and both these gave greater percentage coverages than the other species tested, between which no significant differences were noted. There was a large number of significant differences in clump size (cell-cell attachment) between species-pairs. Among the mutans streptococci, there were significant differences in the percentage coverage between Streptococcus rattus/Streptococcus mutans (p less than 0.05) and Strep. rattus/Streptococcus sobrinus (p less than 0.01), and in clump size between all species-pairs with the exception of Streptococcus cricetus or Strep. sobrinus and Strep. mutans. The rank order of species in relation to fissure caries was mutans streptococci greater than Streptococcus salivarius greater than Streptococcus milleri greater than Strep. sanguis greater than Streptococcus faecalis greater than Streptococcus mitis greater than Streptococcus lactis. There was a significant correlation between percentage coverage in vitro and fissure caries in vivo for strains of Strep. sanguis (p less than 0.05) and pooled strains of Strep. mitis and Strep. sanguis (p less than 0.01). On comparing data for adherence in the presence or the absence of sucrose, the sugar had no effect on the percentage coverage of the seven species tested, but significantly increased the clump size of the mutans streptococci (p less than 0.01) and Strep. sanguis (p less than 0.05).

Several of the cariogenic mutans streptococci produce cell wall-associated glucan-binding lectins (GBLs). The lectins bind alpha-1,6-linked glucans and have no affinity for other polysaccharides or anomeric linkages. When citrate or lactate was included in the growth medium, expression of the activities of the GBLs of Streptococcus cricetus and S. sobrinus was prevented. Furthermore, chelating agents, including citrate, lactate, EDTA, and acetylacetone, were able to reversibly inhibit glucan-induced aggregation of GBL+ streptococci. In addition, the chelating agents prevented sucrose-dependent streptococcal adhesion to glass surfaces and dispersed preformed adherent masses of the streptococci. Neither citrate nor other chelating agents modified the activities of glucosyltransferases. Expression of the lectin could only be achieved by the addition of manganous ion to the growth medium. Chloramphenicol and other metabolic inhibitors prevented synthesis of GBL in cells obtained from manganese-deficient medium and shifted to manganous ion-sufficient medium. The GBL may be a manganoprotein, the manganese of which may be perturbed, but not removed, by chelating agents. During synthesis of the GBL, manganous ion may be required in order for the protein to achieve an active conformation. Citrate or other chelating agents may have promise as anticaries agents.