Interaction of glucosyltransferase from Streptococcus mutans with various glucans

Cell-free glucosyltransferase of Streptococcus mutans strain B13 (serotype d) exclusively synthesized water-insoluble glucan from sucrose. The insoluble glucan possessed strong glucan-associated glucosyltransferase activity even after extensive washing and lyophilization. Furthermore, cell-free glucosyltransferase became bound to heat-treated water-insoluble glucan or to heat-treated S. mutans B13 cells grown in Todd Hewitt broth, and the resulting glucan and cells adhered to a glass surface in the presence of exogenous sucrose. No other water-insoluble glucans bound significant quantities of glucosyltransferase. Glucan synthesis by free or glucan-bound glucosyltransferase was stimulated by low concentrations (1 to 5 mg ml-1) of isomaltose or water-soluble dextrans of various molecular weights, but higher concentrations (10 mg ml-1) inhibited glucan synthesis. The glucan synthesized in the presence of primer dextrans exhibited a reduced ability to adhere to a glass surface. Certain sugars such as maltose and fructose significantly lowered the yield of insoluble glucans. Preincubation of glucosyltransferase with the low molecular weight dextran T10 increased subsequent binding to S. mutans B13 insoluble glucan, whereas preincubation with higher molecular weight dextrans significantly inhibited the glucosyltransferase binding.     

Insoluble glucan synthesis by mutansucrase as a determinant of the cariogenicity of Streptococcus mutans

Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67-1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67-25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.     

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.     

The stimulation of macrophage prostaglandin E2 and thromboxane B2 secretion by Streptococcus mutans insoluble glucans

The effect of insoluble glucan synthesized by Streptococcus mutans on [3H]arachidonate metabolites secretion from peritoneal macrophages was studied. Insoluble glucans stimulated [3H]arachidonate release and secretion of prostaglandin E2 and thromboxane B2 from macrophages. In contrast, commercial soluble glucan (dextran) did not induce [3H]arachidonate release.     

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.     

Free arachidonic acid source for PGE2 and TXB2 production in guinea pig peritoneal macrophages exposed to insoluble glucan from Streptococcus mutans

1. Macrophages are an important source of the lipid mediators arachidonic acid (AA) and its metabolites that are produced during inflammation. 2. Previously, we reported that insoluble glucans from Streptococcus mutans in dental plaque could induce macrophages to secrete PGE2 and TXB2. 3. Studies were undertaken to identify the phospholipid substrates that can serve as a source of AA in macrophages exposed to the insoluble glucan. 4. When macrophage cell prelabelled with [3H]AA, stimulation with insoluble glucan resulted in a loss of label mainly from phosphatidylcholine (PC) and phosphatidylinositol (PI). 5. In addition, the PC-, and PI-specific phospholipase A2-mediated mechanisms for AA release may be activated in guinea peritoneal macrophages exposed to the insoluble glucan from S. mutans.     

Effect of dextran and ammonium sulphate on the reaction catalysed by a glucosyltransferase complex from Streptococcus mutans

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of water-soluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1.55 M-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1.55 M-(MH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1.55 M-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranse, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1.55 M-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1 leads to 3)-alpha bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1 leads to 6)-alpha bond formation.     

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (+/-8) days, and an average of 2.7 x 10(5) CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.     

Effect of oleanolic acid-cyclodextrin inclusion compounds on dental caries by in vitro experiment and rat-caries model.

Earlier work in vitro showed that oleanolic acid (OA) was a potential inhibitor of insoluble glucan (ISG) synthesis from mutans streptococci (MS). In this study, two oleanolic acid-cyclodextrin inclusion compounds (OA-CDs), oleanolic acid-G1-beta-cyclodextrin (OA-G1-beta CD) and oleanolic acid-beta-cyclodextrin (OA-beta CD), were assayed for their effects on ISG synthesis from Streptococcus mutans MT8148R, and on the growth of oral bacteria. OA-beta CD inhibited ISG synthesis by 55.3 and 37.4% at 62.5 and 15.6 micrograms/ml of OA, respectively. Both OA-CDs inhibited the growth of MS, S. sanguis, and S. salivarius at 4 to 8 micrograms/ml of OA. The anticariogenic effect of the OA-beta CD was examined in a rat-caries model. Rats in the infected control groups showed the highest caries score. The infected treatment group B (0.5% OA in diet) showed lower scores than the control group. These results suggest that OA-beta CD is a potential anti-caries agent.     

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.     

Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).     

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.     

Biofilm formation by exopolysaccharide mutants of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Effects of Xylitol on Xylitol-Sensitive Versus Xylitol-Resistant Streptococcus mutans Strains in a Three-Species in Vitro Biofilm

We studied the effects of xylitol on biofilms containing xylitol-resistant (Xr) and xylitol-sensitive (Xs) Streptococcus mutans, Actinomyces naeslundii and S. sanguinis. The biofilms were grown for 8 and 24 h on hydroxyapatite discs. The viable microorganisms were determined by plate culturing techniques and fluorescence in situ hybridization (FISH) was performed using a S. mutans-specific probe. Extracellular cell-bound polysaccharides (EPS) were determined by spectrofluorometry from single-species S. mutans biofilms. In the presence of 5 % xylitol, the counts of the Xs S. mutans decreased tenfold in the young (8 h) biofilm (p < 0.05) but no effect was seen in the mature (24 h) biofilm. No decrease was observed for the Xr strains, and FISH confirmed these results. No differences were detected in the EPS production of the Xs S. mutans grown with or without xylitol, nor between Xr and Xs S. mutans strains. Thus, it seems that xylitol did not affect the EPS synthesis of the S. mutans strains. Since the Xr S. mutans strains, not inhibited by xylitol, showed no xylitol-induced decrease in the biofilms, we conclude that growth inhibition could be responsible for the decrease of the counts of the Xs S. mutans strains in the clinically relevant young biofilms.     

Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme

The nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.     

Metabolism of the polysaccharides of human dental plaque. Part II. Purification and properties of Cladosporium resinae (1 leads to 3)-alpha-D-glucanase, and the enzymic hydrolysis of glucans synthesised by extracellular D-glucosyltransferases of oral streptococci.

Cladosporium resinae (1 leads to 3)-alpha-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1 leads to 3)-alpha-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1 leads to 3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1 leads to 3)-alpha-D-glucosidic linkages that occur in mycodextran, isolichein, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucan synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1 leads to 6)-alpha-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1 leads to 3)-alpha-D-glucanase to determine the relative proportions of (1 leads to 6)-linked to (1 leads to 3)-linked sequences of D-glucose residues in the insoluble glucans produce by oral streptococci. The simultaneous action of the two D-glucanoses was highly effective in solubilizing the glucans.     

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei.

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.     

Serotype-dependent inhibition of glucan synthesis and cell adherence of Streptococcus mutans by antibody against glucosyltransferase of serotype e S. mutans.

A crude glucosyltransferase (GTase) preparation was obtained from the culture supernatant of Streptococcus mutans strain MT703 (serotype e) by 50% ammonium sulphate precipitation. Antiserum specific against the GTase was prepared by immunizing rabbits intramuscularly with the GTase in Freund incomplete adjuvant, followed by GTase without adjuvant intravenously. Gamma globulin fractions of the antiserum and normal serum were partially purified by 1/3 saturated ammonium sulphate precipitation. The antibody strongly inhibited the GTase activity (greater than 90%) of type c, e and f S. mutans, whereas the GTase of type a, d and g was not affected by the antibody. The GTase from type b S. mutans was slightly inhibited. The adherence of viable cells of type c, e, and f S. mutans to a glass surface due to synthesis of glucan by the cell-associated GTase was also significantly inhibited by the antibody to the enzyme. These results suggest that type c, e, and f and types a, d, and g S. mutans can be separated into two major groups in terms of the immunological relationship of GTase.     

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.