Sorbitol inhibition of glucose metabolism by Streptococcus sanguis 160

Clinical studies in Sweden have shown that the proportion of sorbitol-utilizing strains of Streptococcus sanguis increases in dental plaque from individuals using sorbitol-containing products for prolonged periods. We have undertaken to study the metabolism of glucose and sorbitol by S. sanguis 160, isolated from a subject consuming sorbitol-containing chewing-gum 4 times a day for 4 years. Growth on glucose was inhibited by the presence of sorbitol in the growth medium and sorbitol was utilized in the presence of glucose, albeit, at a slower rate than glucose. In addition, pulses of glucose added to cultures growing on sorbitol resulted in the expulsion of sorbitol from the cell. In order to examine further the relationship of sorbitol and glucose, uptake assays were carried out with S. sanguis 160 grown in continuous culture (pH 7.0, dilution rate = 0.1 h-1) with glucose, sorbitol or nitrogen (sorbitol excess) limitations. The uptake of [14C]-glucose by sorbitol-limited cells, but not by glucose-limited cells, was inhibited by sorbitol, as was glycolysis. Kinetic experiments with glucose-limited cells showed 2 transport systems for glucose with Ks values of 5.2 and 40 microM, and glucose phosphorylation activity by decryptified cells indicated transport by the P-enolpyruvate (PEP) phosphotransferase system (PTS) with lesser activity for an ATP-dependent transport process. Transition from glucose-limited growth to sorbitol-limited growth revealed repression of total [14C]-glucose uptake by intact cells and activity for Enzyme II for glucose (Ellglc) of the PTS measured in membrane preparations in the presence of an excess of the soluble PTS proteins in crude cell-free supernatant fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Invertase activity in Streptococcus mutans and Streptococcus sanguis

Toluene treatment of intact cells revealed invertase-like activity in Streptococcus mutans K1-R (cariogenic) and in Streptococcus sanguis 903–1600 (noncariogenic). The activity was identified as a β-d-fructofuranoside fructohydrolase (invertase; EC. 3.2.1.26) by its hydrolysis of sucrose and raffinose and its failure to act on melezitose and other glucoside derivatives. The invertase was induced by sucrose in Strep, sanguis but was a constituent in Strep. mutans. Amylomaltase, a transferase acting on maltosaccharide, was also detected in both organisms.     

Adherence of Streptococcus sanguis

The presence of adhesins on the cell surface of S. sanguis enables the organism to grow and survive in the oral cavity. Many workers are now actively involved in attempting to characterise these adhesins and the molecular basis for adherence of various streptococci. The complexity of the adhesion processes is underlined by the many varied opinions as to the nature of the molecular components and biochemical interactions that are involved. Understanding the molecular mechanisms involved in bacterial adherence and in dental plaque formation will have significant impact on the prevention and treatment of oral diseases.     

Growth of Streptococcus mutans and Streptococcus sanguis in mixed culture

In an anaerobic atmosphere, Streptococcus mutans strain JC 2 and Streptococcus sanguis strain 804 required ammonia, cysteine, B-vitamins, glucose and salts for growth. The only difference in nutritional requirements between the two strains was a requirement for p-aminobenzoic acid by Strep. mutans, but not by Strep. sanguis. In a mixed culture of Strep. sanguis and Strep. mutans in a p-aminobenzoic acid-free medium, Strep. sanguis supported the growth of Strep. mutans to such an extent that the interaction of the organism would be characterized as a parasitism of Strep. mutans on Strep. sanguis. p-Aminobenzoic acid may be one of the substances determining the establishment and population density of Strep. mutans in the microbial aggregations on the tooth surface.     

Nutritional requirements of Streptococcus sanguis

The nutritional requirements of Streptococcus sanguis ATCC strains 10556, 10557 and 10558, Strep. mitis ATCC 9811, Strep. bovis ATCC 9809 and 37 strains isolated from the oral cavity in man and considered to be Strep. sanguis were studied under anaerobic conditions. Streptococcus sanguis ATCC 10558 and 23 of the oral strains required cysteine, glucose, pyridoxine, nicotinic acid, biotin, thiamin, riboflavin, pantothenic acid, ammonia and salts. One oral strain required in addition glutamic acid and Strep. sanguis ATCC 10556 isoleucine and valine. Strep. sanguis ATCC 10557 and 14 of the oral strains required arginine, cysteine, glutamic acid, histidine, glucose, nicotinic acid, biotin, thiamin, riboflavin, pantothenic acid, ammonia and salts. Strep. mitis ATCC 9811 had the same nutritional requirements. One oral strain required adenine in addition. Strep. bovis ATCC 9809 required cysteine, glucose, biotin, thiamin, ammonia and salts.     

Rate-limiting steps of glucose and sorbitol metabolism in Streptococcus mutans cells exposed to air

It has been supposed that rate of sorbitol metabolism in the air-exposed streptococcal cells could be limited by the low capacity to regenerate nicotinamide adenine dinucleotide (NAD) from reduced NAD (NADH) following inactivation of pyruvate formate-lyase by oxygen. The rate-limiting steps, however, have not been identified. The aim of this study was to examine the effect of temporary exposure of the streptococcal cells to air on the intracellular flux of glucose and sorbitol metabolism by measuring acid excretion, fluorescence dependent on cellular level of NADH, glycolytic intermediates and enzyme activities. The exposure of cells to air decreased the acid excretions during glucose and sorbitol metabolism. The analysis of the glycolytic intermediates and the fluorescence suggested that the reduced level of acid excretion in the air-exposed glucose metabolizing cells resulted from the decrease in pyruvate catabolism. In the presence of sorbitol, the decreased acid production resulted from the reduced rates of the reactions catalyzed by sorbitol-phosphoenolpyruvate phosphotransferase and sorbitol 6-phosphate dehydrogenase because of shortage of substrates for these enzymes in addition to the decrease in pyruvate catabolism.     

Membrane-associated and solubilized ATPases of Streptococcus mutans and Streptococcus sanguis

The proton-translocating, membrane ATPases of oral streptococci have been implicated in cytoplasmic pH regulation, acidurance, and cariogenicity. Membranes were isolated from Streptococcus mutans GS-5 and Streptococcus sanguis NCTC 10904 following salt-induced lysis of cells treated with lysozyme and mutanolysin. The ATPase activities of these membranes were 1.8 and 1.1 units per mg membrane protein, respectively. F1 ATPases were washed free from the membranes and purified by fast protein liquid chromatography (FPLC). Hydrolytic activities of the F1 ATPases were maximal at pH values between 6.0 and 6.6, whereas the membrane-bound enzymes had pH maxima of 7.5 (S. sanguis) and 6.0 (S. mutans). The F1 ATPases of the streptococci were similar to the well-characterized enzyme of Escherichia coli; they consisted of five different polypeptides and had apparent, aggregate molecular weights of from 335 to 350 Kd. The membrane-bound ATPases were characterized biochemically and found to be similar to those of proton-translocating ATPases of E. coli and Streptococcus faecalis. Km values for the membranes with respect to ATP were found to be 0.9 and 1.0 mmol/L for S. mutans and S. sanguis, respectively. Both enzymes had specificities for purine triphosphates and were active with a variety of divalent cations, although optimal activity occurred with ATP and Mg. The membrane-associated enzymes were sensitive to the inhibitors dicyclohexylcarbodiimide (DCCD) and azide, but insensitive to ouabain and vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mannitol and sorbitol catabolism in Streptococcus mutans

Mannitol- or sorbitol-adapted Streptococcus mutans NCTC 10449 contained high levels of mannitol-1-phosphate dehydrogenase and sorbitol-6-phosphate dehydrogenase activity. Neither of these activities was present at significant levels in cells grown on glucose or any of several other carbon sources. Moreover, when glucose was added to medium containing mannitol or sorbitol, the cellular level of both mannitol-1-phosphate dehydrogenase and sorbitol-6-phosphate dehydrogenase was reduced to that found in glucose-adapted cells. The two hexitol phosphate dehydrogenase activities were due to distinct enzymes, which could be separated by polyacrylamide gel electrophoresis and by Sephadex G-200 chromatography. Both enzymes were specific for nicotinamide adenine dinucleotide and neither was active with the corresponding non-phosphorylated hexitol substrates. The reaction product for both enzymes appeared to be fructose-6-phosphate. The available data indicate that Strep. mutans NCTC 10449 ferments both mannitol and sorbitol by a pathway that involves phosphorylation of the substrates prior to their oxidation by distinct, inducible enzymes to the common glycolytic intermediate, fructose-6-phosphate. This pathway for hexitol catabolism also appears to be operative in other strains of Strep. mutans.     

Phenotypic and genotypic diversity of Streptococcus sanguis in infants

Streptococcus sanguis comprises a heterogeneous group of oral streptococci indigenous to the oral cavity of humans. A total of 289 isolates from an infant population (n=37) were tentatively identified as S. sanguis on the basis of the distinctive colony morphology as shown on MM10-sucrose non-selective medium. These isolates were divided into four biovars of S. sanguis as determined by an extended panel of biochemical attributes. Chromosomal DNA was extracted from each isolate, and an AP-PCR fingerprint profile was obtained to allow study of the diversity within and among the infants. In this study, all four biovars of S. sanguis were detected in the infants. A wide genotypic diversity of S. sanguis was observed among these isolates; on average, each infant harbored 2.7 unique amplitypes as shown by the AP-PCR fingerprints. To explore the phylogenic relationship among these S. sanguis isolates, 20 strains representing the four biovars were selected at random for sequencing of their 16S rDNA and 16S-23S rDNA intergenic spacer chromosomal loci. Two major sequence patterns were identified within the 16S rDNA sequences. A phylogenic analysis showed that members from each of the four biovars of S. sanguis bore close relationship with the type-strain ATCC 10556 sequence, and that all of the isolates representing the four biovars could be clustered into two main phylotypes. The biovars were distributed throughout the phylotypes, indicating no correlation between the genetic and phenotypic groupings.     

Studies concerning the glucosyltransferase of Streptococcus sanguis

We have shown in previous studies that the glucosyltransferase (Gtf) enzymes of Streptococcus mutans have distinct properties when adsorbed to a surface. In the present study, we compared the activity of Gtf from Streptococcus sanguis, designated GtfSs, in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads, and determined the ability of its product glucan to support the adherence of oral microorganisms. Gtf from S. sanguis 804 NCTC 10904 was purified from culture supernatant fluids by means of hydroxyapatite chromatography. Enzyme and the substrate were prepared in buffers at pH values from 3.5 to 7.5. Maximum activity of GtfSs occurred between pH 5.5 and pH 6.5, whether in solution or adsorbed onto a surface. The solubilized and insolubilized enzymes showed highest activity at 40 degrees C; activity was reduced by 50(+/-2)% at 20 and 30 degrees C. The enzyme did not form glucans in either phase at 10 or 60 degrees C. The K(m), determined from Lineweaver-Burk plots, for the enzyme in solution was 4.3(+/-0.4) mmol/l sucrose, and the K(m) for the enzyme on sHA beads was 5.0(+/-1.0) mmol/l sucrose. The ability of the GtfSs glucan synthesized on the surface of sHA beads to support the adherence of oral bacteria was investigated. (3)H-thymidine-labeled bacteria (S. mutans GS-5, S. sobrinus 6715, S. sobrinus 6716, S. sanguis 10904, Actinomyces viscosus OMZ105E, A. viscosus 2085, and A. viscosus 2086) were incubated with sHA beads coated with GtfSs glucan. S. mutans GS-5 displayed the highest level of binding numerically. These results show that the GtfSs of S. sanguis is active on sHA beads, that the pH optimum for activity on a surface differs slightly from that in solution, and that its product glucan can support the adherence of oral microorganisms.     

Biochemical mechanisms of enhanced inhibition of fluoride on the anaerobic sugar metabolism by Streptococcus sanguis

The effect of fluoride on acid production by Streptococcus sanguis ATCC 10556 was compared under anaerobic and aerobic conditions. The rate of acid production under constant-pH and pH-free-fall conditions was determined during glucose metabolism by resting cells. Anaerobic glycolysis was inhibited more strongly by fluoride than was aerobic glycolysis. Intracellular levels of 3-phosphoglyceric, 2-phosphoglyceric, and phosphoenolpyruvic acids were lower under anaerobic conditions than under aerobic conditions. Thus, S. sanguis had a low phosphoenolpyruvate (PEP) potential under anaerobic conditions. This low PEP potential was suggested to account for the more effective fluoride inhibition of enolase and, consequently, the reduced transport of sugar by the PEP-dependent phosphotransferase system of this micro-organism.     

Antineoplastic agents inhibit the growth of Streptococcus mutans and Streptococcus sanguis in vitro

The effect of methotrexate (MTX) and doxorubicin on the growth, metabolism and ultrastructure of Streptococcus mutans and Streptococcus sanguis was studied in vitro. Both anticancer drugs exerted an inhibitory effect on the oral streptococci. MTX was more inhibitory than doxorubicin. The minimum inhibitory concentrations (MICs) of MTX to S. mutans were 0.25-2.5 micrograms/ml and that of doxorubicin 0.2 mg/ml. The MICs of MTX and doxorubicin to S. sanguis were 0.025 micrograms/ml and 2.0-0.02 mg/ml, respectively. When saliva samples of patients with malignant tumors receiving various doses of MTX were analyzed, MTX was found to be secreted into the oral cavity at concentrations ranging from 0.014 to 4.486 micrograms/ml. The saliva of these patients was also found to inhibit the growth of S. mutans, and the inhibition zones were in accordance with the MIC values observed. The results suggest that anticancer therapy must be taken into account when the salivary microbiological findings of cancer patients are interpreted.     

A charged component in purified polysaccharide preparations from Streptococcus mutans and Streptococcus sanguis

Purified preparations of soluble and insoluble polysaccarides produced in the presence of 5 per cent sucrose by two strains of Streptococcus mutans (OMZ 176 and OMZ 523) and one strain of Streptococcus sanguis (ATCC 10558) contained a negatively charged component. Tracer experiments showed that bound phosphate was present.If bound phosphate is present in dental plaque, it could act as an ion exchanger and influence the flow of ions to and from the tooth surface. Negatively charged polysaccharides may also be of consequence for the stickiness of plaque and its interaction with tooth surfaces.     

Effects of acidification on growth and glycolysis of Streptococcus sanguis and Streptococcus mutans

After carbohydrate intake, pH in dental plaque decreases rapidly and reaches about 4 within a few minutes. The acidification not only promotes demineralization of tooth surface but can also cause damage to bacteria in dental plaque. We, therefore, investigated the effect of acidification on the dental plaque bacteria Streptococcus sanguis and Streptococcus mutans. At pH 4.0 and 4.2, both growth and glycolytic activities in these streptococci were repressed. Prolonged acidification (for 60 min at pH 4.0) not only repressed both growth and glycolytic activities but also impaired them in S. sanguis cells with concomitant inactivation of the glycolytic enzymes, hexokinase, phosphofructokinase, glyceraldehyde-phosphate dehydrogenase and enolase. The impaired abilities of glycolysis and growth recovered following incubation at pH 7.0 for 80–90 min, and this was accompanied by reactivation of the glycolytic enzymes. On the other hand, these impairments were not observed in S. mutans cells exposed to prolonged acidification. These results indicate that the low pH frequently occurring in dental plaque may transiently impair streptococcal glycolysis and growth and that S. mutans is more durable to the acidification than S. sanguis.