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Streptococcus mutans is a component of the dental plaque biofilm and an important aetiological agent in dental caries. Although this organism growing in the suspended (planktonic) state has been well characterized, relatively little is known about its physiology in biofilms, particularly in the acidic environments associated with caries development. The authors determined the effect of biofilm age (1-5 days) and cell density on selected metabolic properties under conditions of glucose limitation in a biofilm-chemostat at pH 7.5 and compared these baseline values with those of 3 day biofilms subjected to acid stress. Biofilm cell biomass more than doubled over the 5 day experimental period under baseline conditions, with the glycolytic rate, glucose uptake, glucose-PTS (phosphotransferase system) activity and protein synthesis maximum at 1-2 days. DNA and RNA synthesis increased for the first 3 days before decreasing in the 5 day biofilms, while H(+)/ATPase activity was higher in 5 day biofilms than 1 day biofilms, with overall activity 5-13-fold higher per cell unit than in the associated planktonic cells. Glucose pulsing (50 mM final concentration) for three consecutive days without pH control for 5 h (pH 4.39+/-0.02) resulted in a progressive decrease in planktonic cell numbers; however, the rate of acid formation and glucose utilization in the chemostat by these cells increased per cell unit. Assays for carbohydrate metabolism in the latter cells showed increased activity, as did an assay for H(+)/ATPase (8-fold); however, DNA, RNA and protein synthesis were repressed (0.3-0.7-fold). Although the 3 day biofilm viable cell counts declined by 51 % on glucose pulsing, all the physiological parameters measured by cell unit increased in activity, with notable increases in RNA and protein synthesis (4.6-7.6-fold). The results indicate that the maintenance of intracellular pH homeostasis is the basis of the enhanced physiological status and acid tolerance of biofilm cells.

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.

Previous research with Streptococcus mutans and other oral streptococci has demonstrated that the acid shock of exponential-phase cells (pH 7.5 to 5.5) resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3.5-3.0). The current study was designed to determine whether two fresh isolates, H7 and BM71, and two laboratory strains, Ingbritt and LT11, were capable of a stationary-phase ATR as estimated by a survival test at pH 3.5 for 3 h. All four strains were unable to generate a stationary-phase ATR under control conditions at pH 7.5, with the exception of a burst of survivors in the transition between the exponential and stationary phases when the carbon source (glucose) was depleted. Adaptation at pH 5.5 resulted in the expected pH-dependent exponential-phase ATR, but only the fresh isolates exhibited a stationary-phase ATR at this pH. Glucose starvation of cells in complex medium was shown to enhance acid tolerance for the fresh isolates, but not the laboratory strains. This tolerance was, however, greatly diminished for all strains in a defined medium with a low concentration of amino acids. Growth of strain H7 in complex medium resulted in the formation of at least 56 extracellular proteins, nine of which were degraded in the early stationary phase following the induction of proteolytic activity during the transition period. No proteolytic activity was observed with strain LT11 and only 19 extracellular proteins/peptides were apparent in the medium with only one being degraded in the early stationary phase. Strain H7 was also shown to have two- to fourfold higher levels of intracellular glycogen in the stationary phase than strain LT11. These results suggest that S. mutans H7 possessed the required endogenous metabolism to support amino acid/peptide uptake in the early-stationary phase, which resulted in the formation of basic end products that, in turn, contributed to enhanced intracellular pH homeostasis.

An essential protein translocation pathway in Escherichia coli and Bacillus subtilis involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon of Streptococcus mutans resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in S. mutans after construction of bona fide sat operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of ylxM that was responsible for upregulating the coordinate expression of all five genes of the sat operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of satD and one just 3' to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the sat locus resulted in viable mutants. Most striking, however, was the finding that a mutation in ffh did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in S. mutans.

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[(14)C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.

Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol P-enolpyruvate phosphotransferase transport system (PTS) of Streptococcus mutans LT11. The first gene, srlD, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by srlR, coding for a transcriptional regulator; srlM, coding for a putative activator; and the srlA, srlE, and srlB genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the srlD gene is found after the genes coding for the EII components; thus, the location of the gene in S. mutans is unique. The SrlR protein is similar to several transcriptional regulators found in Bacillus spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865-874, 1998), and its gene overlaps the srlM gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria.

Our previous research has demonstrated that with the more aciduric oral bacteria, an acid shock to sub-lethal pH values results in the induction of an acid tolerance response that protects the cells at extremely low pH (pH 3.0-4.0) that kills unadapted control cells maintained at pH 7.5 (Oral Microbiol Immunol 1997: 12: 266-273). In this study, we were interested in comparing the protein profiles of acid-shocked and control cells of nine organisms from three acid-ogenic genera that could be categorized as strong, weak and non-acid responders in an attempt to identify proteins that could be classified as acid-regulated proteins and which may be important in the process of survival at very low pH. For this, log-phase cultures were rapidly acidified from pH 7.5 to 5.5 in the presence of [14C]-amino acids for varying periods up to 2 h, the period previously shown to be required for maximum induction of the acid response. The cells were extracted for total protein and subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide chromatography with comparable control and acid-shocked protein profiles compared by scanning and computer analysis. Of particular interest were the proteins in the acid-shocked cells that showed enhanced labeling (i.e., synthesis) over the control cells, since these were considered acid-regulated proteins of importance in pH homeostasis. Streptococcus mutans LT11 generated the most rapid and complex pattern: a total of 36 acid-regulated proteins showing enhanced synthesis, with 25 appearing within the first 30 min of acid shock. The enhanced synthesis was transient with all proteins, with the exception of two with molecular weights of 50/49 and 33/32 kDa. Within the acid-regulated proteins were proteins having molecular weights comparable to the heat shock proteins and the various subunits of the membrane H+/ATPase. By comparison, the strong responder, Lactobacillus casei 151, showed the enhanced formation of only nine proteins within the first 30 min of the acid shock, with a total of 11 acid-regulated proteins formed during the 2-h adaptation period with enhanced synthesis transient for seven of these proteins. Streptococcus salivarius AT2 and Streptococcus gordonii TH12 had the formation of 6 and 8 proteins enhanced, while the weakly responding organisms, Streptococcus sanguis ATCC 10,556 and Streptococcus oralis ATCC 10,557, exhibited 8 and 6 such proteins, respectively. Even non-responding strains unable to survive at very low pH, such as Streptococcus sobrinus CH125/43, Streptococcus mitis ATCC 12,261 and Actinomyces naeslundii 301-13 showed the initial formation of 3-9 acid-regulated proteins, but protein synthesis was not sustained over the entire adaptation period. Clearly, the survival of oral bacteria at very low pH is related, not to the total number of the acid-regulated proteins induced per se but to the formation of key proteins that function to augment normal pH homeostasis.

Experiments were conducted with Streptococcus mutans NCTC 10449 and Streptococcus sanguis ATCC 10556 to determine whether the acid end-products, lactate and acetate, were involved in the regulation of cellular growth and metabolism. The growth rate and culture biomass of both organisms was inhibited by the addition of lactate and acetate at concentrations as high as 200 mM to the cultures, although the final pH values of the lactate and acetate cultures were similar. In addition, the metabolic conversion of glucose to lactate was decreased by external lactate but stimulated by acetate. In spite of this, calculation of the yield of cell biomass per mole of ATP (YATP) showed that the yield of both organisms actually increased in the presence of added lactate, but decreased with acetate. This indicates that the two acids interacted with the cells of the organisms by different mechanisms. For both organisms, the final external undissociated lactic acid was relatively constant at concentrations between 0 and 200 mM added lactate, 24.9-32.5 mM for S. mutans and 8.0-11.5 mM for S. sanguis. On the other hand, the final concentration of undissociated acetic acid in the S. mutans cultures increased from 2.9 to 83.7 mM as the medium acetate concentration increased, and from 1.0 to 36.0 mM with the S. sanguis cultures. Counterflow experiments provided evidence for a lactate carrier in both S. mutans and S. sanguis, but an acetate carrier in these organisms could not be demonstrated. [14C]-lactate and [14C]-acetate were taken up into de-energized, chemostatgrown cells of S. mutans and S. sanguis in response to an artificially generated pH gradient but not by an imposed electrical gradient. Thus, under these conditions lactate uptake occurred via a symport process with only one proton. Growth of both organisms in the presence of increasing concentrations of acetate resulted in a small reduction (27%) in the transmembrane pH gradient (delta pH) as measured by the permeant acid, [14C]-salicylate. However, the uptake of [14C]-acetate for the estimation of delta pH revealed significant inhibition of the acetate concentration gradient in the presence external acetate, indicating that the cells expelled the acetate anion. The results indicate that, unlike acetate uptake, lactate transport by S. mutans and S. sanguis was strictly regulated via the lactate carrier in order to prevent excessive dissipation of the pH gradient. Clearly, the formation of acetate by oral streptococci is more problematic for cellular homeostasis than the formation of lactate.

In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose.

Streptococcus mutans transports glucose via the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS). Earlier studies indicated that an alternate glucose transport system functions in this organism under conditions of high growth rates, low pH, or excess glucose. To identify this system, S. mutans BM71 was transformed with integration vector pDC-5 to generate a mutant, DC10, defective in the general PTS protein enzyme I (EI). This mutant expressed a defective EI that had been truncated by approximately 150 amino acids at the carboxyl terminus as revealed by Western blot (immunoblot) analysis with anti-EI antibody and Southern hybridizations with a fragment of the wild-type EI gene as a probe. Phosphotransfer assays utilizing 32P-PEP indicated that DC10 was incapable of phosphorylating HPr and EIIAMan, indicating a nonfunctional PTS. This was confirmed by the fact that DC10 was able to ferment glucose but not a variety of other PTS substrates and phosphorylated glucose with ATP and not PEP. Kinetic assays indicated that the non-PTS system exhibited an apparent Ks of 125 microM for glucose and a Vmax of 0.87 nmol mg (dry weight) of cells-1 min-1. Sugar competition experiments with DC10 indicated that the non-PTS transport system had high specificity for glucose since glucose transport was not significantly by a 100-fold molar excess of several competing sugar substrates, including 2-deoxyglucose and alpha-methylglucoside. These results demonstrate that S. mutans possesses a glucose transport system that can function independently of the PEP PTS.

We report the sequencing of a 2,019-bp region of the Streptococcus mutans NG5 genome which contains a 1,428-bp open reading frame (ORF) whose putative translation product had 50% identity to the amino acid sequences of the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenases (GAPN) from maize and pea. This ORF is located approximately 200 bp downstream of the ptsI gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system. Mutant BCH150, in which the putative gapN gene had been inactivated, lacked GAPN activity that was present in the wild-type strain, thus positively identifying the ORF as the S. mutans gapN gene. Another strain of S. mutans, DC10, which contains an insertionally inactivated ptsI gene, still possessed GAPN activity, as did S. salivarius ATCC 25975, which contains an insertion element between the ptsI and gapN genes. Since the wild-type S. mutans NG5 lacks both glucose-6-phosphate dehydrogenase and NADH:NADP oxidoreductase activities, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is important as a means of generating NADPH for biosynthetic reactions.

We report the sequencing of a 2,242-bp region of the Streptococcus mutants NG5 genome containing the genes for ptsH and ptsI, which encode HPr and enzyme I (EI), respectively, of the phosphoenolpyruvate-dependent phosphotransferase transport system. The sequence was obtained from two cloned overlapping genomic fragments; one expresses HPr and a truncated EI, while the other expresses a full-length EI in Escherichia coli, as determined by Western immunoblotting. The ptsI gene appeared to be expressed from a region located in the ptsH gene. The S. mutans NG5 pts operon does not appear to be linked to other phosphotransferase transport system proteins as has been found in other bacteria. A positive fermentation pattern on MacConkey-glucose plates by an E. coli ptsI mutant harboring the S. mutans NG5 ptsI gene on a plasmid indicated that the S. mutans NG5 EI can complement a defect in the E. coli gene. This was confirmed by protein phosphorylation experiments with 32P-labeled phosphoenolpyruvate indicating phosphotransfer from the S. mutans NG5 EI to the E. coli HPr. Two forms of the cloned EI, both truncated to varying degrees in the C-terminal region, were inefficiently phosphorylated and unable to complement fully the ptsI defect in the E. coli mutant. The deduced amino acid sequence of HPr shows a high degree of homology, particularly around the active site, to the same protein from other gram-positive bacteria, notably, S. salivarius, and to a lesser extent with those of gram-negative bacteria. The deduced amino acid sequence of S. mutans NG5 EI also shares several regions of homology with other sequenced EIs, notably, with the region around the active site, a region that contains the only conserved cystidyl residue among the various proteins and which may be involved in substrate binding.

Our previous continuous culture studies with strains of Streptococcus mutans have indicated that the organism has the capacity of adapt to growth in acidic environments. This study was undertaken to examine this question in more detail. S. mutans Ingbritt and the phosphotransferase system (PTS)-defective mutant, S. mutans DR0001/6, were grown in continuous culture at pH 7.5 and 5.5 or 5.1, and the pH optimum for glucose uptake and glycolysis and the capacity of the cells to generate pH gradients were determined over the pH range 4.5 to 8.0 with steady state, washed cells. In addition, the proton permeability of the cells was measured over the pH range by an acid pulse technique. The results indicate that the pH optimum for glucose uptake by S. mutans Ingbritt grown at pH 7.5 was 7.5 and this optimum shifted to 7.0 and 6.0 for cells grown at pH 5.5 and 5.1, whereas with the S. mutans DR0001/6, the optimum shifted from 7.5 to 7.0 for the pH 5.5 cells. A similar shift in the pH optimum for glycolysis was observed for the 2 strains, and this was particularly pronounced for cells incubated with glucose in the presence of gramicidin to dissipate proton gradients. The capacity of the cells to generate pH gradients was related to their metabolic activity, and although larger gradients were not formed by the pH 5.5 cells, these cells were nevertheless capable of maintaining gradients at a lower pH; S. mutans DR0001/6 generated 2-fold larger pH gradients at pH 5.5 than S. mutans Ingbritt.(ABSTRACT TRUNCATED AT 250 WORDS)

The growth of Streptococcus mutants Ingbritt in continuous culture at low pH or high growth rates repressed the biosynthesis of the components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The cellular concentrations of the soluble components HPr, enzyme I (EI), and EIII for mannose (IIIman) and EII activity for glucose, mannose, 2-deoxyglucose (2DG), and fructose were determined in membrane preparations from cells grown at pHs from 8.0 to 5.0 and at dilution (D) or growth rates from 0.1 to 1.0 h-1. The cellular levels of HPr and EI varied less than threefold under all of the growth conditions tested. On the other hand, EII activity in membranes from cells grown at D = 0.1 h-1 was repressed by growth at pHs below 8.0, with cells grown at pH 5.0 completely devoid of EII activity. In addition, cells grown at D = 0.5 and 1.0 h-1 exhibited little PTS activity for glucose, mannose, and 2DG and twofold-lower activity for fructose. These activities were stimulated by the addition of a membrane-free cytoplasmic fraction, and this activating activity was shown to be due to the presence of IIIman. Estimation of the cellular content of IIIman indicated that the synthesis of this factor was repressed by growth above and below pH 7.0 and was particularly sensitive to growth at high rates. These results indicate that with S. mutans Ingbritt, both pH and growth rate regulate the genes for the synthesis of EIIs involved in the phosphorylation of glucose, mannose, 2DG, and fructose and the gene for the formation of IIIman.

The components of the transmembrane electrochemical proton gradient, or proton motive force (PMF, delta p), were determined in cells of Streptococcus mutans Ingbritt and Streptococcus sobrinus ATCC 27352 growing in continuous culture under conditions of changing glucose concentration, growth rate and growth pH. The pH gradient (delta pH) and membrane electrical potential (delta psi) were assayed with the weak acid, salicyclic acid, and the lipophilic cation, methyltriphenylphosphonium iodide, respectively. S. mutans Ingbritt growing in continuous culture (pH 7.0, dilution rate (D) = 0.1 h-1) at 8 glucose concentrations ranging from 2.8 to 288 mM maintained a relatively constant delta p of 58.3 mV (SD +/- 5.8) in spite of a transition from glucose to nitrogen-limited growth and significant changes in cell physiology. Changes included a decreasing yield constant, increasing glucose uptake rates in the chemostat, repression of Ellglc of the PEP phosphotransferase sugar transport system and decreasing glycolytic capacity of the cells as the medium glucose concentration increased. Changes in the dilution or growth rate of S. mutans Ingbritt from 0.1 to 1.0 h-1 and S. sobrinus from 0.1 to 0.8 h-1, when growing at pH 7.0 with limited glucose and lactose, respectively, resulted in significantly lower delta p values due to the dissipation of the delta psi. When the cells of S. mutans Ingbritt were grown with excess glucose (nitrogen limitation), lower delta p values were observed at pH 5.5, but not at pH 7.0.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison was made of the properties of Streptococcus mutans Ingbritt grown in continuous culture under conditions of excess glucose (nitrogen limitation) and limiting glucose at mean generation times of 1.7 to 14 h. Only low levels of glucoamylase-specific glycogen were formed in cells from either culture, and the total carbohydrate content of the cells under excess glucose was only at most 1.6-fold higher than in the glucose-limited culture. A negligible amount of cell-free polysaccharide was formed in either culture, although a significant level of glucosyltransferase activity was observed in both, with the highest activity at D = 0.2 and 0.4 h(-1) with a glucose limitation. Other differences were observed. (i) Lactate was the main end product of the glucose-excess culture, whereas acetate, formate, and ethanol were the main products of the glucose-limited culture except at a mean generation time of 1.5, when lactate represented 30% of the products. (ii) The yield (in grams per mole of glucose) of the latter culture was 2.6- to 4.0- fold higher than the yield of the glucose-excess culture. (iii) Washed cells from the glucose-limited culture were much more acidogenic (1.7- to 6.2-fold) than the glucose-excess cells when incubated with glucose, sucrose, and fructose. Endogenous glycolytic activity by the latter cells was significant, being 31 to 92% of the exogenous glucose rate at the four dilution rates. (iv) Cells from the glucose-excess culture were more insensitive to fluoride than cells from the glucose-limited culture. The NaF 50% inhibition dose values for the effect of fluoride on the metabolism of glucose, sucrose, and fructose were calculated for the four dilution rates at four pH values. This analysis indicated that rapidly metabolizing cells were more sensitive to fluoride than cells that metabolized the sugars more slowly.

A study was undertaken to compare the effects of pH and fluoride on the growth and metabolic properties of Streptococcus mutans 2452 and Streptococcus mitior 572, strains recently isolated from 8-year-old school children and grown in continuous culture with a glucose limitation. Each experiment had four consecutive stages of growth: (i) pH 7.0, (ii) no pH control, (iii) pH 7.0, and (iv) no pH control plus 50 mug of fluoride per ml in the medium. At a dilution rate (D) of 0.13 h (-1), cells of S. mitior possessed high glycolytic activity at pH 7.0 in the initial stage, but were washing out of the chemostat within 24 h after the pH control was shut off and the pH fell to 5.1. Once the culture was reestablished at pH 7.0, fluoride (50 mug/ml) was added to the medium and the pH control was again turned off. Whereas cell numbers fell from 24.0 x 10(8) to 0.9 x 10(8)/ml within 24 h, the culture remained relatively constant during the following 6 days despite the fall in pH to 5.4. The cells from this culture also maintained an intermediate glycolytic rate of 0.44 mumol mg(-1) min(-1). The cells in this latter stage developed phenotypic resistant to fluoride at concentrations up to 16 mM. Growth of S. mitior at D = 0.034 h(-1) resulted in a slower response to environmental change such that cells were able to grow to pH values as low as 5.2 in the absence of fluoride. In contrast to S. mitior, S. mutans 2452 under the same conditions at D = 0.13 h(-1) grew to higher cell numbers and higher yields and was able to maintain significant cell numbers to pH 4.8 once the pH control was shut off in the presence and absence of fluoride. S. mutans had 40% less glycolytic activity but was fourfold more resistant to fluoride at the start of the experiment, and cells were shown to adapt to growth at low pH and to fluoride at levels as high as 20 mM. This fluoride resistance by freshly isolated S. mutans 2452 was significantly higher than that of S. mutans DR0001 grown under identical conditions in the chemostat. S. mutans DR0001 is a strain which has been subcultured in vitro for several years. This study demonstrated that S. mutans 2452 was more aciduric than S. mitior 572 and, unlike the latter organism, could grow at pH values below 5.1. The addition of fluoride to the medium stabilized the S. mitior culture in the absence of pH control, indicating that whereas fluoride does suppress growth and glycolytic activity it also results in higher environmental pH values, which permit the survival of the less aciduric bacteria.

Streptococcus mutans Ingbritt was grown anaerobically in a chemostat with a glucose limitation, as well as with an excess of glucose (amino acid limitation) at dilution rates (D) between 0.05 and 0.4 h(-1) (mean generation time = 12 to 1.5 h). The glucose-limited culture produced cells having 1.5- to 6.0-fold greater glycolytic activity than the cells from the glucose-excess culture. The preferred substrate for these cells was glucose, with the glycolytic rate for sucrose being only slightly lower; the rate for fructose was half that of glucose. The glycolytic rate of the glucose-limited cells was maximum at D = 0.1 h(-1), with a decline in rate as the growth rate approached D = 0.4 h(-1). A comparison of the activity of phosphoenolpyruvate phosphotransferase system (PTS) in the two types of cells showed that the glucose-limited cells had 1.7- to 5.6-fold greater PTS activity for the three sugars than the glucose-excess-grown cells. Whereas little difference was seen between the three sugars with the latter cells, the glucose-PTS had the greatest activity with glucose-limited cells, with the maximum in cells grown at D = 0.1 h(-1). Comparison of the rate of sugar uptake in the chemostat with the rate of PTS transport activity in the cells at each growth rate demonstrated that only under conditions of slow growth with a glucose limitation was the PTS system capable of supporting growth on glucose. Furthermore, PTS activity in cells grown with an excess of glucose was insignificant when compared with glucose uptake during growth in the chemostat. This evidence supports the observation that S. mutans possesses at least one other system, in addition to the PTS, for the transport of glucose into the cell. The organism was, however, devoid of glucose-proton symport transport activity.

Strains of Streptococcus mutans (biotype 1), Streptococcus sanguis, and Streptococcus mitior have been grown in mixed continuous culture in a semidefined medium under glucose limitation at a growth rate of D = 0.1 h-1. The effect of varying the environmental pH on the proportions of the different populations within the community has been determined. Initially the populations were allowed to reach steady state at pH 7.0 when S. sanguis was dominant with S. mutans and "S. mitior" maintaining similar populations. The medium pH was then lowered in steps of 0.5 pH units from pH 7.0 to 4.5, and the community was grown at each step for at least 15 generations. Viable counts of each species were made at 24-h intervals. The population ratios established at pH 7.0 remained relatively stable when the environmental pH was set at pH 6.5. However, after the medium pH was lowered to 6.0 (days 18-27), the population of S. mutans began to increase and the S. mitior population began to decline. A further change was seen at pH 5.5 (days 27-34) when S. mutans became dominant, S. sanguis declined, and S. mitior was not detectable. At pH 4.5, both S. mutans and S. sanguis were reduced in numbers, but survived until the experimental run was terminated (44 days). Samples of culture fluid were taken throughout the experiment and analyzed for the presence of the acid products of glucose metabolism. The amounts of lactic acid produced by the community increased as the environmental pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

Streptococcus mutans DR0001 and a glucose-phosphotransferase (PTS)-defective mutant, DR0001/6, were grown anaerobically in a chemostat with a glucose limitation at dilution rates (D) of 0.04 to 0.6 h(-1) (mean generation time, 17 to 1.2 h). The mutant possessed only 15% of glucose-PTS activity of the wild type and gave cell yields (19%) less than those of the wild type. Glucose-PTS activity in strains DR0001 was maximum at D = 0.1 h(-1) and was adequate to account for transport in the chemostat at all dilution rates except D = 0.6 h(-1), at which it was 80% of the actual glucose uptake activity. The mutant DR0001/6, on the other hand, possessed only sufficient glucose-PTS activity to sustain growth at below D = 0.1 h(-1), indicating the presence of an alternate transport activity. This was confirmed in glycolytic rate experiments with washed cells, which demonstrated that the mutant showed rates 11- to 27-fold higher than that accountable via glucose-PTS activity alone. The wild-type organism contained both a high (K(s), 6.7 to 8.0 muM)- and a low (K(s), 57 to 125 muM)-affinity transport system, whereas the glucose-PTS-defective mutant contained only the low-affinity system (K(s), 62 to 133 muM). The glucose-PTS was shown to be the high-affinity system. Glucose uptake by the mutant was unaffected by 8 mM sodium arsenate, 10 mM azide, and 10 mM dinitrophenol but was completely inhibited by 0.05 mM sodium iodoacetate. Glycolysis in the organism was almost completely inhibited by 0.25 mM N',N' -dicyclohexylcarbodiimide (DCCD), indicating the involvement of an ATPase in glucose uptake. The ionophores carbonylcyanide-m-chlorophenylhydrazone and tetrachlorosali-cylanilide were inhibitory at concentrations of 10 muM, suggesting that a proton gradient was important in the transport process. Higher levels of DCCD and the ionophores were required to inhibit the wild-type organism to the same degree. A mechanism is proposed for the alternative transport system whereby proton motive force is created by the extrusion of protons by the DCCD-sensitive ATPase and glucose is transported down a proton gradient in a symport with protons.

Polyvinyl chloride membrane electrodes sensitive to tetraphenyl phosphonium (TPP+), butyltriphenyl phosphonium ( bTPP +), and methyltriphenyl phosphonium ( mTPP +) ions have been compared for the determination of the electrical potential difference (delta psi) of the oral bacteria, Streptococcus mutans DR0001 /6 and Lactobacillus casei RB1014 . All three types of electrode proved suitable for determining delta psi, although the TPP+-sensitive electrode was particularly susceptible to interference by protonmotive force (delta p) dissipators known to inhibit sugar uptake by the bacteria. The mTPP +-sensitive electrode was the least affected. Similarly, both strains had a high nonspecific binding capacity for TPP+ and bTPP + ions, and this increased for all three ions when the bacteria were heated to 80 degrees C for 1 h to abolish glucose uptake and metabolism. This heat-treatment procedure is therefore not a suitable control for determination of nonspecific binding to cells. However, 1% (v/v) toluene, 20 microM gramicidin, or 10 microM valinomycin effectively depolarized the bacteria without interfering with nonspecific binding. The ionophores were therefore used subsequently for the determination of nonspecific binding of the lipid-soluble cations. The mTPP + ion and corresponding electrode proved the most effective system, and delta psi values of -89 and -107 mV were obtained for S. mutans and L. casei, respectively, harvested from glucose-limited continuous cultures and incubated in 100 mM Hepes-KOH buffer (pH 7.0), containing 1 mM dithiothreitol and 10 mM glucose. Although the delta psi of S. mutans decreased significantly in the presence of Mes-KOH and potassium phosphate buffers at pH 7.0, it increased to -119 mV in Tris-HCl buffer (pH 7.0).(ABSTRACT TRUNCATED AT 250 WORDS)

In natural oral ecosystems, succession of Streptococcus mutans and Lactobacillus species occurs in bacterial communities which can be associated with the development of carious lesions. The objective of this study was to explore the interaction between 2 strains of S. mutans (SF2452) and Lactobacillus casei (RB1014) when grown together in continuous culture under conditions of varying environmental pH and in the presence of fluoride. When the strains, isolated from approximal carious lesions, were grown initially at a dilution rate of 0.1 h-1 under a glucose limitation at pH 7.0, a stable mixed community developed (Days 5-10) with S. mutans as the dominant population. When the pH control was removed (Day 10), the binary culture lowered the pH to 5.1 causing a small reduction in the numbers of S. mutans and allowing L. casei to predominate. A more dramatic effect was seen when the environmental pH was rapidly lowered to 4.8 by the addition of acid. In this case, the S. mutans population was significantly reduced and the L. casei population rose to the level previously held by S. mutans. Re-adjustment of the pH to 7.0 resulted in a re-establishment of the population ratios prior to the addition of acid. The possibility that S. mutans was capable of aciduric adaptation during a biologically-generated pH reduction was examined by mixing cultures of both organisms after each had been grown to steady state at pH 5.5 in separate chemostats.

The addition of lactose, galactose, or isopropyl-beta-D-thiogalactoside (IPTG) to glucose-grown cells of Streptococcus salivarius 25975 resulted in the co-induction of both the lactose-P-enolpyruvate phosphotransferase system (lactose-PTS) and beta-galactosidase, with the latter the predominant metabolic system. With various strains of Streptococcus mutans and Streptococcus sanguis 10556, on the other hand, the lactose-PTS was the major metabolic pathway with beta-galactosidase induced either to low or negligible levels. In all cases, induction of the lactose-PTS resulted in the concomitant induction of 6-P-beta-galactosidase. The induction by lactose of both the lactose-PTS and beta-galactosidase in all strains was repressed by glucose and other catabolites, notably, fructose. Induction of beta-galactosidase in S. salivarius 25975 by IPTG was, however, relatively resistant to glucose repression. Induction experiments with IPTG and lactose suggested that a cellular metabolite of lactose metabolism was a repressor of enzyme activity. Exogenous cAMP was shown to reverse the transient repression by glucose of beta-galactosidase induction in cells of S. salivarius 25975 receiving lactose, provided the cells were grown with small amounts of toluene to overcome the permeability barrier to this nucleotide, cAMP, was however, unable to overcome the permanent repression of beta-galactosidase activity to a significant extent under these conditions.

Streptococcus mutans Ingbritt was grown anaerobically in a chemostat, at a rate (mean generation time, 13 h) similar to that in dental plaque, in a complex medium with excess glucose and at pH values of 6.5, 6.0, and 5.5. The yield of cells was constant at pH 6.5 and 6.0 (2.00 mg/ml) but fell to 1.25 at pH 5.5; Y(glucose) was relatively constant under all conditions. Lactic acid was the major end product. Amino acid analysis of the culture supernatants indicated that growth was probably limited by the availability of cysteine. Cells were harvested and monitored for their capacity to produce acid from endogenous polysaccharide and exogenous sugars in the presence and absence of NaF, as well as for their glucose phosphoenolpyruvate (PEP)-phosphotransferase activity. Surprisingly, cells grown at pH 5.5 possessed two to three times more glycolytic activity, as measured by the rate of acid production, than cells grown at pH 6.5 and 6.0 when incubated in a washed suspension at constant pH with a sugar source. Furthermore, the cells grown at pH 5.5 were about twice as resistant to the effect of NaF in reducing the rate of acid production in this system. Fluoride inhibition could be reversed by increasing the pH of the system. Cells grown at all three pH values showed significant acid production from endogenous reserves, despite the fact that the glucoamylase-specific glycogen content of the cells dropped from 33% of the total carbohydrate during pH 6.5 growth to only 3% after growth at pH 6.0 and 6.5. Incubation of washed cells for 18 h in phosphate buffer resulted in the loss of 62% of the total carbohydrate, indicating that nonglycogen cellular polysaccharide was metabolized. A comparison of the fluoride effect on endogenous and exogenous metabolism under pH fall conditions showed that, with pH 6.5- and 6.0-grown cells, the inhibitor was more effective in the presence of an exogenous carbon source than in its absence. This effect was not seen with pH 5.5-grown cells. The decreased sensitivity of the pH 5.5-grown cells to fluoride was probably associated with the decreased glucose PEP-phosphotransferase activity (11%) in these cells compared with the activity of those grown at pH 6.5. This evidence supports the hypothesis that S. mutans possesses at least two glucose transport systems, one of which is relatively fluoride insensitive.

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway.

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-1) at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme IIGlc (EIIGlc) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGlc increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.

Growth of Streptococcus mutans Ingbritt on limiting sucrose in a chemostat at dilution rates of 0.05 to 0.4 h-1 (mean generation time, 14 to 1.7 h) resulted in a heterofermentative pattern of metabolic end products. During fast growth, lactic acid was the major end product, whereas at slower growth rates, acetic and formic acids, as well as ethanol, increased to be major end products. The patterns obtained were similar to those seen with the same organism growing on glucose. The glycolytic rate by washed cells was maximum at the lowest dilution rates and decreased as the cells were made to grow faster. Transport of sucrose, glucose, and fructose via the phosphoenolpyruvate phosphotransferase system (PTS) was repressed during growth on sucrose after growth on glucose. Uptake rates suggested that sucrose was transported in the PTS as the intact disaccharide. Comparison of the rate of sugar uptake in the chemostat with the rate of PTS activity in the cells at each growth rate indicated that the PTS was capable of supporting growth only at a dilution rate of 0.05 h-1. Growth on sucrose at faster growth rates required the activity of a second transport system, supporting our earlier observations with glucose that S. mutans contains at least two sugar transport systems.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.

Our previous continuous culture studies with strains of Streptococcus mutans have indicated that the organism has the capacity of adapt to growth in acidic environments. This study was undertaken to examine this question in more detail. S. mutans Ingbritt and the phosphotransferase system (PTS)-defective mutant, S. mutans DR0001/6, were grown in continuous culture at pH 7.5 and 5.5 or 5.1, and the pH optimum for glucose uptake and glycolysis and the capacity of the cells to generate pH gradients were determined over the pH range 4.5 to 8.0 with steady state, washed cells. In addition, the proton permeability of the cells was measured over the pH range by an acid pulse technique. The results indicate that the pH optimum for glucose uptake by S. mutans Ingbritt grown at pH 7.5 was 7.5 and this optimum shifted to 7.0 and 6.0 for cells grown at pH 5.5 and 5.1, whereas with the S. mutans DR0001/6, the optimum shifted from 7.5 to 7.0 for the pH 5.5 cells. A similar shift in the pH optimum for glycolysis was observed for the 2 strains, and this was particularly pronounced for cells incubated with glucose in the presence of gramicidin to dissipate proton gradients. The capacity of the cells to generate pH gradients was related to their metabolic activity, and although larger gradients were not formed by the pH 5.5 cells, these cells were nevertheless capable of maintaining gradients at a lower pH; S. mutans DR0001/ 6 generated 2-fold larger pH gradients at pH 5.5 than S. mutans Ingbritt. In addition, activity for the membrane-associated proton-extruding ATPase in S. mutans Ingbritt increased 4-fold as the pH of growth was decreased from 8.0 to 5.0. The most striking difference between cells grown at pH 7.5 and 5.5 was observed in the proton permeability profiles for the 2 organisms. The pH for minimum proton permeability for the pH 7.5-grown cells of S. mutans Ingbritt was 6.4, whereas that for pH 5.5 cells was 5.0; he corresponding values for S. mutans DR0001/6 were pH 5.6 and 4.6 for the pH 7.5 and 5.5 cells, respectively. This indicates that the cells compensate for the lower external pH by decreasing the permeability of protons into the cell at the lower pH values. Collectively, these results indicate that prolonged growth of S. mutans in an acidic environment, Such as might be found in fissures or carious lesions, results in significant changes in cell physiology that confer increased acidurance. [ABSTRACT FROM AUTHOR]

The components of the transmembrane electrochemical proton gradient, or proton motive force (PMF, Δp), were determined in cells of Streptococcus mutans Ingbritt and Streptococcus sobrinus ATCC 27352 growing in continuous culture under conditions of changing glucose concentration, growth rate and growth pH. The pH gradient (ΔpH) and membrane electrical potential (Δψ) were assayed with the weak acid, salicylic acid, and the lipophilic cation, methyltriphenylphosphonium iodide, respectively. S. mutans Ingbritt growing in continuous culture (pH 7.0, dilution rate (D) = 0.1 h-1) at 8 glucose concentrations ranging from 2.8 to 288 mM maintained a relatively constant Δp of 58.3 mV (SD ± 5.8) in spite of a transition from glucose to nitrogen-limited growth and significant changes in cell physiology. Changes included a decreasing yield constant, increasing glucose uptake rates in the chemostat, repression of Ellglc of the PEP phosphotransferase sugar transport system and decreasing glycolytic capacity of the cells as the medium glucose concentration increased. Changes in the dilution or growth rate of S. mutans Ingbritt from 0.1 to 1.0 h-1 and S. sobrinus from 0.1 to 0.8 h-1, when growing at pH 7.0 with limited glucose and lactose, respectively, resulted in significantly lower Δp values due to the dissipation of the Δψ. When the cells of S. mutans Ingbritt were grown with excess glucose (nitrogen limitation), lower Δp values were observed at pH 5.5, but not at pH 7.0. These results indicate that the generation of proton motive force is maintained by s. mutans and S. sobrinus during growth in continuous culture, except when the organisms are stressed at high growth rates and with excess glucose at low pH. Furthermore, the data indicate that, unlike other bacteria, these oral streptococci do not maintain significant pH gradients (i.e.<0.74) even when growing in continous culture at pH values as low as 5.0. [ABSTRACT FROM AUTHOR]

In natural oral ecosystems, succession of Streptococcus mutans and Lactobacillus species occurs in bacterial communities which can be associated with the development of carious lesions. The objective of this study was to explore the interaction between 2 strains of S. mutans (SF2452) and Lactobacillus casei (RB1014) when grown together in continuous culture under conditions of varying environmental pH and in the presence of fluoride. When the strains, isolated from approximal carious lesions, were grown initially at a dilution rate of 0.1 h-1 under a glucose limitation at pH 7.0, a stable mixed community developed (Days 5-10) with S. mutans as the dominant population. When the pH control was removed (Day 10), the binary culture lowered the pH to 5.1 causing a small reduction in the numbers of S. mutans and allowing L. casei to predominate. A more dramatic effect was seen when the environmental pH was rapidly lowered to 4.8 by the addition of acid. In this case, the S. mutans population was significantly reduced and the L. casei population rose to the level previously held by S. mutans. Re-adjustment of the pH to 7.0 resulted in a re-establishment of the population ratios prior to the addition of acid. The possibility that S. mutans was capable of aciduric adaptation during a biologically-generated pH reduction was examined by mixing cultures of both organisms after each had been grown to steady state at pH 5.5 in separate chemostats. In this case S. mutans maintained its population and was dominant in the community at pH 5.5 indicating that adaptation by S. mutans had given it an ecological advantage over L. casei at this pH. The addition of fluoride (20 mM) to the mixed culture favored L. casei since the growth of S. mutans was greatly inhibited at this fluoride level. Nevertheless S. mutans was able to survive at low levels in the chemostat, presumably in surface films, for at least 5 days, since removal of fluoride from the medium resulted in the re-establishment of S. mutans in a competitive position with L. casei. These experiments indicate that S. mutans possesses adaptive and survival characteristics which would be of considerable advantage in an ecological system such as dental plaque. [ABSTRACT FROM AUTHOR]

Experiments were conducted with Streptococcus mutans NCTC 10119 and Streptococcus sanguis ATCC 10556 to determine whether the acid end-products, lactate and acetate, were involved in the regulation of cellular growth and metabolism. The growth rate and culture biomass of both organisms was inhibited by the addition of lactate and acetate at concentrations as high as 200 mM to the cultures, although the final pH values of the lactate and acetate cultures were similar. In addition, the metabolic conversion of glucose to lactate was decreased by external lactate but stimulated by acetate. In spite of this, calculation of the yield of cell biomass per mole of ATP (YATP) showed that the yield of both organisms actually increased in the presence of added lactate, but decreased with acetate. This indicates that the two acids interacted with the cells of the organisms by different mechanisms. For both organisms, the final external undissociated lactic acid was relatively constant at concentrations between 0 and 200 mM added lactate, 24.9-32.5 mM for S. mutans and 8.0-11.5 mM for S. sanguis. On the other hand, the final concentration of undissociated acetic acid in the S. mutans cultures increased from 2.9 to 83.7 mM as the medium acetate concentration increased, and from 1.0 to 36.0 mM with the S. sanguis cultures. Counterflow experiments provided evidence for a lactate carrier in both S. mutans and S. sanguis, but an acetate carrier in these organisms could not be demonstrated. [14C]-lactate and [14C]-acetate were taken up into de-energized, chemostat-grown cells of S. mutans and S. sanguis in response to an artificially generated pH gradient but not by an imposed electrical gradient. Thus, under these conditions lactate uptake occurred via a symport process with only one proton. [ABSTRACT FROM AUTHOR]