Transcriptional Profiling Reveals the Importance of RcrR in the Regulation of Multiple Sugar Transportation and Biofilm Formation in Streptococcus mutans

The human oral cavity is a constantly changing environment. Tooth decay is a commonly prevalent chronic disease mainly caused by the cariogenic bacterium Streptococcus mutans . S. mutans is an oral pathogen that metabolizes various carbohydrates into extracellular polysaccharides (EPSs), biofilm, and tooth-destroying lactic acid.

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Understanding the Basis of METH Mouth Using a Rodent Model of Methamphetamine Injection, Sugar Consumption, and Streptococcus mutans Infection

mBio ◽

10.1128/mbio.03534-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Hiu Ham Lee ◽

Preethi Sudhakara ◽

Shreena Desai ◽

Kildare Miranda ◽

Luis R. Martinez

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Human Saliva ◽

Tooth Decay ◽

Content Type ◽

Oral Rinse ◽

Tooth Grinding ◽

Oral Tissue ◽

Public Health Strategies ◽

The Impact

ABSTRACT “METH mouth” is a common consequence of chronic methamphetamine (METH) use, resulting in tooth decay and painful oral tissue inflammation that can progress to complete tooth loss. METH reduces the amount of saliva in the mouth, promoting bacterial growth, tooth decay, and oral tissue damage. This oral condition is worsened by METH users’ compulsive behavior, including high rates of consumption of sugary drinks, recurrent tooth grinding, and a lack of frequent oral hygiene. Streptococcus mutans is a Gram-positive bacterium found in the oral cavity and associated with caries in humans. Hence, we developed a murine model of METH administration, sugar intake, and S. mutans infection to mimic METH mouth in humans and to investigate the impact of this drug on tooth colonization. We demonstrated that the combination of METH and sucrose stimulates S. mutans tooth adhesion, growth, and biofilm formation in vivo. METH and sucrose increased the expression of S. mutans glycosyltransferases and lactic acid production. Moreover, METH contributes to the low environmental pH and S. mutans sucrose metabolism, providing a plausible mechanism for bacterium-mediated tooth decay. Daily oral rinse treatment with chlorhexidine significantly reduces tooth colonization in METH- and sucrose-treated mice. Furthermore, human saliva inhibits S. mutans colonization and biofilm formation after exposure to either sucrose or the combination of METH and sucrose. These findings suggest that METH might increase the risk of microbial dental disease in users, information that may help in the development of effective public health strategies to deal with this scourge in our society. IMPORTANCE “METH mouth” is characterized by severe tooth decay and gum disease, which often causes teeth to break or fall out. METH users are also prone to colonization by cariogenic bacteria such as Streptococcus mutans. In addition, this oral condition is aggravated by METH users’ compulsive behavior, including the consumption of beverages with high sugar content, recurrent tooth grinding, and a lack of frequent oral hygiene. We investigated the effects of METH and sugar consumption on S. mutans biofilm formation and tooth colonization. Using a murine model of METH administration, sucrose ingestion, and oral infection, we found that the combination of METH and sucrose increases S. mutans adhesion and biofilm formation on the teeth of C57BL/6 mice. However, daily chlorhexidine-based oral rinse treatment reduces S. mutans tooth colonization. Similarly, METH has been associated with dry mouth or hyposalivation in users. Hence, we assessed the impact of human saliva on biofilm formation and demonstrated that surface preconditioning with saliva substantially reduces S. mutans biofilm formation. Our results are significant because to our knowledge, this is the first basic science study focused on elucidating the fundamentals of METH mouth using a rodent model of prolonged drug injection and S. mutans oral infection. Our findings may have important translational implications for the development of treatments for the management of METH mouth and more effective preventive public health strategies that can be applied to provide effective dental care for METH users in prisons, drug treatment centers, and health clinics.

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Disruption of L-Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

Journal of Bacteriology ◽

10.1128/jb.00728-19 ◽

2019 ◽

Vol 202 (6) ◽

Cited By ~ 1

Author(s):

Andrew P. Bischer ◽

Christopher J. Kovacs ◽

Roberta C. Faustoferri ◽

Robert G. Quivey

Keyword(s):

Cell Wall ◽

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Etiologic Agent ◽

Growth Defect ◽

Cell Wall Polysaccharide ◽

Content Type ◽

Severe Growth

ABSTRACT The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses. IMPORTANCE Streptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

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Lemon myrtle extract inhibits lactate production by Streptococcus mutans

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab147 ◽

2021 ◽

Vol 85 (10) ◽

pp. 2185-2190

Author(s):

Yukinori Yabuta ◽

Yui Sato ◽

Arisu Miki ◽

Ryuta Nagata ◽

Tomohiro Bito ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Virulence Factors ◽

Biofilm Formation ◽

Tooth Enamel ◽

Lactate Production ◽

Tooth Decay ◽

Lactate Dehydrogenase Activity

ABSTRACT Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.

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Molecule Targeting Glucosyltransferase Inhibits Streptococcus mutans Biofilm Formation and Virulence

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00919-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 126-135 ◽

Cited By ~ 44

Author(s):

Zhi Ren ◽

Tao Cui ◽

Jumei Zeng ◽

Lulu Chen ◽

Wenling Zhang ◽

...

Keyword(s):

Streptococcus Mutans ◽

Dental Plaque ◽

Biofilm Formation ◽

Extracellular Polysaccharides ◽

Content Type ◽

Oral Infections ◽

Concomitant Reduction ◽

New Strategies

ABSTRACTDental plaque biofilms are responsible for numerous chronic oral infections and cause a severe health burden. Many of these infections cannot be eliminated, as the bacteria in the biofilms are resistant to the host's immune defenses and antibiotics. There is a critical need to develop new strategies to control biofilm-based infections. Biofilm formation inStreptococcus mutansis promoted by major virulence factors known as glucosyltransferases (Gtfs), which synthesize adhesive extracellular polysaccharides (EPS). The current study was designed to identify novel molecules that target Gtfs, thereby inhibitingS. mutansbiofilm formation and having the potential to prevent dental caries. Structure-based virtual screening of approximately 150,000 commercially available compounds against the crystal structure of the glucosyltransferase domain of the GtfC protein fromS. mutansresulted in the identification of a quinoxaline derivative, 2-(4-methoxyphenyl)-N-(3-{[2-(4-methoxyphenyl)ethyl]imino}-1,4-dihydro-2-quinoxalinylidene)ethanamine, as a potential Gtf inhibitor.In vitroassays showed that the compound was capable of inhibiting EPS synthesis and biofilm formation inS. mutansby selectively antagonizing Gtfs instead of by killing the bacteria directly. Moreover, thein vivoanti-caries efficacy of the compound was evaluated in a rat model. We found that the compound significantly reduced the incidence and severity of smooth and sulcal-surface cariesin vivowith a concomitant reduction in the percentage ofS. mutansin the animals' dental plaque (P< 0.05). Taken together, these results represent the first description of a compound that targets Gtfs and that has the capacity to inhibit biofilm formation and the cariogenicity ofS. mutans.

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Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan

Applied and Environmental Microbiology ◽

10.1128/aem.00869-17 ◽

2017 ◽

Vol 83 (15) ◽

Cited By ~ 11

Author(s):

Ryo Nagasawa ◽

Tsutomu Sato ◽

Hidenobu Senpuku

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Cell Surface Hydrophobicity ◽

Extracellular Dna ◽

Regulatory Function ◽

Extracellular Polysaccharides ◽

Oral Flora ◽

Content Type ◽

Positive Effects ◽

Low Concentrations

ABSTRACT Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan. IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and other carbohydrates on biofilm formation. Since raffinose has been reported to have positive effects on enterobacterial flora, research on the effects of raffinose on the oral flora are required prior to its use as a beneficial sugar for human health. Here, we showed that raffinose induced biofilm formation by S. mutans in low concentrations of sucrose. The induction of biofilm formation generally generates negative effects on the oral flora. Therefore, we believe that this finding will aid in the development of more effective oral care techniques to maintain oral flora health.

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A New Small Molecule Specifically Inhibits the Cariogenic Bacterium Streptococcus mutans in Multispecies Biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01496-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2679-2687 ◽

Cited By ~ 47

Author(s):

Chang Liu ◽

Roberta J. Worthington ◽

Christian Melander ◽

Hui Wu

Keyword(s):

Dental Caries ◽

Small Molecules ◽

Streptococcus Mutans ◽

Small Molecule ◽

Biofilm Formation ◽

Content Type ◽

Multispecies Biofilm ◽

Small Molecule Library ◽

Cariogenic Bacterium ◽

Multispecies Biofilms

ABSTRACTStreptococcus mutansis a major cariogenic bacterium. It has adapted to the biofilm lifestyle, which is essential for pathogenesis of dental caries. We aimed to identify small molecules that can inhibit cariogenicS. mutansand to discover lead structures that could give rise to therapeutics for dental caries. In this study, we screened a focused small-molecule library of 506 compounds. Eight small molecules which inhibitedS. mutansat a concentration of 4 μM or less but did not affect cell growth or biofilm formation of commensal bacteria, represented byStreptococcus sanguinisandStreptococcus gordonii, in monospecies biofilms were identified. The active compounds share similar structural properties, which are characterized by a 2-aminoimidazole (2-AI) or 2-aminobenzimidazole (2-ABI) subunit. In multispecies biofilm models, the most active compound also inhibited cell survival and biofilm formation ofS. mutansbut did not affect commensal streptococci. This inhibitor downregulated the expression of six biofilm-associated genes,ftf,pac,relA,comDE,gbpB, andgtfB, in planktonicS. mutanscells, while it downregulated the expression of onlyftf,pac, andrelAin the biofilm cells ofS. mutans. The most potent compound also inhibited production of two key adhesins ofS. mutans, antigen I/II and glucosyltransferase (GTF). However, the compound did not alter the expression of the corresponding genes in bothS. sanguinisandS. gordonii, indicating that it possesses a selective inhibitory activity againstS. mutans.

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Nitrite Production from Nitrate and Its Link with Lactate Metabolism in Oral Veillonella spp.

Applied and Environmental Microbiology ◽

10.1128/aem.01255-20 ◽

2020 ◽

Vol 86 (20) ◽

Cited By ~ 1

Author(s):

Dimas Prasetianto Wicaksono ◽

Jumpei Washio ◽

Yuki Abiko ◽

Hitomi Domon ◽

Nobuhiro Takahashi

Keyword(s):

Lactic Acid ◽

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

General Health ◽

Pathogenic Bacteria ◽

Reducing Power ◽

Lactate Metabolism ◽

Content Type ◽

Biochemical Characteristics

ABSTRACT Veillonella species are among the major anaerobes in the oral cavity and are frequently detected in both caries lesions and healthy oral microbiomes. They possess the ability to utilize lactate and convert nitrate (NO3–) into nitrite (NO2–). Recently, interest in NO2– has increased rapidly because of its beneficial effects on oral and general health; i.e., it inhibits the growth and metabolism of oral pathogenic bacteria, such as Streptococcus mutans, and lowers systemic blood pressure. However, there is only limited information about the biochemical characteristics of NO2– production by Veillonella species. We found that NO3– did not inhibit the growth of Veillonella atypica or Veillonella parvula, and it inhibited the growth of Streptococcus mutans only at a high concentration (100 mM). However, NO2– inhibited the growth of Streptococcus mutans at a low concentration (0.5 mM), while a higher concentration of NO2– (20 mM) was needed to inhibit the growth of Veillonella species. NO2– production by Veillonella species was increased by environmental factors (lactate, acidic pH, and anaerobic conditions) and growth conditions (the presence of NO3– or NO2–) and was linked to anaerobic lactate metabolism. A stoichiometric evaluation revealed that NO3– is reduced to NO2– by accepting reducing power derived from the oxidization of lactate. These findings suggest that the biochemical characteristics of NO2– production from NO3– and its linkage with lactate metabolism in oral Veillonella species may play a key role in maintaining good oral and general health. IMPORTANCE The prevalence of dental caries is still high around the world. Dental caries is initiated when the teeth are exposed to acid, such as lactic acid, produced via carbohydrate metabolism by acidogenic microorganisms. Veillonella species, which are among the major oral microorganisms, are considered to be beneficial bacteria due to their ability to convert lactic acid to weaker acids and to produce NO2– from NO3–, which is thought to be good for both oral and general health. Therefore, it is clear that there is a need to elucidate the biochemical characteristics of NO2– production in Veillonella species. The significance of our research is that we have found that lactate metabolism is linked to NO2– production by Veillonella species in the environment found in the oral cavity. This study suggests that Veillonella species are potential candidates for maintaining oral and general health.

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Competence-Stimulating-Peptide-Dependent Localized Cell Death and Extracellular DNA Production in Streptococcus mutans Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.02080-20 ◽

2020 ◽

Vol 86 (23) ◽

Author(s):

Ryo Nagasawa ◽

Tatsuya Yamamoto ◽

Andrew S. Utada ◽

Nobuhiko Nomura ◽

Nozomu Obana

Keyword(s):

Cell Death ◽

Single Cell ◽

Streptococcus Mutans ◽

Structural Stability ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Imaging Techniques ◽

Extracellular Dna ◽

Content Type ◽

Cariogenic Bacterium

ABSTRACT Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm. IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

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Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance

Journal of Bacteriology ◽

10.1128/jb.00813-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1087-1100 ◽

Cited By ~ 14

Author(s):

Gursonika Binepal ◽

Kamal Gill ◽

Paula Crowley ◽

Martha Cordova ◽

L. Jeannine Brady ◽

...

Keyword(s):

Stress Tolerance ◽

Streptococcus Mutans ◽

Transport System ◽

Biofilm Formation ◽

Cellular Response ◽

Pathogenic Bacteria ◽

Transport Systems ◽

Content Type ◽

Growth And Survival ◽

Dental Biofilm

ABSTRACTPotassium (K+) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K+and a variety of K+transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K+acquisition inStreptococcus mutansand the importance of K+homeostasis for its virulence attributes. TheS. mutansgenome harbors four putative K+transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K+cotransporter (GlnQHMP), and a channel-like K+transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K+] less than 5 mM eliminated biofilm formation inS. mutans. The functionality of the Trk2 system was confirmed by complementing anEscherichia coliTK2420 mutant strain, which resulted in significant K+accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K+-dependent cellular response ofS. mutansto environment stresses.IMPORTANCEBiofilm formation and stress tolerance are important virulence properties of caries-causingStreptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment ofS. mutans. K+is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K+transporters inS. mutans. We identified the most important system for K+homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K+for the activity of biofilm-forming enzymes, which explains why such high levels of K+would favor biofilm formation.

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A Novel Gene Involved in the Survival of Streptococcus mutans under Stress Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.02549-13 ◽

2013 ◽

Vol 80 (1) ◽

pp. 97-103 ◽

Cited By ~ 11

Author(s):

Dan Li ◽

Yukie Shibata ◽

Toru Takeshita ◽

Yoshihisa Yamashita

Keyword(s):

Streptococcus Mutans ◽

Streptococcus Pyogenes ◽

Biofilm Formation ◽

Acid Tolerance ◽

Stress Conditions ◽

Insertion Mutagenesis ◽

Trna Modification ◽

Content Type ◽

Virulence Proteins ◽

Random Insertion

ABSTRACTAStreptococcus mutansmutant defective in aciduricity was constructed by random-insertion mutagenesis. Sequence analysis of the mutant revealed a mutation ingidA, which is known to be involved in tRNA modification inStreptococcus pyogenes. Complementation ofgidAbyS. pyogenesgidArecovered the acid tolerance ofS. mutans. Although thegidA-inactivatedS. pyogenesmutant exhibited significantly reduced expression of multiple extracellular virulence proteins, theS. mutansmutant did not. On the other hand, thegidAmutant ofS. mutansshowed reduced ability to withstand exposure to other stress conditions (high osmotic pressure, high temperature, and bacitracin stress) besides an acidic environment. In addition, loss of GidA decreased the capacity for glucose-dependent biofilm formation by over 50%. This study revealed thatgidAplays critical roles in the survival ofS. mutansunder stress conditions, including lower pH.

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