Relationship between growth rate and phagocytosis susceptibility in Streptococcus mutans

Streptococcus mutans strain GS-5 was grown in a variety of carbon sources in order to achieve different balanced growth rates, ranging from 61 to 383 minutes. The influence of the S. mutans growth rate on mouse phagocyte activity against these bacteria has been evaluated. The percentages of bacteria phagocytized and intracellularly killed by macrophages rose to 60-80% and 85-95% respectively when the doubling time was longer, showing that S. mutans is particularly sensitive to nonspecific immune defence mechanisms when cultured under conditions similar to those of its natural ecosystem.     

Enhanced glucosyltransferase activity in penicillin-treated cultures of Streptococcus mutans.

Penicillin-treated cultures of Streptococcus mutants GS-5 produced elevated levels of extracellular glucosyltransferase activity.     

Teichoic acids from chemostat-grown cultures of Streptococcus mutans and Lactobacillus plantarum.

We examined the effect of growth conditions in chemostat culture on the quantity and composition of the cell wall teichoic acids of Streptococcus mutans BHT and Lactobacillus plantarum NCIB 7220 and the membrane lipoteichoic acid from S. mutans Ingbritt. With the cell wall teichoic acids, which are covalently linked to peptidoglycan, the amount of teichoic acid is independent of the growth conditions employed. However, the extent of glucosyl substitution of the polymer from L. plantarum was dependent on growth conditions. S. mutans Ingbritt lipoteichoic acid, on the other hand, was little affected by growth conditions in terms of composition or serological activity, but the amount produced was markedly affected by changes in growth conditions.     

Effects of oxygen on glucose-limited growth of Streptococcus mutans.

Aerotolerance in the growth of Streptococcus mutans and related streptococci was examined under glucose-limited conditions. The growth rate of all strains tested was more or less retarded when they were transferred from anaerobic to aerobic conditions. As for growth yield, however, some strains (group 1) showed reduced values for the increment of cellular dry weight change (in grams per mole of glucose), whereas others showed either unaltered (group 2) or increased (group 3) yields. The characteristic feature of strains in groups 2 and 3 was their high activity of both glucose- and pyruvate-dependent oxygen uptake when strains were grown under aerobic conditions. By contrast, the activity of pyruvate-dependent oxygen uptake by group 1 strains was negligibly low, whether grown under aerobic or anaerobic conditions. Some strains (group 1a) consumed oxygen in the presence of glucose at a much faster rate than others did (group 1b). There seems to be a good correspondence of these aerotolerance groupings to those based on serotypes of S. mutans. Thus, the groups 1a, 1b, 2, and 3 correspond to serotypes g, a+d, c+f, and b, respectively.     

Unbalanced growth and macromolecular synthesis in Streptococcus mutans FA-1.

The continued synthesis of deoxyribonucleic acid, protein, cell wall peptidoglycan and intracellular iodophilic polysaccharide (IPS) by Streptococcus mutans strain FA-1 after several treatments intended to inhibit protein synthesis was studied. Exponential-phase cultures were: (i) simultaneously deprived of two required amino acids (cystine and leucine) that are not present in the cell wall peptidoglycan of this species; (ii) depreived of required amino acids (lysine or glutamate plus glutamine ) that are present in both peptidoglycan and protein; or (iii) treated with tetracycline. Each of these three types of treatment was accompanied by a different pattern of unbalanced growth. The patterns of unbalanced growth that accompanied treatments (i) or (ii) differed substantially from the patterns observed previously for Streptococcus faecalis ATCC 9790, a noncariogenic organism that does not contain IPS. In contrast to S. faecalis 9790, S. mutans FA-1 failed to accumulate peptidoglycan and thicken its wall when deprived of non-wall amino acids. Instead, S. mutans FA-1 continued to accumulate IPS to levels substantially higher than those found in exponential-phase cells. Again, in contrast to S. faecalis, S. mutans FA-1 failed to autolyze upon deprivation of essential precursors of wall peptidoglycan. Under conditions of lysine of glutamate/glutamine deprivation, S. mutans FA-1 continued to accumulate IPS to very high levels. Treatment with tetracycline did result in peptidoglycan accumulation and wall thickening in a manner very similar to that observed previously for inhibition of protein synthesis in S. faecalis. Realtively little IPS synthesis continued after tetracycline treatment. Accumulation of IPS appeared to occur when both ribonucleic acid and peptidoglycan synthesis were severely inhibited. The observations are discussed in terms of the survival of cariogenic organisms in the oral environment.     

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.     

Growth rate and biofilm thickness of Streptococcus sobrinus and Streptococcus mutans on hydroxapatite

Bacteria in biofilm and planktonic bacteria exhibit different properties. The objective of the present study was to compare the growth rates of Streptococcus sobrinus and Streptococcus mutans on different types of biofilm with their planktonic growth rate. Our experimental model consisted of hydroxyapatite beads coated with human saliva (sHA). Glucans or fructans were synthesized in situ on sHA by immobilized cell-free glucosyltransferase or fructosyltransferase isolated from oral bacteria. S. sobrinus or S. mutans was then adsorbed onto the glucan- or fructan-coated sHA and incubated for different time intervals. The depth of the developing biofilm was measured. Our results show that growth rates of S. sobrinus and S. mutans on both fructan- and glucan-coated sHA were similar during a 23 h period. In addition, the profile was similar to the growth profile of the same planktonic bacteria. The resemblance in growth rates between planktonic and biofilm bacteria may be attributed to the thin and non-dense biofilm formed in the initial stages of the biofilm formation. The thin biofilm coat, reaching a maximal depth of 11 microm, has only imposed limited diffusion restrictions, thus not affecting the growth of the bacteria in the biofilm. Our study shows that growth of bacteria on surfaces may resemble their growth in suspension if the bacteria are not embedded in a thick dense biofilm.     

Effect of growth conditions on sucrose phosphotransferase activity of Streptococcus mutans.

Sucrose and glucose phosphoenolpyruvate-dependent phosphotransferase (PTS) activities were studied in growing cultures of Streptococcus mutans serotype c and d/g cells adapted to either glucose or sucrose. Both acid production and optical absorbance were used to monitor growth in pH-controlled defined growth medium. The sucrose PTS activity appeared to be significant only under conditions of substrate limitation or slow growth as a result of low environmental pH. However, under environmental conditions which permitted rapid growth sucrose PTS activity appeared to be repressed, and only when the cells approached substrate-limited stationary phase after growth on high sucrose-supplemented medium was significant sucrose PTS activity again observed. A mutant apparently defective in sucrose PTS activity grew rapidly and produced acid under conditions of high environmental sucrose level but showed no sucrose PTS activity when the culture approached stationary phase. The mutant, however, after adaptation to glucose, demonstrated significant glucose PTS once the culture had attained the stationary growth phase. During diauxie growth in the presence of glucose and sucrose, there were sequential apparent inductions and repressions of glucose and sucrose PTS activities corresponding to decreases and increases of growth rate on the two substrates. Thus, S. mutans possesses at least two transport mechanisms for each substrate studied. One system (PTS) functions under conditions permitting slow growth and another functions under conditions permitting rapid growth.     

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.     

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.     

Pyruvate dehydrogenase activity in Streptococcus mutans.

Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.     

Effect of trace metals on growth of Streptococcus mutans in a teflon chemostat.

Correlations between the presence of certain trace metals in dental enamel or in drinking water and the incidence of human dental caries have been demonstrated; therefore, the effects of several trace metals on growth of the cariogenic organism Streptococcus mutans OMZ176 were determined. For continuous growth in a chemically defined medium (treated with Chelex-100 to lower trace metal contamination and supplemented with high-purity trace metal salts) used in a chemostat constructed of Teflon, S. mutans required input of carbon dioxide and supplementation with magnesium (126 microM) and manganese (18 to 54 microM). Addition of iron (3.6 microM) increased the level of steady-state growth by a factor of 2.8 (stimulation index [SI]); zinc at 0.4 microM nearly doubled equilibrium growth (SI = 0.9). Higher concentrations of iron and zinc (5.4 and 0.8 microM, respectively) were less stimulatory (SI values of 1.95 and 0.3, respectively). Small (but statistically significant) increases in steady-state growth were effected by cobalt (SI = 0.3 at 5.1 to 20.4 microM) and tin (SI = 0.4 at 5.1 to 10.2 microM). These data suggest nutritional requirements for these metals. Copper at a concentration of 0.16 microM was inhibitory. These results show significant effects of these metals on growth of S. mutans and may confirm epidemiological evidence suggesting a role for certain trace metals in the incidence of dental caries.     

Linkage of sucrose-metabolizing genes in Streptococcus mutans.

Antibiotic resistance markers inserted adjacent to different cloned genes from Streptococcus mutans were used to determine the relative positions of these genes on the chromosome. The results showed that these genes, fru-1 and gbp, are closely linked to the gtfA-ftf-scrB cluster. However, gtfD was linked neither to this cluster nor to gtfB-gtfC.     

Virulence of Streptococcus mutans: cariogenicity of S. mutans in adult gnotobiotic rats.

Gnotobiotic rats infected with Streptococcus mutant 6715, mutans C211 at 45 days of age on provided a purified diet containing 5% sucrose developed carious lesions on buccal, sulcal, and proximal molar surfaces within 15 days (60 days of age). The level of caries increased significantly (P less than or equal to 0.01) within the next 15 days (by day 75), an extensive decay was observed on all three molar surfaces of 90-day-old infected rats (45 days after challenge). Mutant C211 was previously shown to exhibit increased glucosyltransferase activity and greater adherence and virulence than S. mutans 6715 wild type (wt). Gnotobiotic rats (90 days of age) infected with either S. mutans AHT or S. mutans 6715 (wt) at 45 days of age developed significantly (P less than or equal to 0.01) fewer caries on all molar surfaces than rats of the same age that were infected with S. mutans 6715, mutant C211. The level of plaque increased 2-fold, and the number of viable S. mutans in plaque increased 10-fold between days 60 and 90 in rats infected with S. mutans 6715, mutant C211. Ninety-day-old rats infected with either S. mutans AHT or S. mutans 6715 (wt) had similar levels of plaque and numbers of S. mutans in plaque; however, these values were two- to fourfold lower than those observed in rats of the same age that were infected with S. mutans 6715, mutant C211.     

Influence of growth conditions on adherence of Streptococcus mutans ingbritt to saliva-coated hydroxyapatite.

Streptococcus mutans Ingbritt grown under standardized conditions adhered less effectively to saliva-coated hydroxyapatite beads than did Streptococcus sanguis G9B, and there was competition for binding. The results with Ingbritt were influenced by the generation time, the pH of growth, and the carbohydrate source as shown by studies on organisms grown in continuous culture.     

Fluoride uptake by Streptococcus mutans 6715.

The short-term kinetics of fluoride uptake by cells from 20- to 22-h cultures of Streptococcus mutans strain 6715 were studied using rapid filtration and centrifugation techniques. Saline-suspended organisms were diluted with fluoride-containing solutions buffered at four different pH values (2.0, 4.0, 5.5, and 8.2). Fluoride disappearance from the medium was inversely related to pH and to the duration of the exposure at any given pH. The uptake was rapid and extensive at the lower pH values and decreased as the pH increased. Media fluoride concentrations subsequently increased; i.e., fluoride was released from the cells. The presence of glucose, cyanide, or iodoacetate did not influence the results. However, preincubation of the cells in fluoride-free buffers, followed by the addition of fluoride, reduced fluoride uptake markedly. Cell-to-media pH gradients were determined by the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione. Fluoride uptake was found to be a function of the magnitude of the pH gradient (P less than 0.001). It is hypothesized that fluoride uptake occurs by the diffusion of hydrogen fluoride and the subsequent trapping of ionic fluoride.     

Transformation of fluoride resistance genes in Streptococcus mutans.

To study the nature of fluoride resistance in Streptococcus mutans, we transformed DNA extracted from fluoride-resistant mutants of S. mutans GS-5 into fluoride-sensitive cells of the same strain. Transformation with DNA from first-step mutants produced transformants with resistance to either 600 or 1,000 micrograms of sodium fluoride per ml, both of which are within the first-step resistance range (400 to 1,000 micrograms/ml). In five of six of these transformation experiments, however, the transformant resistance levels were greater than those of their respective DNA donors. Transformation with DNA from a second-step mutant resistant to 1,600 micrograms/ml resulted in transformants resistant to 600 micrograms/ml, similar to some transformants receiving DNA from first-step mutants. When a second-step mutant resistant to 3,000 micrograms/ml was used as a DNA donor, four different levels of resistance were seen in the transformants (600, 1,000, 1,500, and 2,000 micrograms/ml). In many cases, the growth rates of the transformants (first and second step) were faster than those of the DNA donors. Additionally, many of the transformants demonstrated abrupt shifts in growth rates at relatively low culture densities.     

Bacteriophage deoxyribonucleic acid-induced mutation of Streptococcus mutans.

A cariogenic strain, Streptococcus mutans PK 1, has been demonstrated to have prophage by observation of phage particles with an electron microscope and by induction with mitomycin C. The phage particles could not be detected in a mutant strain which lost the characteristic adhesive nature on glass surfaces and exhibited diminished ability to synthesize insoluble polysaccharide. By infecting the mutant cells with the phages or with free phage deoxyribonucleic acid isolated from the parent strain of S. mutans PK1, the mutant cells were transformed to the cariogenic strain with adhesive nature. The transformants retained the general characteristics of S. mutans PK 1, but in addition all transformants showed a new character; namely, the transformant cell could produce ammonia from arginine, whereas neither the parent nor mutant strains of S. mutans PK 1 had such a property;     

Streptococcus mutans dextransucrase: availability of disaggregated enzyme after growth in a chemically defined medium.

The soluble dextransucrase (EC 2.4.1.5) activity produced by Streptococcus mutans strain 6715 during growth on a chemically defined synthetic medium (FMS) was compared to enzyme from glucose broth cultures (TSB). Growth on the two media was similar. The specific activity of ammonium sulfate-precipitated FMC enzyme was 17 times greater than similar TSB enzyme preparations. The FMC enzyme was stimulated 11-fold, whereas the TSB enzyme was stimulated 1.2-fold by the addition of exogenous primer dextran. In contrast to the TSB enzyme, the FMC activity could be disaggregated to a low-molecular-weight form by 1 M salt. Thus, low-molecular-weight S. mutans dextransucrase activity free of contaminating primer glucan may be readily obtained after growth of the bacterium in a chemically defined sucrose-free medium.