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J Bacteriol. Oct 2009; 191(20): 6415–6424.
Published online Aug 14, 2009. doi:  10.1128/JB.00793-09
PMCID: PMC2753040

Inactivation of VicK Affects Acid Production and Acid Survival of Streptococcus mutans [down-pointing small open triangle]

Abstract

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

In many bacteria, the ability to survive in the presence of a lethal acidic pH is enhanced if the cells are first exposed to sublethal acidic conditions for a period of time prior to a subsequent acid shock (15-17, 24, 33, 40, 51). When cells are stressed with a relatively milder acidic pH, the physiology of the cells undergoes an adaptive process, known as the acid tolerance response (ATR), which is a process that facilitates enhanced production of various proteins (e.g., acid pumps, chaperones, DNA repair and membrane proteins) required to efficiently counteract acid damage and maintain viability during exposure to low pH (9, 15, 17, 41, 51). The molecular basis of the ATR has been studied extensively in Escherichia coli (44), Salmonella enterica serovar Typhimurium (16, 18), and oral streptococcal species (51).

In Streptococcus mutans, a primary etiological agent of dental caries, ATR-mediated acid adaptation has been considered a key survival mechanism used to combat acid stress (9, 24, 51, 55). S. mutans is able to rapidly metabolize dietary sugars, producing lactic acid as a metabolic end product, which can lower the plaque pH and erode the tooth enamel, thereby initiating cavity formation. Hence, to circumvent the detrimental effects of acidic pH and to facilitate proper enzyme functions (e.g., functions of glycolytic enzymes required for ATP production), defense mechanisms that counter the negative effects of intracellular acid stress must be activated. Studies have shown that if S. mutans cultures are preexposed to an adaptive acidic environment (approximately pH 5.0 to 5.5), the survival rates of the bacteria are dramatically enhanced at sublethal pH values (approximately pH 3.5) (24, 25, 41, 52). Several proteins important in acid tolerance have been characterized and are considered essential components of the ATR in S. mutans; at least 30 proteins have been shown to be upregulated during acid stress (51). Although previously bacterial survival under acid stress conditions was attributed mainly to the function of F1F0-ATPase proton pumps, it has become apparent that a combination of defense mechanisms is required to contend with acid stress. These mechanisms include constitutive and inducible strategies that facilitate expulsion of protons (H+), alkalization of the extracellular environment, alteration of the cell envelope composition, and production of general shock proteins, transcriptional factors, and factors involved in DNA and protein repair (9). The ability of pathogenic bacteria such as S. mutans to use ATR systems to withstand highly acidic environments has enormous implications for the ability of these organisms to survive, persist, and cause disease.

A fundamental step in responding to an environmental insult is to first detect the threat so that necessary adaptive measures can be initiated. Bacteria can detect transient environments, such as fluctuations in pH, temperature, nutrient levels, etc., using two-component signal transduction systems (TCSTSs) comprised of a histidine kinase located in the membrane and its cognate intracellular response regulator (49, 57, 58). When a change in the environment is sensed, the histidine kinase undergoes autophosphorylation at a conserved histidine residue; subsequent transfer of the phosphate to the cognate response regulator transmits the message into the cell. Phosphorylation-induced “activation” of this responder protein can affect its binding affinity for DNA, which then directs binding of the RNA polymerases at various promoter sites of genes, thereby regulating transcription of genes under control of these enzymes (50). There are genes encoding 14 TCSTSs in the S. mutans genome (3), and one of our goals in this study was to understand the contribution of the VicK sensor kinase, which is part of the VicK/VicR signal transduction system, to the ATR of this bacterium. Previously, we reported that the VicR/VicK system was important for growth, adhesion, biofilm formation, oxidative stress tolerance, and development of genetic competence in S. mutans (45, 46). Loss of VicK resulted in a clumping phenotype in broth culture, which in combination with the defective growth observed under normal unstressed conditions made it difficult to reach conclusions based on growth kinetics under acid stress conditions. However, initial growth analyses suggested that the VicK mutant had higher growth rates under low-pH conditions than the wild-type strain (unpublished results). Also, interestingly, VicK was previously shown to have a positive regulatory role in gbpB expression (45). S. mutans GbpB shares extensive homology with putative peptidoglycan hydrolases from Streptococcus agalactiae and Streptococcus pneumoniae (36, 37, 42, 43) and has demonstrated roles in cell wall homeostasis and acid stress tolerance in S. mutans (23, 32).

In the present study, we investigated the contribution of VicK to acidogenicity and aciduricity and to elucidate the VicK transcriptome profile under low-pH conditions by assessing a Vick-deficient mutant relative to its parent strain. In this report, we present evidence that deletion of VicK severely compromised acid production by S. mutans, whereas the acid survival of VicK-deficient mutants was significantly enhanced compared to the acid survival of the wild type. We also present the global transcriptome profiles of S. mutans UA159 and a VicK-deficient mutant of this strain for low-pH conditions, and the results of this study not only provide insight into how S. mutans can adapt to low pH but also expand the role of VicK as a putative pH sensor that is important for the ATR of this bacterium.

MATERIALS AND METHODS

Strains and growth media.

S. mutans wild-type strain UA159 (provided by J. Ferretti, University of Oklahoma) and a VicK-deficient mutant derivative (SmuvicK) (45) were used in this study. All S. mutans strains were routinely grown in Todd-Hewitt yeast extract (THYE) (Becton Dickinson, Sparks, MD) broth as standing cultures or on THYE medium solidified with 1.5% (wt/vol) agar (Bioshop, Burlington, Ontario, Canada) at 37°C with 5% (vol/vol) CO2 in air. Tryptone yeast extract medium (TYE) (10% tryptone, 5% yeast extract, 17.2 mM K2HPO4) was utilized for ATR assays (tryptone was obtained from Bioshop, Burlington, Ontario, Canada). For glycolysis experiments and ATR assays, NaOH or HCl was added to THYE medium or TYE to adjust final the pH to pH 7.5 or pH 5.5. To grow mutant strains, erythromycin was added to a final concentration of 10 μg/ml when necessary.

ATR assay.

Overnight cultures grown in THYE medium were diluted 1:20 using sterile prewarmed TYE at pH 7.5 supplemented with 1% (wt/vol) glucose. Cultures were then grown until mid-logarithmic phase (optical density at 600 nm [OD600], ~0.4), equally divided into two aliquots, and pelleted by centrifugation. To facilitate adaptation to low pH, one aliquot was first resuspended in TYE at pH 5.5 for 2 h. The resulting “adapted” cells were then exposed to a “killing” pH, pH 3.5. The second pelleted aliquot of each culture was used to assay the survival of “unadapted” cells by directly resuspending them in TYE at a lethal pH, pH 3.5. Following incubation at 37°C, cell fractions were removed from these “adapted” and “unadapted” cultures every hour starting at time zero until 3 h, gently sonicated, and serially diluted in 10 mM potassium phosphate buffer (pH 7.2). Each dilution was then spotted in triplicate (20 μl each) onto THYE agar plates and incubated at 37°C for 2 days, and the CFU were counted. For “adapted” and “unadapted” cells of each strain, the ATR was calculated by dividing the number of CFU obtained at pH 3.5 (at 1 h, 2 h, or 3 h) by the number of CFU present at time zero and multiplying the result by 100.

Growth kinetics.

S. mutans strains were grown overnight in THYE medium, diluted 1:20 in fresh sterile THYE medium, and grown until the OD600 was approximately 0.3 to 0.4. Growth kinetics experiments were performed as previously described using an automated growth monitor (45). Briefly, the growth of each strain was monitored in quadruplicate under the following stress conditions: pH 5.5, 0.4 M NaCl, 0.003% H2O2, and 25 mM paraquat. Uninoculated wells and wells containing THYE medium alone were used as controls, and pH-buffered THYE medium adjusted to pH 7.0 was used as a control in acid stress experiments. No antibiotics were used in growth assays to avoid additional stress. Following incubation, absorbance measurements were exported and graphed over time to obtain growth curves.

Temporal pH measurement.

Overnight cultures of S. mutans strains UA159 and SmuvicK were diluted 1:20 using sterile prewarmed THYE medium and incubated at 37°C. During incubation, terminal pH measurements were obtained for aliquots of each culture from time zero to 8 h using a benchtop pH meter (SympHony model SB20; VWR International). Two independent experiments were carried out, and results were obtained in triplicate.

Glycolytic rate measurement.

To harvest cells for glycolytic rate measurement, overnight cultures were diluted 1:10 using sterile prewarmed THYE medium and incubated at 37°C until the OD600 was ~0.4 to 0.5. The cultures were then pelleted by centrifugation, and cells were washed three times in 10 ml of cold suspension solution (1% KCl, 1% peptone) adjusted to either pH 5.5 or pH 7.5. Following the final centrifugation, cells were resuspended in suspension solution at the relevant pH to obtain a final OD600 of ~1.0. Subsequently, 18-ml aliquots of each cell suspension were equilibrated in a reaction vessel at 37°C until the residual glycolytic activity diminished. To initiate glycolysis, 2 M glucose was added at a final concentration of 200 mM, and glycolysis was monitored utilizing a Radiometer ABU901 autoburette and a PHM290 pH controller (Radiometer, Denmark), which recorded the rate of addition of potassium hydroxide (10 mM KOH at pH 7.0 and 2 mM KOH at pH 5.0) required to keep the pH at a constant value. The glycolytic rate was expressed in micromoles of acid neutralized per milligram (dry weight) of cells per minute. For controls sterile water was added to the cell suspension instead of glucose. The average dry weight per milliliter of cell suspension was determined by drying cells on preweighed filters overnight in a 60°C oven. Results were obtained in triplicate using cells derived from three independent experiments. A statistical analysis was performed using single-factor analysis of variance; a P value of <0.05 was considered statistically significant.

Lactate assay.

Overnight cultures were grown in THYE medium and later diluted 1:20 using sterile TYE supplemented with 0.1% or 1% glucose and incubated until the OD600 was ~0.4. To monitor lactate production, 1 or 2 ml of a culture was centrifuged, the cell-free supernatant was diluted 10% in sterile water, and the lactate assay was performed using an EnzyChrome lactate assay kit (ECLC-100) according to the manufacturer's instructions (BioAssay Systems). Experiments were conducted in triplicate, and optical densities were used to normalize the lactate production results. Student's t test was used to analyze the data, and a P value of <0.05 was considered statistically significant.

Microarray analysis.

Cells of S. mutans UA159 (control strain) and SmuvicK (experimental strain) obtained from four independent overnight cultures were diluted 10 times in sterile prewarmed TYE supplemented with 0.5% glucose at pH 7.5. The cells were then incubated at 37°C with 5% CO2, grown to mid-log phase (OD600, ~0.4), and divided into two aliquots. Following centrifugation, cells from each aliquot were resuspended in 0.5% glucose-supplemented TYE adjusted to pH 7.5 or 5.5. The cells were subsequently incubated for 1 h at 37°C, harvested by centrifugation, and snap-frozen in liquid nitrogen until they were needed. When cells were needed, they were immediately resuspended in Trizol reagent (Invitrogen), and total RNA was isolated using the FastPrep system (Bio 101 Savant) as specified by the manufacturer. To eliminate genomic DNA, the samples were then treated with RQ1 DNase (Promega) according to the supplier's instructions. As adapted from Abranches et al. (1), RNA derived from wild-type strain UA159 was used as a reference for the control and experimental samples. The total RNA was converted to cDNA using a First-Strand synthesis kit (MB1 Fermentas) as specified in the manufacturer's protocol. Control and experimental cDNAs were labeled with cyanine 3 (Cy3), whereas reference cDNAs were labeled with cyanine 5 (Cy5), as outlined in the aminoallyl labeling protocol available at the Craig Venter Institute website (http://pfgrc.jcvi.org/index.php/microarray/protocols.html) (previously The Institute for Genomic Research [TIGR]). The following changes were made in this protocol: the ratio of aminoallyl-dUTP to dTTP was changed to 3:2, and the total amount of RNA used for cDNA synthesis was increased to 5 μg. A MAUI hybridization chamber (Bio Microsystems Inc.) was used for hybridization, and slides were scanned using a GenePix scanner (Axon Instruments Inc., Union City, CA) at 532 nm for the Cy3 channel and at 635 nm for the Cy5 channel. Next, the scanned images were processed using TIGR's Spotfinder program with default settings. The output data set was then normalized using software available in TM4 Microarray Software Suite (http://www.tm4.org/index.html) using TIGR's MIDAS program, and an analysis was performed using the Biometric Research Branch microarray tool (http://linus.nci.nih.gov/BRB-ArrayTools.html). A Class Prediction analysis was first performed to validate the reproducibility of each biological replicate. The statistical algorithms used for data analysis included the Compound Covariate Predictor, Diagonal Linear Discriminant Analysis, 1-Nearest Neighbor, 3-Nearest Neighbors, Nearest Centroid, and Support Vector Machines analysis. Statistically significant genes were then identified using a Class Comparison analysis. A two-sample t test (with a random-variance model) with the parametric P value cutoff set at <0.001 was used for statistical analysis. Subsequently, a selected number of genes showing differential expression under acid conditions were validated utilizing quantitative real-time PCR (rtPCR).

rtPCR.

Overnight cells were diluted 10-fold in sterile prewarmed TYE supplemented with 0.5% glucose (pH 7.5) and grown to mid-log phase (OD600, ~0.4). Cultures were then divided into two aliquots, and cells were collected by centrifugation. After pellets were resuspended in 0.5% glucose-supplemented TYE adjusted to pH 7.5 or pH 5.5, cultures were incubated for 1 h at 37°C with 5% CO2. Cells were then harvested by centrifugation, snap-frozen in liquid nitrogen, and stored at −80°C until they were needed. When they were needed, cells were immediately resuspended in Trizol reagent (Invitrogen), and total RNA was isolated using the FastPrep system (Bio 101 Savant) according to the supplier's instructions. DNase treatment of RNAs, cDNA synthesis, rtPCR, and analysis of expression were carried out as previously described (46). Gene expression was normalized to 16S rRNA gene expression, which did not vary under the test conditions used (data not shown). A statistical analysis was conducted using single-factor analysis of variance; a P value of <0.05 was considered significant.

RESULTS AND DISCUSSION

VicK affects acidogenicity of S. mutans.

S. mutans, one of the most acidogenic species found in dental plaque, can produce acid from fermentable carbohydrates at a higher rate and over a wider pH range than other oral streptococcal strains (12). To gain insight into the role of VicK in the acidogenicity of S. mutans, we first monitored the pH drop over time using S. mutans wild-type strain UA159 and a VicK-deficient mutant derivative of this strain grown in THYE medium that contained 0.2% glucose. Temporal pH measurements indicated that the rate of pH drop was lower in the SmuvicK mutant strain culture than in the UA159 culture (Fig. (Fig.1A).1A). Since this observation indicated a possible defect in the mutant's ability to produce acid via glycolysis, the acidogenic potential of this organism was evaluated using glycolytic rate measurement. The results of triplicate experiments comparing the wild type with SmuvicK indicated that the absence of VicK significantly reduced the glycolytic rate of the mutant strain at both pH 7.0 and pH 5.0 (P < 0.05) (Fig. (Fig.1B),1B), suggesting that VicK has a novel role in the acidogenicity of S. mutans.

FIG. 1.
(A) pH values for S. mutans strains UA159 and SmuvicK grown in THYE medium at 37°C with an air mixture containing 5% CO2 over time. (B) Glycolytic rates of UA159 and SmuvicK in the presence of 200 mM glucose as the sugar source in media ...

VicK mutant has reduced lactate production.

The ability of S. mutans to readily utilize glucose to produce lactate as an end product of carbohydrate metabolism is a primary virulence factor associated with this pathogen. Since the glycolytic rates of the SmuvicK mutant were drastically reduced and temporal pH measurements indicated a lower rate of pH drop for this mutant, we tested the ability of SmuvicK to produce lactic acid in the presence of glucose as the sole carbohydrate source. As shown in Fig. Fig.2,2, compared with UA159, in TYE supplemented with a low concentration of glucose (0.1%, wt/vol) and in TYE supplemented with a high concentration of glucose (1%, wt/vol), the VicK-deficient mutant showed significant 28% and 20% reductions in lactic acid production, respectively (P < 0.05, Student's t test), further expanding the role of VicK in producing a key metabolic end product that contributes to the virulence of S. mutans.

FIG. 2.
Lactate production by S. mutans wild-type strain UA159 and the SmuvicK mutant strain. Cells grown to mid-log phase in TYE supplemented with 0.1% glucose (low sugar) and in TYE supplemented with 1% glucose (high sugar) were used to assay ...

Survival of the VicK mutant is enhanced at low pH.

Since the VicK mutant aggregated in THYE medium (45), analysis of the growth rate of this mutant at a low pH proved to be challenging. Moreover, interpretation of this mutant's acid sensitivity was also impeded by the fact that its doubling time was altered at a neutral pH compared with the doubling time of the wild-type parent strain (45). Therefore, we utilized an ATR assay to examine acid stress-mediated killing of SmuvicK and the wild-type strain by exposing cells grown at pH 7.5 to a killing pH, pH 3.5, with and without preexposure to a signal pH, pH 5.5. As judged by the increased percentage of survivor CFU in acid-habituated cultures of both strains, it was evident that for cells that were allowed to adapt to low pH by preexposure to pH 5.5 there was a dramatic increase in the ability to survive in the presence of a lethal pH (Fig. (Fig.3A3A and and3B).3B). Moreover, compared to the data for the parent strain, the percentage SmuvicK survivors was significantly increased irrespective of prior adaptation to low pH (P < 0.001, Student's t test).

FIG. 3.
ATR of SmuvicK and UA159 determined using adapted (A) and unadapted (B) cells. The data are the means ± standard errors of three independent experiments. ***, differences are statistically significant (P < 0.05, ...

Repeated pulses of sugar can decrease the plaque pH to values beyond easily recoverable levels managed by the buffering capacity of saliva, which can result in exposure of plaque microbes to low pH values (e.g., pH ≤5.5) for prolonged periods of time (11, 48). The dramatically enhanced resistance to acid killing exhibited by the VicK-deficient strain suggested that VicK has a novel role in the acid tolerance of S. mutans. However, it is important to note that the enhanced acid survival of S. mutans facilitated by the loss of VicK did not depend on preexposure to an adaptive pH. Hence, it remains to be investigated whether the enhanced acid resistance seen in the mutant was mediated by the acid-inducible ATR, by other means that involved pH-independent constitutive mechanisms that can combat environmental stressors, or by both means.

Expression of vicR and vicK is enhanced at low pH.

Since the ability of S. mutans to survive at low pH was remarkably enhanced in the absence of VicK, we asked whether vicK expression was controlled by low pH. Using cDNAs derived from S. mutans wild-type strain UA159 grown in TYE supplemented with 0.5% glucose, we assessed the relative expression of vicR and vicK at pH 5.5 and pH 7.5. Not surprisingly, in UA159, both the transcription of vicR and the transcription of vicK were significantly increased (2.2-fold; P < 0.05) when the organism was exposed to acid compared to the transcription at pH 7.5 (Table (Table1),1), suggesting that the VicR/VicK signal transduction system responds to acid challenge.

TABLE 1.
Analysis of relative expression of selected genes derived from cDNAs of S. mutans UA159 grown in TYE at pH 7.5 and pH 5.5

The responses of TCSTSs to low pH and their involvement in acid tolerance have been widely examined for numerous bacteria (4, 5, 10, 38, 56). In S. mutans, the involvement of RR11, ComD/ComE, CiaH, and GcrR in acid response pathways has been demonstrated previously (6, 7, 26, 29, 30, 39). To better understand the role of VicK in an ATR in S. mutans, we next examined the global transcription profile of the VicK mutant and UA159 wild-type strains under acidic conditions.

Global transcriptional analysis of UA159 and SmuvicK at low pH.

To examine the transcriptome of S. mutans at low pH and to examine the VicK regulon under acid conditions, we performed a microarray analysis using mid-log-phase cells of the UA159 and SmuvicK mutant strains that were resuspended in 0.5% glucose-supplemented TYE adjusted to pH 7.5 or pH 5.5 and incubated for 1 h at 37°C. Exposure of the S. mutans wild-type strain to pH 5.5 significantly affected expression of 347 transcripts representing ~17.8% the genes in the genome (P < 0.001). Table S1 in the supplemental material shows values for predicted functional categories of genes that were significantly up- or downregulated more than twofold in the parent strain at the low pH. An overview of important cellular functions and their underlying genetic loci affected by growth at pH 5.5 is shown in Fig. Fig.4.4. Not surprisingly, the upregulated genes included genes with known or predicted functions in proton expulsion (F1F0-ATPases) and the protection or repair of DNA or protein macromolecules and genes involved in combating environmental stresses (e.g., nox-1, dpr, and satE) (Fig. (Fig.44 and Table S1 in the supplemental material). The overlap of resistance to acid stress with tolerance to other environmental stressors, such as temperature and osmotic stress, has been demonstrated previously (53). Also, notably, low-pH-induced transcription loci included genes encoding putative sodium and potassium transport systems. In fact, the contribution of cation transporters, especially those that take up potassium, and their influence on intracellular pH homeostasis are not novel and have been extensively studied in Escherichia coli and other aciduric bacterial strains (8, 13, 34, 47). Among the components of signal transduction systems affected by low pH were CiaR (SMu1129), ScnK (SMu1814), and RelR/RelS (SMu927/SMu928), in addition to VicR/VicK. Although the S. mutans ComD/ComE system that constituted a peptide-dependent quorum-sensing system was not found in the microarray study, we were able to confirm upregulation of this system using cDNAs derived from acid-induced UA159 cells and quantitative rtPCR (Table (Table1).1). Among other genes, genetic loci responsible for sugar transport, genetic transformation, and bacteriocin production were found to be downregulated in the UA159 microarray study.

FIG. 4.
Overview of transcripts representing genes that were differentially regulated when S. mutans UA159 was switched from TYE at pH 7.5 to TYE at pH 5.5. ↑, upregulated genes; ↓, downregulated genes. Gene identities and putative functions were ...

Compared with the expression of transcripts of the wild-type strain, exposure of the VicK-deficient strain to low pH affected 121 transcripts representing genes comprising 6.2% of the genome (Table (Table2).2). Eighty-nine of these transcripts were significantly up- or downregulated more than twofold compared to the UA159 transcripts (P < 0.001). A large number of these affected transcripts had no known function, warranting further investigation to determine their roles in the cell and in acid tolerance. Two gene categories that were prominently affected by acid following VicK mutagenesis included genes involved in (i) fatty acid and phospholipid metabolism and (ii) transport and binding. Studies aimed at understanding the role of VicR/VicK in S. mutans, as well as the roles of orthologs in other closely related streptococci, have established that these proteins are strongly involved in dealing with stresses that include oxidative stress, osmotic stress, antibiotic stress, and high temperature (14, 27, 31, 37, 54). For Streptococcus pneumoniae, evidence from both proteomic and microarray transcriptome studies, as well as analyses of membrane fatty acid composition, indicated that the VicR/VicK homologs are involved in regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids (35). Hence, it is possible that membrane alterations enabled by the loss of VicK had a profound impact on the response to environmental stresses, as demonstrated by VicR/VicK mutants of other bacteria. Our VicK-deficient microarrays for nonstress conditions indicated that a number of transcripts (SMu20, SMu22, SMu609, and SMu1344) shown to be affected by loss of VicK had putative roles in cell wall metabolism (data not shown).

TABLE 2.
Microarray results for strains SmuVicK and UA159 grown at pH 5.5

In a study conducted by Fozo and Quivey (19), it was demonstrated that S. mutans must make major alterations to its membrane to survive at low pH and that the action of an enzyme designated FabM is required, a characteristic that is shared with Staphylococcus aureus (9, 19). In fact, alteration of cell membrane fluidity by adjustment of the membrane fatty acid composition plays a significant role in the acid resistance phenotype of S. mutans, which in turn has been shown to influence the virulence of this organism (19-22). More specifically, Fozo et al. showed that at low pH, shifts in the membrane fatty acid composition involved a higher proportion of long-chain, monounsaturated fatty acids, which resulted in a membrane that was less permeable to protons, thereby facilitating survival in highly acidic environments (20). In a study that examined the fatty acid profiles of S. mutans, Streptococcus gordonii, Streptococcus salivarius, and Lactobacillus casei in response to environmental acidification, it was shown that aciduric oral bacteria, but not a nonaciduric S. salivarius strain, were able to increase levels of long-chain, monounsaturated membrane fatty acids at low pH (20). While these observations suggest that membrane fatty acid alteration is a common mechanism utilized by bacteria to withstand environmental stress, the role of the VicK protein of S. mutans in membrane modification under acid conditions remains to be investigated.

Conclusions.

We have predicted that VicK has only a single transmembrane domain, and with exception of a few amino acids that extend outside the cell, the bulk of the protein (including the kinase and PAS domains) is predicted to be localized intracellularly. Hence, it is quite possible that VicK acts as a sensor that responds to cytoplasmic physiological changes such as intracellular pH. In this report, we present evidence demonstrating the role of VicK not only in the response to acid stress but also in facilitating the ATR and acid production of S. mutans. While we have also determined the VicK regulon induced at low pH, whether the response of VicK to acid is due to direct sensing of intracellular pH or is an indirect general response initiated by acid stress has not been investigated yet.

In a recent review of the Bacillus subtilis VicR/VicK homologs (designated YycF/YycG), Dubrac et al. emphasized the role of this highly conserved TCSTS as a major regulator of cell wall metabolism (14). In addition to regulation of gbpB, which is responsible for normal cell wall synthesis (32), a link between VicK and cell wall metabolism was previously established; disruption of VicK caused inhibition of processing of AtlA, an autolysin required for biofilm maturation and biogenesis of a normal cell surface in S. mutans (2). Notably, loss of VicK resulted in a mutant that was hyperresistant to autolysis. It has been previously suggested that the Vic system may have a broad regulatory function in governing lytic pathways that modulate the cell surface composition and architecture of S. mutans (2). Previously, we showed that the VicK mutant had a markedly higher growth rate and yield than the wild type when the organisms were exposed to 25 mM paraquat (46), likely indicating that there was better survival under intracellular oxidative stress induced by this reagent. Hence, the hyperresistant phenotype of the VicK mutant in the presence of stresses induced by acid and paraquat may be attributed to the enhanced resistance of this mutant to autolysis. While we have discovered a novel role for VicK in metabolic “tuning,” as well as in tolerating acid stress, the underlying physiological and genetic basis of this process warrants further investigation.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Elena Voronejskaia and Jennifer Lamonaca Bada for technical assistance with ATR and glycolytic rate assays, respectively.

This study was supported by NIH grant RO1DE013230, by CIHR grant MT-15431 to D.G.C., who is a recipient of a Canada Research Chair in Microbiology, and by NIDCR grant DE13239 to R.A.B.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 August 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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