Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Malolactic fermentation by Streptococcus mutans

Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation of L-malate to yield L-lactic acid and concomitant reduction in acidity. The activity was inducible by L-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add standardized HCl solution to maintain a set pH value in pH-stat experiments. The net conclusion is that malate has the potential to be effective for alkalinization of dental plaque, although the fermentation is sensitive to fluoride and triclosan, which are commonly added to oral care products.     

Transport and metabolism of citrate by Streptococcus mutans

Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Two virulence determinants of S. mutans are its acidogenicity and aciduricity (the ability to produce acid and the ability to survive and grow at low pH, respectively). Citric acid is ubiquitous in nature; it is a component of fruit juices, bones, and teeth. In lactic acid bacteria citrate transport has been linked to increased survival in acidic conditions. We identified putative citrate transport and metabolism genes in S. mutans, which led us to investigate citrate transport and metabolism. Our goals in this study were to determine the mechanisms of citrate transport and metabolism in S. mutans and to examine whether citrate modulates S. mutans aciduricity. Radiolabeled citrate was used during citrate transport to identify citrate metal ion cofactors, and thin-layer chromatography was used to identify metabolic end products of citrate metabolism. S. mutans was grown in medium MM4 with different citrate concentrations and pH values, and the effects on the growth rate and cell survival were monitored. Intracellular citrate inhibited the growth of the bacteria, especially at low pH. The most effective cofactor for citrate uptake by S. mutans was Fe(3+). The metabolic end product of citrate metabolism was aspartate, and a citrate transporter mutant was more citrate tolerant than the parent.     

Cloning,Nucleotide Sequence,and Disruption of Streptococcus mutans Glutathione Reductase Gene (gor)

We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Clustered Genes for Galactose Metabolism from Streptococcus mutans Cloned in Escherichia coli

DNA cloned into Escherichia coli from a serotype c strain of Streptococcus mutans allowed a galKTE mutant to utilize galactose for growth. However, the DNA does not appear to encode enzymes of the Leloir pathway used by E. coli, but rather appears to encode enzymes of the tagatose phosphate pathway.     

Production of mutacin-like substances by Streptococcus mutans

Production of inhibitory substances by strains of the Streptococcus mutans group is well documented, but the nature of the substances implied is often unknown. Of nine laboratory strains known to produce inhibitory substances, the optimal conditions for producing inhibition zones on solid media were found to vary between strains but good production was generally obtained on all-purpose media with Tween 80 at 37 degrees C after 2-4 days of aerobic incubation. Streptococcus sanguis Ny101 was found to be more sensitive than Streptococcus rattus LG-1 to all inhibitory substances produced by the S. mutans strains tested. While all strains showed some inhibition, only six showed inhibition after neutralization; arginine incorporated in agar at 0.75% completely eliminated all inhibition zones. However 1% arginine in the overlays did not affect the production of inhibition zones by strains of S. mutans C67-1, Ny257, Ny266, and T8. These strains were shown to elaborate (in a reproducible fashion) inhibitory substances which were not organic acids. Inhibitory activity was never obtained in liquid preparations, except for strains Ny257 and T8 where it was found to be very unstable.     

Disulfide reduction and sulfhydryl uptake by Streptococcus mutans

Incubation of Streptococcus mutans cells with certain disulfide compounds resulted in accumulation of reduced sulfhydryl compounds in the extracellular medium or in both the medium and the cells. Oxidized lipoic acid and lipoamide competed for reduction. At high concentrations, these compounds were reduced at rates comparable to that of glucose metabolism, and all of the increase in sulfhydryls was in the medium. Cystamine did not compete with these compounds for reduction but was also reduced at high rates and low apparent affinity, and all of the cysteamine produced from cystamine accumulated in the medium. In contrast, glutathione disulfide (GSSG) and L-cystine were reduced slowly but with high apparent affinity, and 60 to 80% of the increase in sulfhydryls was intracellular. NADH-dependent lipoic acid or lipoamide reductase activity was present in the particulate (wall-plus-membrane) fraction, whereas NADPH-dependent GSSG reductase activity was present in the soluble (cytoplasmic) fraction. Two transport systems for disulfide and sulfhydryl compounds were distinguished. GSSG, L-cystine, and reduced glutathione competed for uptake. L-Cysteine was taken up by a separate system that also accepted L-penicillamine and D-cysteine as substrates. Uptake of glutathione or L-cysteine, or the uptake and reduction of GSSG or L-cystine, resulted in up to a 10-fold increase in cell sulfhydryl content that raised intracellular concentrations to between 30 and 40 mM. These reductase and transport systems enable S. mutans cells to create a reducing environment in both the extracellular medium and the cytoplasm.     

Inactivation of D-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation

Cell-free D-glucosyltransferase of D-glucose-grown Streptococcus mutans AHT was completely inactivated in the presence of 0.002% of Methylene Blue at 25 degrees and pH 7.0 after illumination with a 150-W incandescent lamp. The rate of inactivation was decreased at pH values less than 7.0. Histidine was the only amino acid residue modified to a significant extent, and the rates of oxidation of histidine residues and loss of enzyme activity closely agreed. Production of both water-insoluble and -soluble D-glucan fractions from sucrose by the oxidized D-glucosyltransferase preparations was significantly inhibited. Photooxidation with 0.002% of Rose Bengal at pH 7.0 or higher also induced complete inactivation of the D-glucosyltransferase. These results strongly suggest that the imidazole portion of histidine may function as part of the active sites of both D-glucosyltransferase isozymes of S. mutans AHT, which are responsible for the synthesis of (1 goes to 3)- and (1 goes to 6)-alpha-D-glucosidic linkages. The D-glucosyltransferases from S. mutans 6715 and AHT-mutant M1, and Streptococcus sanguis ATCC 10558 were also almost completely inactivated by Methylene Blue-sensitized photooxidation.     

Streptococcus mutans murein hydrolase

Allelic replacement of the C terminus of a Streptococcus mutans surface protein affects murein hydrolase activity. The targeted open reading frame encodes a 67-kDa protein (SmaA) with an N-terminal signal sequence and cleavage site, three 46-amino-acid (aa) direct repeats, and two 88-aa direct repeats. The identical autolytic profile was obtained using a sortase mutant (SrtA(-)).     

Functions of Two Types of NADH Oxidases in Energy Metabolism and Oxidative Stress of Streptococcus mutans

We have previously identified two distinct NADH oxidases corresponding to H(2)O(2)-forming oxidase (Nox-1) and H(2)O-forming oxidase (Nox-2) induced in Streptococcus mutans. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component of Salmonella typhimurium alkyl hydroperoxide reductase; an open reading frame upstream of nox-1 also showed homology to AhpC, the direct peroxide-reducing component of S. typhimurium alkyl hydroperoxide reductase. To determine their physiological functions in S. mutans, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD(+) but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of Escherichia coli TA4315 (ahpCF-defective mutant) transformed with pAN119 containing both nox-1 and ahpC genes was not only restored but enhanced relative to that of E. coli K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of S. mutans to cumene hydroperoxide and H(2)O(2), implying that the existence of some other antioxidant system(s) independent of Nox-1 in S. mutans compensates for the deficiency.     

Sucrose selectively regulates Streptococcus mutans polysaccharide by GcrR

Streptococcus mutans (S. mutans) is the principal etiological agent in cariogenesis because of its ability to metabolize sucrose into extracellular polysaccharides (EPS). The response regulators GcrR and VicR could regulate EPS metabolism, but with opposing regulatory functions. However, the cooperative interactions between gcrR and vicR regulating sucrose-selective EPS metabolism have not been fully elucidated. First, we constructed several dual-mutant strains (vicR + gcrR+, vicR and gcrR overexpression; vicR + gcrR-, vicR overexpression and gcrR deficient; ASvicRgcrR+, vicR low-expression and gcrR overexpression; ASvicRgcrR−, vicR low-expression and gcrR deficient) to clarify gtfB/gtfC expression levels were modulated by gcrR regardless of the vicR gene expression levels. Next, we found gcrR deletion mutant (SmugcrR) displayed obvious auto-aggregation and bacterial cells were densely packed in enriched EPS induced by sucrose. In the contrast, SmugcrR biofilm showed very little carbohydrate-dependent aggregation in the absence of sucrose. The presence of sucrose amplifies the negative regulation of gcrR acting as a ‘switch-off’. After sucrose induction, dexA gene expression was significantly enhanced in gcrR overexpression mutant (SmugcrR+). Furthermore, GcrR was shown to directly bind to the promoter region of the dexA gene. Taken together, our results reveal that GcrR interacts with VicR to block EPS biosynthesis via polysaccharide digestion by DexA, and that this process is induced in a sucrose-selective manner. Hence, targeting GcrR is a potential strategy for the management of dental caries.     

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.