Role of the dpr product in oxygen tolerance in Streptococcus mutans

We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H(2)O(2), implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc(r) mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc(r) mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc(r) ahpC::Em(r)::nox-1 triple mutant nor the dpr::Spc(r) sod::Em(r) double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.     

Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans.

We had isolated three genes (rmlA, rmlB, and rmlC) involved in dTDP-rhamnose synthesis in Streptococcus mutans and found that three genes were insufficient for dTDP-rhamnose synthesis (Y. Tsukioka, Y. Yamashita, T. Oho, Y. Nakano, and T. Koga, J. Bacteriol. 179:1126-1134, 1997). The rmlD gene of S. mutans, encoding the enzyme which catalyzes the last step of dTDP-rhamnose synthesis, has been cloned and sequenced. The cell extract of Escherichia coli expressing the rmlD gene of S. mutans exhibited enzymatic activity corresponding to its counterpart in Shigella flexneri, a gram-negative bacterium. Rhamnose was not detected in the cell wall preparation purified from the mutant in which the cloned gene was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with autoclaved extracts from the mutant. The rmlD gene product of S. mutans compensated for the incompleteness of dTDP-rhamnose synthesis by the three previously isolated genes. These results indicate that the rmlD gene product is indispensable for the dTDP-rhamnose pathway and subsequently for the synthesis of serotype-specific antigen in S. mutans. Furthermore, conservation of the rmlD gene in Streptococcus species was demonstrated by Southern blot analysis.     

Regulation of the intracellular free iron pool by Dpr provides oxygen tolerance to Streptococcus mutans

Dpr is an iron-binding protein required for oxygen tolerance in Streptococcus mutans. We previously proposed that Dpr could confer oxygen tolerance to the bacterium by sequestering intracellular free iron ions that catalyze generation of highly toxic radicals (Y. Yamamoto, M. Higuchi, L. B. Poole, and Y. Kamio, J. Bacteriol. 182:3740-3747, 2000; Y. Yamamoto, L. B. Poole, R. R. Hantgan, and Y. Kamio, J. Bacteriol. 184:2931-2939, 2002). Here, we examined the intracellular free iron status of wild-type (WT) and dpr mutant strains of S. mutans, before and after exposure to air, by using electron spin resonance spectrometry. Under anaerobic conditions, free iron ion concentrations of WT and dpr strains were 225.9 +/- 2.6 and 333.0 +/- 61.3 microM, respectively. Exposure of WT cells to air for 1 h induced Dpr expression and reduced intracellular free iron ion concentrations to 22.5 +/- 5.3 microM; under these conditions, dpr mutant cells maintained intracellular iron concentration at 230.3 +/- 28.8 microM. A decrease in cell viability and genomic DNA degradation was observed in the dpr mutant exposed to air. These data indicate that regulation of the intracellular free iron pool by Dpr is required for oxygen tolerance in S. mutans.     

Identification and Analysis of a Collagenolytic Activity in Streptococcus mutans

Streptococcus mutans is an important pathogen in coronal caries and is implicated in dental root decay by its ability to bind collagen from various sources. In the present study, electron microscopic analysis demonstrated the ability of S. mutans to bind and to disrupt collagen fibrils of the amniotic membrane. The synthetic peptide FALGPA, which is similar in structure to collagen, was degraded by S. mutans, with a lower level of FALGPA hydrolytic activity observed in sucrose-grown cells compared with cells grown in the absence of sucrose. Inhibition studies of FALGPA hydrolytic activity showed a pattern characteristic of collagenase activity, with inhibition by 1,10-phenanthroline and EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Additionally, immunological cross-reactivity was observed between proteins from disrupted cells of S. mutans and antiserum to collagenase from Clostridium histolyticum. Gelatinolytic activity was demonstrated by gelatin zymogram analysis. These findings suggest that collagenolytic activity by S. mutans may be an important virulence factor in dental root decay. Received: 8 April 1996 / Accepted: 10 July 1996     

Acid tolerance of biofilm cells of Streptococcus mutans

Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.     

Acid tolerance response of biofilm cells of Streptococcus mutans

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

Expression of a gene for glucan-binding protein from Streptococcus mutans in Escherichia coli

The structural gene for a glucan-binding protein (GBP) of Streptococcus mutans has been inserted into a bacteriophage lambda vector and expressed in Escherichia coli K12. Lysates of E. coli infected with the recombinant phage contain an antigenic protein of the same size as S. mutans GBP. The GBP synthesized in E. coli can be affinity-purified on immobilized glucan and antiserum raised against it has been shown to precipitate fructosyltransferase activity from S. mutans.     

Involvement of sensor kinases in the stress tolerance response of Streptococcus mutans

The gram-positive bacterium Streptococcus mutans is the primary causative agent in the formation of dental caries in humans. The ability of S. mutans to adapt and to thrive in the hostile environment of the oral cavity suggests that this cariogenic pathogen is capable of sensing and responding to different environmental stimuli. This prompted us to investigate the role of two-component signal transduction systems (TCS), particularly the sensor kinases, in response to environmental stresses. Analysis of the annotated genome sequence of S. mutans indicates the presence of 13 putative TCS. Further bioinformatics analysis in our laboratory has identified an additional TCS in the genome of S. mutans. We verified the presence of the 14 sensor kinases by using PCR and Southern hybridization in 13 different S. mutans strains and found that not all of the sensor kinases are encoded by each strain. To determine the potential role of each TCS in the stress tolerance of S. mutans UA159, insertion mutations were introduced into the genes encoding the individual sensor kinases. We were successful in inactivating all of the sensor kinases, indicating that none of the TCS are essential for the viability of S. mutans. The mutant S. mutans strains were assessed for their ability to withstand various stresses, including osmotic, thermal, oxidative, and antibiotic stress, as well as the capacity to produce mutacin. We identified three sensor kinases, Smu486, Smu1128, and Smu1516, which play significant roles in stress tolerance of S. mutans strain UA159.     

A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.     

Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.     

Transcriptional analysis of the bglP gene from Streptococcus mutans

Background  

An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II.     

Identification of essential amino acids in the Streptococcus mutans glucosyltransferases.

A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.     

Identification and Functional Analysis of Genome Mutations in a Fluoride-Resistant Streptococcus mutans Strain

It is known that fluoride-resistant microorganisms are different from fluoride-sensitive ones in growth, adherence and metabolic activity. It was hypothesized that these phenotypic differences were due to stable genotypic changes in the fluoride-resistant strains. However, until now, no studies have reported these genotypic changes. The aim of this study is to identify such changes in a fluoride-resistant Streptococcus mutans strain (C180-2FR) using whole-genome shotgun (WGS) sequencing and to examine the potential function of the identified mutations by comparing gene expression between the fluoride-sensitive (C180-2) and C180-2FR strains. We performed 50 bp paired-end Illumina shotgun sequencing for both strains. Through extensive bioinformatic analysis, we were able to identify 8 single nucleotide polymorphisms (SNPs) in the genome of C180-2FR, which were further confirmed by Sanger sequencing. Expression of the genes containing or in proximity to the SNPs in C180-2 and C180-2FR was then quantified by real-time PCR. A gene cluster containing genes coding for fluoride antiporters was up-regulated 10-fold in C180-2FR when compared to that in C180-2, independent of growth phase. Two SNPs are located in this gene cluster, one in its promoter region and the other in its protein-coding region. In addition, one gene, which codes for a putative glycerol uptake facilitator protein, was found to be down-regulated by 60% in C180-2FR at an early growth phase. The promoter region of this gene contained a SNP. No difference in expression was found for the other SNP-containing genes. In summary, using WGS sequencing, we were able to uncover genetic changes in the genome of a fluoride-resistant strain. These findings can provide new insights into the mechanism of microbial fluoride resistance.