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Electron microscopy revealed that Streptococcus mitis ATCC 903 bound gold probes conjugated with goat IgG by non-immune mechanisms. Only a few of the cells and the cell wall fragments could bind IgG, in contrast to Staphylococcus aureus and Streptococcus group G which showed a more homogeneous binding to nearly all cells or cell wall fragments.

Cell wall extracts from nine strains of viridans streptococci representing five species were analyzed for aminopeptidases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with zymogram procedures revealed a common pattern of aminopeptidases in six strains, all having enzyme bands at 89 and 45 kDa. Three strains had an additional band at 200 kDa. Crossreactivity between aminopeptidases of all active strains was shown with crossed immunoelectrophoresis. Strains of Streptococcus mutans and S. sobrinus were without detectable cell wall aminopeptidases.

Scanning electron (SEM) microscopy of epoxy replicas made from dental impressions has shown that in buccal gingival recession the root surfaces are devoid of cementum, leaving the dentin exposed. In this study replication techniques were applied to correlate the micromorphology of the buccocervical region with early streptococcal attachment. The subjects were 27 healthy young adults. The buccocervical surfaces of all the premolars were meticulously cleaned. The subjects fasted for 2 h before impression-taking. Replicas were made from impressions in hydrophilic A-silicone, and streptococcal attachment was visualized by light microscopy of mitis-salivarius agar replicas incubated anaerobically for 48 h. The surface micromorphology was documented by SEM of corresponding epoxy replicas. Colonization only 2 h after cleaning was very sparse. Sites with healthy or inflamed gingivae had markedly different colonization patterns in the sulcular region. In 4 subjects with a total of 12 sites where gingival recession, undetected clinically, was disclosed by SEM, representative colonies were retrieved and identified microbiologically to species level. Two healthy sites per subject were also sampled. Streptococcus mutans and S. sobrinus were identified from eight sites with exposed root dentin. S. oralis predominated on the enamel surfaces. The method offers a valuable complement to in situ and in vitro microbiologic studies of exposed dentin and a novel technique for sampling clinical isolates of streptococci.

Arginine catabolism via the arginine deiminase pathway was found in Streptococcus sanguis 903. Citrulline and ornithine were released from resting cells incubated with arginine, arginine-containing peptides, or saliva. Maximum arginine catabolism by resting cells of S. sanguis 903 was found in the pH range 7-8 and at 45-48 degrees C. Arginine deiminase activity was found in the cytoplasm and in the cell-wall extract of this strain, while ornithine carbamoyltransferase activity was found in the cytoplasm and in extracts of cell walls and cytoplasmic membranes. Streptococcus mutans GS-5 and Streptococcus sobrinus strains OMZ 176 and 6715 could release arginine from salivary peptides but were incapable of significant arginine catabolism.

Streptococcus mitis ATCC 903 and Streptococcus sanguis 804 could utilize large peptides present in proteose peptone as their sole source of nitrogen but grew poorly in amino acid medium. By contrast, Streptococcus mutans BHT and S. mutans IB grew in amino acid medium but could not utilize large peptides. Streptococcus sp. strain Bravato had requirements for both peptides and amino acids. Peptide size did not seem important for the utilization of peptides by S. mitis except that dipeptides were not utilized. Large peptides were essential for depressed hyaluronidase synthesis in nongrowing S. mitis. Intracellular aminopeptidase activities were high in S. mitis ATCC 903 and Streptococcus sp. strain Bravato, intermediate in S. sanguis 804, and low in S. mutans BHT and S. mutans IB.

Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.

Cells of Streptococcus mitis ATCC 903 were converted to stable protoplasts by the cell wall-degrading M-1 enzyme of the mutanolysin complex isolated from Streptomyces globisporus. Over 90% of total glucokinase (EC 2.7.1.2), aminopeptidase (EC 3.4.11.1), and dextranglucosidase (EC 3.2.1.70) was recovered in the cytoplasmic fraction, whereas over 20% of total invertase (beta-fructofuranosidase: EC 3.2.1.26) was released during protoplast formation. ATPase (EC 3.6.1.3). chymotrypsin-like protease (EC 3.4.21.1), arginine aminopeptidase (EC 3.4.11.6), and lactate dehydrogenase (EC 1.1.1.27) were detected in Triton X-100 extracts of the cytoplasmic membrane fraction by crossed immunoelectrophoresis in combination with enzyme-staining procedures. By these methods, NADH dehydrogenase (EC 1.6.99.3), aminopeptidase, and lactate dehydrogenase were detected in the cytoplasmic fraction. Aminopeptidases in the cytoplasmic fraction differed from this activity in the membrane fractions in electrophoretic mobility and substrate specificity.

beta-Fructofuranosidase activity was found to be cell-bound in Streptococcus mitis ATCC 903. The following evidence suggests that induction functions as a regulatory mechanism for beta-fructofuranosidase in S. mitis: (1) on transfer of glucose-grown exponential phase bacteria to sucrose medium, the specific activity of beta-fructofuranosidase increased fourfold in the course of one generation; (2) other sugars had no stimulatory effect on the rate of synthesis of beta-fructofuranosidase; (3) the effect of sucrose on the rate of synthesis of beta-fructofuranosidase could be measured within a few minutes. Glucose, fructose and mannose repressed beta-fructofuranosidase. The addition of glucose to bacteria growing on sucrose repressed beta-fructofuranosidase for about one generation. The intracellular concentration of glucose was considerably increased during repression, while the intracellular concentration of glycolytic intermediates did not vary significantly.

Extracts of Streptococcus mitis ATCC 903 were analysed for beta-fructofuranosidase and alpha-glucosidase activities by isoelectric focusing in thin-layer polyacrylamide gels combined with zymogram procedures. Three bands of activity were visualized in the gels after incubation with sucrose (pI 4.05, 4.25 and 4.85) and three other bands after incubation with p-nitrophenyl alpha-D-glucopyranoside (pI 3.90, 4.45 and 4.65). The enzymes responsible for the reaction with sucrose were identified as beta-fructofuranosidases (EC 3.2.1.26) for the following reasons: identical enzyme bands were visualized in the gels after incubation with raffinose; no enzyme bands appeared in the gel after incubation with the alpha-glucosides maltose, turanose, trehalose and melezitose; and the soluble fraction hydrolysed sucrose to equimolar amounts of glucose and fructose.

A constitutive phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system was found in decryptified cells of Strep. mitis. The system displayed saturation kinetics and the apparent Km value for the non-fermentable analogue 2-deoxyglucose (2DG) was 0.4 mM. Sodium fluoride inhibited the activity when 2-phosphoglycerate was used as the energy source instead of PEP. Intact cells accumulated 2DG and also a derivative that behaved chromatographically like 2DG-6-phosphate. PEP-dependent glucose phosphotransferase activity was also demonstrated in freshly collected plaque.

The adhesion of Streptococcus rattus BHT and Streptococcus mutans IB to metal specimens of amalgam, silver, tin and copper was studied using (6-3H)thymidine labeled cells. In the standard assay the metal specimens were suspended by a nylon thread in an adhesion solution containing a chemically defined bacterial growth medium (FMC), sucrose, and radiolabeled bacteria. Maximum amounts of adhering bacteria were obtained after about 100 min of incubation. Saturation of the metal specimens with bacteria was not observed. Both strains also adhered in the absence of sucrose, indicating that glucan formation was not necessary for adhesion. However, in the presence of glucose, adhesion was only 26-45% of that observed in the presence of equimolar sucrose. Sucrose-dependent stimulation of adhesion seemed to be due to increased cell-to-cell adhesion capacity. Isolated radiolabeled water-insoluble and water-soluble polysaccharides produced from sucrose by S. rattus BHT were not adsorbed to the metal surfaces.