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The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 μg ml(-1) of MDPB for 60 s. At a concentration of 4-8 μg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 μg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.

In this study, the bactericidal activity of antibacterial monomer MDPB (12-methacryloyloxydodecylpyridinium bromide) against Streptococcus mutans was tested by a rapid method for monitoring viability. To S. mutans culture containing fluorescence staining solution that distinguishes live from dead cells, MDPB was added at a concentration of 250, 100, 50, or 10 microg/ml. Bacterial cells were observed by fluorescence microscopy and the percentage of dead cells was calculated. After 10, 20, or 30 minutes' contact with MDPB, the live/dead ratio was measured by fluorometry and viable counts (CFU) determined by the conventional plating method. Viability staining revealed that MDPB exhibited significant bactericidal effects at 50 microg/ml or greater (ANOVA, Fisher's PLSD test), and complete killing of the cells at 250 microg/ml of MDPB was demonstrated in conjunction with a plating method. The staining method thus provided a sensitive means to determine loss of viability, and indicated the strong killing effects of MDPB on S. mutans.

The basis for genotypic and phenotypic variation within Streptococcus mutans is poorly understood but the availability of the genome sequence of strain UA159 provides an opportunity for comparative studies. Genomic DNA prepared from nine strains of S. mutans was used to probe a microarray consisting of oligonucleotides representing 1948 open reading frames of S. mutans UA159. A total of 385 (20%) of the UA159 open reading frames were found to be absent from one or more of the test strains. Absent open reading frames frequently occurred in blocks of adjacent open reading frames and represented regions previously experimentally detected by polymerase chain reaction, predicted genomic islands and insertion sequence elements as well as novel open reading frames. Approximately half appear to involve foreign DNA acquired by horizontal transmission. The results indicate the existence of distinct core and dispensable genomes and may help explain the phenotypic and genotypic variation within S. mutans.

The genome of Streptococcus mutans UA159 contains two phospho-beta-glucosidase genes, bglA and celA, which occur in operon-like arrangements along with genes for components of phosphotransferase transport systems and a third phospho-beta-glucosidase encoded by the arb gene, which does not have its own associated transport system but relies on uptake by the bgl or cel systems. Targeted inactivation of each of the phospho-beta-glucosidase genes revealed that bglA is involved in aesculin hydrolysis, celA is essential for utilisation of cellobiose, amygdalin, gentobiose and salicin, and arb is required for utilisation of arbutin. Inactivation of genes for the phosphotransferase systems revealed an overlap of specificity for transport of beta-glucosides and also indicated that further, unidentified transport systems exist. The cel and arb genes are subject to catabolite repression by glucose, but the regM gene is not essential for catabolite repression. Screening a collection of isolates of S. mutans revealed strains with deletions affecting the msm, bgl and/or cel operons. The phenotypes of these strains could largely be explained on the basis of the results obtained from the knockout mutants of S. mutans UA159 but also indicated the existence of other pathways apparently absent from UA159. The extensive genetic and phenotypic variation found in beta-glucoside metabolism indicates that there may be extensive heterogeneity in the species.

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei . Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly α-1,3 or α-1,6 links, as well as alternating α-1,3 and α-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

Streptococcus mutansproduces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called ‘A’ repeats. TheS. mutansgenome sequence was searched for ORFs containing ‘A’ repeats, and one novel gene,gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three ‘A’ repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with aKDof 2–3 nM. Construction of truncated derivatives of GbpD confirmed that the ‘A’ repeat region was essential for binding. Furthermore, agbpDknockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to theα/βhydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site ‘lipase box’ and an ‘oxyanion hole’. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. ThegbpDmutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity ofS. mutansand another plaque bacterium,Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) ofS. sanguinis, but had no effect on LTA fromS. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm.

Many isolates of Streptococcus mutans lack the ability to ferment melibiose and other sugars. We previously reported that this was commonly due to a chromosomal deletion and, in the present study, sequence information from the S. mutans genome project was used to design PCR primers to explore the nature and extent of the deletion. In all melibiose-negative strains examined, there was an incomplete insertion element, ISSmu3, in place of the 18-kb stretch of chromosome encoding the msm and gal operons. Strains that were also unable to utilise β-glucosides were found to have a separate 4 kb deletion in the bgl regulon that is proposed to be due to homologous recombination between two short stretches of identical sequence. The evidence is consistent with all the melibiose-negative strains examined being derived from a common ancestor.

Porphyromonas gingivalis produces virulence factors which can modify the molecular and cellular components of the host immune response. In the present work we investigated the role of specific virulence factors from P. gingivalis in the induction of apoptosis in Jurkat T cells. P. gingivalis culture supernatants mimicked the effect of butyric acid on T-cell apoptosis and this effect was associated with an increase in histone H4 acetylation. A role for proteases was excluded in experiments which demonstrated that neither protease inhibitors nor use of P. gingivalis mutants defective in protease synthesis had any effect on the stimulation of T-cell apoptosis in this system.

The ability of a range of potential inhibitors to affect the catalytic activity or binding of dextran by a glucosyltransferase (GTF-I) that synthesises insoluble α1,3-linked glucan was tested. Acarbose, deoxynojirimycin, N-dodecyldeoxynojirimycin and Tris, which are thought to interfere with the active site of the enzyme of GTF and related glycosidases, inhibited glucan synthesis but not glucan binding. Tris was found to act as a competitive inhibitor of GTF-I. The effectiveness of the active site inhibitors was not altered by immobilisation of GTF-I on saliva-coated hydroxyapatite. In contrast, three amine hydrofluorides were markedly less effective against immobilised GTF than soluble GTF. The pH of the reaction mixture was found to have a strong influence on inhibition by acarbose, Tris and amine hydrofluorides, a finding that is of direct relevance to use of inhibitors in vivo.

A homologue of the HtrA family of stress-response proteases was detected by analysis of the Streptococcus mutans genome sequence. Disabling of the S. mutans htrA gene by insertional inactivation resulted in bacterial clumping in liquid medium, altered colony morphology and a reduced ability to withstand high temperature, extremes of pH or oxidative stress. Seven different extracellular or wall-associated proteins that are known to be subject to post-translational proteolysis were examined in cultures of wild-type S. mutans and an htrA mutant. Inactivation of the htrA protease had no effect on degradation of the proteins.

Glucosyltransferases of oral streptococci, dextransucrases and alternansucrase of Leuconostoc mesenteroides, collectively referred to as glucansucrases, are large extracellular enzymes that synthesise glucans with a variety of structures and properties. A characteristic of all these glucansucrases is the possession of a C-terminal domain consisting of a series of tandem amino acid repeats. These repeat units are thought to interact with glucan but closely resemble the cell wall binding domain motif found in choline binding proteins in Streptococcus pneumoniae and surface-located proteins in a range of other bacteria. Analysis of dextransucrase and alternansucrase sequences has now shown that they also contain these repeat motifs in the N-terminal region, raising questions about their evolutionary origin and functional importance.

Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the alpha-amylase family (glycoside hydrolase family 13), which all have a (beta/alpha)8 barrel structure. In the glucansucrases, however, the alpha-helix and beta-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In alpha-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis-Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (beta/alpha)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which alpha1,3-linked glucosyl units are joined sequentially to maltose.

Glucansucrases of oral streptococci and Leuconostoc mesenteroides are enzymes of medical and biotechnological interest that synthesize α-glucans. They can also synthesize oligosaccharides in the presence of a sugar acceptor. Previous reports have identified an amino acid residue that may affect the structure of the glucan product; therefore, random mutagenesis of the corresponding Asp-569 of Streptococcus downei glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanism and to evaluate how different amino acids can modulate glucan and oligosaccharide synthesis. GTF-I variants were obtained where Asp-569 was replaced by each of the different possible classes of amino acids. These were expressed in Escherichia coli and purified by means of a His 6 tag. The results showed that the amino acid in position 569 influences the structure of the glucan and the size of the oligosaccharides produced by GTF-I. The results suggest that the amino acid occupying this position is more likely to interact with the acceptor molecules (oligosaccharides or elongating glucan chain) than to be directly involved in glucosyl transfer from sucrose. Engineering of the equivalent position in glucansucrases thus appears to be a good target to expand the range of oligosaccharides synthesized.

Multiple sequence alignment and structure prediction of glucansucrases produced by oral streptococci and Leuconostoc mesenteroides showed that all have common structural features, with three major domains. There is no conservation of primary sequence or structure in the N-terminal variable region. Sequence-based structure prediction combined with circular dichroism spectrum analysis of purified truncated forms of Streptococcus downei GTF-I revealed that the core catalytic region has a defined structure consistent with the proposed (α/β)8-barrel structure. The C-terminal domain is a mixed structure with significant amounts of β-sheet and random-coil. This information contributes to the development of our understanding of structure-function relationships in glucansucrases.

The activity of glucosyltransferases (GTF), a group of enzymes that synthesize water-soluble and -insoluble glucans from sucrose, significantly contributes to the cariogenicity of mutans streptococci. Streptococcus downei produces four glucosyltransferases, GTFI, which produces insoluble glucan, and GTFS, GTFT, and GTFU, which synthesize soluble glucans. We have previously reported that inactivation of gtfS results in altered adherence and have now examined its interaction with other enzymes by constructing mutants which were gtfS, gtfS/gtfT, gtfS/gtfI and gtfI. The mutants were tested for their ability to accumulate on wires and on plastic microtiter trays in the presence of sucrose. The gtfS mutant displayed a reduced ability to adhere compared to the wild type but there was no further reduction of adherence in a gtfS/gtfT mutant. In contrast, the gtfS/gtfI double mutant showed a drastic reduction in adherence and when gtfI alone was inactivated, bacteria were unable to adhere to a hard surface. The results confirmed that insoluble glucan is required for strong adherence to a smooth surface but that the amount and structure of this glucan is dependent upon the availability of soluble glucans to act as primer molecules.

Representative strains of oral streptococci, lactobacilli and bifidobacteria were incubated overnight with lactulose or other carbohydrates and the final pH recorded. Most bacteria tested were able to metabolize lactulose with the exception of strains of Streptococcus salivarius, Lactobacillus acidophilus and Lact. fermentum. Streptococcus mutans produced most acid overnight but the initial rate of acid production from lactulose by uninduced cultures was very low. Plaque pH was monitored in 12 volunteers following rinsing the mouth with lactulose, sucrose or sorbitol or Lactulose BP. These studies in vivo showed both lactulose and Lactulose BP to exhibit low acidogenic potential. Thus, although plaque bacteria are capable of fermenting lactulose, the results suggest that lactulose is likely to pose a small acidogenic challenge to teeth under normal conditions of use.

A locus containing a gene with homology to ccpA of other bacteria has been cloned from Streptococcus mutans LT11, sequenced, and named regM . Upstream of the regM gene, on the opposite strand, is a gene encoding an X-Pro dipeptidase, pepQ . A 14-bp palindromic sequence with homology to the consensus catabolite-responsive element sequence lay in the promoter region between the two genes. To study the function of regM , the gene was inactivated by insertion of an antibiotic resistance marker. Diauxic growth of S. mutans on a number of sugars in the presence of glucose was not affected by disruption of regM . The loss of RegM increased glucose repression of α-galactosidase, mannitol-1-P dehydrogenase, and P-β-galactosidase activities. These results suggest that while RegM can affect catabolite repression in S. mutans , it does not conform to the model proposed for CcpA in Bacillus subtilis.

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of α-galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.

Summary: Streptococcus mutans is able to synthesize extracellular glucans from sucrose which contribute to adherence of these bacteria. Extracellular dextranase can partially degrade the glucans, and may therefore affect virulence of S. mutans. In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random Sau3AI-digested fragments of chromosomal DNA from S. mutans into the BamHI site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia coli but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into S. mutans LT11, allowing insertional inactivation through homologous recombination. Two transformants were identified which did not possess dextranase activity. Integration of a single copy of the plasmid into the chromosome of these transformants was confirmed by Southern hybridization analysis. Chromosomal DNA fragments flanking the plasmid were recovered using a marker rescue technique, and sequenced. Comparison with known sequences using the blastx program showed 56% homology at the amino acid level between the sequenced gene fragment and dextranase from Streptococcus sobrinus, strongly suggesting that the S. mutans dextranase gene (dexA) had been inactivated. The colony morphology of the dextranase mutants when grown on Todd-Hewitt agar containing sucrose was altered compared to the parent strain, with an apparent build-up of extracellular polymer. The mutants were also more adherent to a smooth surface than LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation. In contrast to LT11, neither the dexA mutants nor a mutant in the dexB gene, which encodes a dextran glucosidase, were able to ferment dextran to produce acid, supporting an earlier hypothesis that both enzymes are required for metabolism of dextran. From the results obtained by inactivating the dexA gene, a role for dextranase is suggested in controlling the amount and nature of extracellular glucans, in adherence of S. mutans, and in the utilization of glucans as a carbohydrate source.

Attempts to produce resin composite with antibacterial properties by incorporation of an antibacterial agent such as chlorhexidine have been reported, but problems can arise due to release of the inhibitory agent from the composite. Such problems may include toxic effects, influence on mechanical properties, and loss of effectiveness. A new monomer, methacryloyloxydodecylpyridinium bromide (MDPB), was synthesized by combining an antibacterial agent and methacryloyl group. The monomer was incorporated into resin composite to develop a non-releasing antibacterial composite. The ability of composite incorporating MDPB to inhibit growth and plaque accumulation by Streptococcus mutans in vitro was assayed, elution of antibacterial components from the material was investigated, and the influence of incorporation of MDPB on the mechanical properties of composite was studied. Uncured MDPB revealed antibacterial activity against S. mutans and six other species of oral streptococci, with the minimum inhibitory concentration for S. mutans being comparable with that of triclosan. After composite incorporating MDPB was cured, no elution of the antibacterial components was observed from the material, even after 90 days' immersion in water or other solvents. Growth of S. mutans on agar under specimens of MDPBcontaining composite was inhibited compared with controls. In a bacterial accumulation study, S. mutans accumulated to a lesser degree on the surface of composite incorporating MDPB (p < 0.05) than on control. Incorporation of MDPB had no significant influence on the mechanical properties of the composite.

The introduction of techniques for the manipulation of DNA in vitro has had an enormous impact on progress in every area of biological research. In the case of oral microbiology, the first reports on the application of molecular genetics to streptococci started to appear in the early 1980s, and it is now more than 10 years since the first paper describing cloning and expression of a gene from Streptococcus mutans in another bacterium was published. The purpose of this review is not to provide a resumé of all the work that has been done on the molecular biology of oral bacteria; indeed, we are approaching the stage when a comprehensive survey of work on all oral bacteria is no longer feasible--for instance, over 40 genes have now been cloned and sequenced from S. mutans alone--but rather to illustrate examples of how the new techniques have been applied to give novel approaches and insights into old problems, or expand into new directions. The great majority of published work so far relates to streptococci, and many aspects have been covered in previous review articles [Curtiss, 1985; Russell, 1991], the most recent thorough review being that by Kuramitsu [1993]. Discussion of molecular biology is now so all-pervasive that most readers will be familiar with many of the basic terms, but it may be worth clarifying some of the main points during the course of this article. For total novices, the short book by Brown [1990] forms a useful introduction. A number of recent articles have reviewed recent research on plaque formation [Kolenbrander and London, 1993] and the mutans streptococci in particular [Loesche, 1986], while Freedman et al. [1981] and Tanzer [1992] have summarised the information derived from a genetic approach to oral microbiology before the advent on in vitro manipulation of DNA.

The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 μg ml(-1) of MDPB for 60 s. At a concentration of 4-8 μg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 μg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.

The Streptococcus mutans dexB gene, a member of the multiple sugar metabolism (msm) operon, encodes an intracellular glucan 1,6-alpha-glucosidase which releases glucose from the non-reducing terminus of alpha-1,6-linked isomaltosaccharides and dextran. Comparison of primary amino acid sequences showed strong homology to Bacillus oligo-1,6-glucosidases and, like these enzymes, DexB was able to release free glucose from the alpha-1,4,6-branch point in panose. This suggested a role for DexB in the metabolism of either starch or intracellular polysaccharide, which contain such branch points. However, purified intracellular polysaccharide from the wild-type S. mutans strain LT11 and a mutant deficient in dexB revealed no substantial differences in the extent of branching as demonstrated by iodine staining spectra and the degree of polymerization. Furthermore, thin layer chromatography of radiolabelled intracellular polysaccharide digested with S. mutans wild-type and mutant cell extracts showed no differences in the products obtained. The involvement of DexB in dietary starch metabolism was investigated using alpha-limit dextrins produced from the action of alpha-amylase on starch. These induced the msm operon, including dexB, and the DexB enzyme was able to act on the alpha-limit dextrins to give further fermentable substrates. The transport system encoded by the msm operon can also transport alpha-limit dextrin. DexB may therefore be important in the metabolism of extracellular starch.

Extraction of Streptococcus mutans with the detergents HECAMEG and lauroyl sarcosinate preferentially extracted antigen D, previously identified as a low molecular mass wall-associated protein. Western blotting with specific antisera was used to demonstrate that this antigen is the HPr component of the sugar-phosphotransferase transport system. Non-denaturing electrophoresis indicated that HECAMEG selectively extracted only one of the two forms of HPr. It is suggested that this form of HPr may have a specific cell surface location.

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/ II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However p, nucleotide diversity per site, was low for all loci being in the range 0.019–0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3–1.15] compared to 8.3 [95% confidence interval 5.0–14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

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.

Insertion sequence (IS) elements are widely distributed selfmobilizing genetic elements that can affect genetic changes by integrating into the chromosome, mediating genetic rearrangements and facilitating horizontal gene transfer. Three members of the IS3 family have been identified in Streptococcus mutans: IS199 was discovered by its transposition while ISSmu1 and ISSmu2 were predicted from the genome sequence of S. mutans UA159. Sixty-eight strains of S. mutans were screened by PCR for carriage of the IS elements IS199, ISSmu1 and ISSmu2. Twenty-seven (30%) of the strains were positive for IS199, 33 (49%) were positive for ISSmu1 and 51 (75%) carried ISSmu2. All three IS were found in 11 strains, two were found in various combinations in 31, one was found in 16, while only 10 strains had none of the three IS for which we screened. ISSmu1 was demonstrated to be capable of transposition at a low frequency but no transposition of ISSmu2 was observed.

Genome sequencing and other molecular techniques for the identification and characterisation of bacteria have provided us with a vast amount of new information, both on the detailed properties of certain species and on the total plaque microflora. Thus, from genomics, we have gained many valuable insights into the biology of Streptococcus mutans and the diversity within the species, while molecular techniques of identification have prompted a heightened awareness of the importance of species other than S. mutans to the caries process. S. mutans remains deserving of detailed study, both as an exemplar of an acidogenic, aciduric plaque bacterium and because it shows a strong association with caries. Nevertheless, its value as a target for inhibitors and its useful- ness as an indicator organism for monitoring the value of caries prediction or prevention need to be evaluated in the light of new discoveries.

A range of properties, including the ability to utilize various sugars, bind macromolecules and produce mutacins, are known to vary in their occurrence in different strains of Streptococcus mutans. In addition, insertion-sequence elements show a limited distribution and sequencing of the genome of S. mutans UA159 has revealed the presence of putative genomic islands of atypical base composition indicative of foreign DNA. PCR primers flanking regions suspected of having inserted DNA were designed on the basis of the genome sequence of S. mutans UA159 and used to explore variation in a collection of 39 strains isolated in various parts of the world over the last 40 years. Extensive differences between strains were detected, and similar insertion/deletion events appear to be present in the genomes of strains with very different origins. In two instances, insertion of foreign DNA appears to have displaced original S. mutans genes. Together with previous results on the occurrence of deletions in genes associated with sugar metabolism, the results indicate that S. mutans has a core genome and a dispensable genome, and that dispensable genes have become widely distributed through horizontal transfer.

Ethyleneglycol dimethacrylate monomers have been previously reported to stimulate the growth of certain caries-associated bacteria on the basis of turbidity measurements. To elucidate the detail of this effect, we examined the influence of resin monomers on the growth of Streptococcus sobrinus and Streptococcus sanguis by determination of bacterial numbers (colony-forming units), morphological observation, and chemical analysis. Although the absorbance values in the stationary phase of bacterial suspension were increased in the presence of ethyleneglycol monomers, no significant differences were observed for bacterial numbers throughout the incubation period. Scanning electron microscopy observation revealed the formation of sparse vesicular material surrounding bacterial cells when incubated with ethyleneglycol monomers, and these products were proved to be resin polymers. The results demonstrate that the apparent biomass increase during incubation with ethyleneglycol monomers is due not to promotion of bacterial multiplication, but to the polymerization of resin monomers to form vesicular structures attached to cells.

This study examined the antibacterial activity of prepolymerized resin filler, in which the bactericide quaternary ammonium was immobilized. The experimental filler was prepared by grinding prepolymerized resin blocks of methacrylate monomers, silica particles, and the antibacterial monomer methacryloyloxydodecylpyridinium bromide (MDPB). The number of Streptococcus mutans after incubation for 18h in contact with the experimental filler with or without protein adsorption by saliva treatment was determined, and adherence of bacteria to the filler surface was evaluated by scanning electron microscopy. Elution of unpolymerized MDPB from the filler and its influence on bacterial growth were also investigated. The growth of S. mutans was completely inhibited by contact with the experimental filler without saliva treatment. Although the effects were attenuated, the saliva-treated filler still exhibited growth inhibition at >99.9%. Less bacteria attached to the experimental filler than the control filler without MDPB, indicating that the reduction in bacterial number after contact with the experimental filler was not due to bacterial adherence to the particles. Unpolymerized MDPB at 1 microg/ml was eluted from the filler particles but was confirmed to have little effect on bacterial growth. The results indicate that the bactericide-immobilized filler containing MDPB shows significant bacteriostatic effects without releasing antibacterial components, and is useful for incorporation into various resin-based restoratives.

To determine and compare the intrinsic antibacterial activity of commercially available simplified adhesive systems.The antibacterial activity of five self-etching/priming one-step adhesives and three priming/bonding adhesives against Streptococcus mutans, Lactobacillus casei, and Actinomyces viscosus was assessed by the agar disc-diffusion test and determination of the minimum inhibitory/bactericidal concentration (MIC/MBC). Twenty microl of each adhesive was impregnated into a paper disc and placed on an agar plate inoculated with a bacterial suspension, with or without light-irradiation. The size of inhibition zones produced around the specimens was measured after 48 hours of incubation. The MIC values were measured by serial microdilution assays, visually examining the growth of bacteria after incubation with each adhesive for 24-48 hours. The subcultures were made on BHI agar plates from the wells showing no visible growth of bacteria, and the MBC values were determined based on production of colonies. Two primers in two-step self-etching systems and an experimental primer containing chlorhexidine were tested for reference. The results of disc-diffusion tests were analyzed by ANOVA and Fisher's PLSD test.The size of inhibition zones produced by adhesives varied among the brands. None of the available commercial adhesives showed significant inhibition against all three of the bacterial species tested. Light-irradiation reduced the size of inhibition zones for a few materials, but a similar trend to the effectiveness of non-irradiated specimens was observed. The antibacterial activity of commercial products determined in terms of MIC/MBC values were different from the results by disc-diffusion tests. The self-etching adhesives with low pH were not necessarily more effective than priming/bonding solutions, and no significant relationships between the acidity and antibacterial effects were found. Compared with commercial products, an experimental primer containing chlorhexidine produced significantly larger inhibition zones against all species (P0.05) and exhibited greater bacteriostatic/bactericidal activity, demonstrating lower MIC/MBC values.

Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.

The aim of this study was to investigate bactericidal characteristics and cytotoxicity of the newly developed antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB). To evaluate the bactericidal activity of MDPB against oral streptococci, the minimum bactericidal concentration (MBC) for seven species and time-kill kinetics against Streptococcus mutans were determined. The cytotoxic effects of MDPB on human pulpal cells were assessed by [3H]-thymidine uptake after contact with MDPB solutions at various concentrations. MDPB showed strong bactericidal activity against seven streptococci, the MBC value ranging from 31.1 to 62.5 micrograms ml-1. Time-kill determination indicated a rapid killing effect of MDPB at 250 micrograms ml-1 or over, and all cells were killed within 1 min by MDPB at 500 micrograms ml-1 or over. No cytotoxic effect was observed on contact with MDPB at concentrations of 10 micrograms ml-1 or less, and the toxicity of MDPB was considered to be similar to those of other monomers used for dental materials. These results suggest that MDPB can be effectively incorporated in dental resin-based materials to provide bactericidal activity against oral bacteria.

Truncated variants of GTF-I from Streptococcus downei MFe28 were purified by means of a histidine tag. Sequential deletions showed that the C-terminal domain was not directly involved in the catalytic process but was required for primer activation. A fully active catalytic core of only 100 kDa was isolated.

Dental caries arises as a consequence of enamel demineralization by organic acid end products formed by metabolism of dietary carbohydrates by bacteria in dental plaque. The organism believed to be the principal etiological agent of dental caries is Streptococcus mutans, and high levels of S. mutans at a tooth site are indicative of a high risk of subsequent caries development (15). Factors thought to determine the levels of S. mutans include the ability of this species to ferment a wide range of substrates and to withstand conditions of low pH. S. mutans can also accumulate a glycogen-like intracellular polysaccharide (IPS) containing mainly α-1,4-linked glucose units (11). This stored IPS is believed to be of significance, in the absence of fermentable dietary carbohydrates, in production of acid which can lead to enamel demineralization and contribute to S. mutans cariogenicity (35). Dietary availability of carbohydrates is thus the major influence upon levels of S. mutans, and since starch is a major constituent of the human diet, it is important to investigate its metabolism by S. mutans. There are conflicting bodies of evidence from epidemiological studies on the cariogenicity of starch (25), though there is increasing concern that modern high-temperature processes for manufacturing starch-based food products may increase their fermentability. In North African countries, where the diet is extensively starch based, high numbers of S. mutans are found in plaque, though the fact that caries levels are generally low in these countries suggests that the means by which starch encourages S. mutans accumulation is not related to its fermentability (40). Dental plaque exhibits starch-degrading activity, though this ability may be largely due to bound salivary α-amylase (8). In one report, S. mutans was demonstrated to have some starch-degrading activity but the enzymes involved were not characterized (10). Another study indicated that utilization of starch by S. mutans was dependent on the addition of exogenous α-amylase (4). In this study, we report the nucleotide sequence of an intracellular α-amylase of S. mutans and our attempt to determine its function in the utilization of starch and in the synthesis and breakdown of IPS.

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site-directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, alpha-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function.

The polymerizable monomer methacryloyloxydodecylpyridinium bromide (MDPB) shows antibacterial activity when immobilized in a resin-based material. In this study, the antibacterial effect of a dentin primer incorporating MDPB was investigated. The influence of incorporation of MDPB on bond strength to dentin and on the curing performance of the adhesive system was also evaluated. Experimental primers were prepared by addition of MDPB into a proprietary primer at 1, 2, or 5%. Antibacterial effects of experimental primers were compared with those of control primer and two other proprietary primers by an agar disc-diffusion method and bactericidal activity test. Experimental primers produced greater inhibition zones against Streptococcus mutans, Actinomyces viscosus, and Lactobacillus casei than any of three proprietary primers, and inhibition increased as the concentration of MDPB was increased. Bactericidal activity of MDPB-containing primers against Streptococcus mutans was greater than those of the other three primers, with incorporation of MDPB at 5% showing complete killing of bacteria after 30 s contact. No decrease in tensile bond strength was observed for materials containing MDPB. On the contrary, the primer incorporating 1 and 2% MDPB showed higher bond strength than all the others, including the control (p < 0.05). When the degree of conversion of the complex of primer and adhesive resin was determined with Fourier Transform Infrared Spectroscopy, there were no significant differences between any of the experimental primers and the control (p > 0.05). These results indicate that incorporation of the antibacterial monomer MDPB enhanced the antibacterial effect of a proprietary dentin primer before curing, and had no adverse influence on bond strength to dentin and curing of the adhesive system.

The oral bacterium Streptococcus mutans possesses the ability to ferment a wide range of carbohydrates, which results in the production of acids that can cause demineralisation of tooth enamel and subsequent dental caries. However, it has been shown that approximately 11% of independent isolates of S. mutans from around the world lack the ability to ferment melibiose, raffinose, and on occasion β-glucosides (1,4). This is due to a deletion which extends through a region of the genome that includes the multiple sugar metabolism (msm) operon involved in the uptake and metabolism of a number of sugars (3). The msm region has been reintroduced to the deletion mutants by transformation and the ability to grow on galactose and β-glucosides was also restored, showing the linkage of the respective genes. The extent of the deletion is further being investigated with the use of pulsed field gel electrophoresis and Southern hybridizations with known S. mutans genes as probes.

Objectives: Previously, we have reported that dental composite incorporating the new monomer methacryloy-loxydodecylpyridinium bromide (MDPB) showed no release of antibacterial components after being cured but still exhibited antibacterial activity against Streptococcus mutans on its surface. In this study, in order to elucidate the mechanism of the antibacterial effect of immobilized MDPB, the bactericidal activity of MDPB polymer in water-soluble and -insoluble form was investigated, and the effect of MDPB polymer on bacterial attachment was estimated. Methods: Solutions of homo-polymer of MDPB and co-polymer of MDPB with acrylamide were prepared and the viability of seven major oral streptococci was determined after incubation with each polymer solution. For the estimation of bactericidal activity of insolubilized MDPB polymer, bacteria were kept in contact with cured unfilled Bis-GMA-based resin discs with or without MDPB, and the recovery of viable cells was measured. Attachment of streptococci to cured resin discs with or without MDPB was also compared using radiolabelled bacteria. Results: Water-soluble homo-polymer of MDPB and co-polymer with acrylamide showed bactericidal activity against oral streptococci. However, cured resin incorporating MDPB, which is in water-insoluble form, had little bactericidal activity. Attachment of streptococci, including species which are early colonizers in dental plaque formation, to the cured resin containing MDPB was significantly less than to the control without MDPB. Conclusions: These results indicate that the bactericidal activity of MDPB polymer is reduced after immobilization, but MDPB on the surface of a resin-based material still shows a bacteriostatic effect and antiadhesion property against oral streptococci.

Streptococcus mutans is frequently identified on the basis of phenotypic characteristics such as the ability to ferment carbohydrates. The usefulness of some of these identification tests may be limited in the case of isolates which are atypical with regard to their fermentation properties. We previously identified isolates of S. mutans which were unable to ferment melibiose, a characteristic which is included in some typing schemes. In all of these isolates there was a large chromosomal deletion which included the multiple sugar metabolism (msm) operon which encodes several genes involved in the uptake and metabolism of a number of sugars including melibiose. In the present study, sugar fermentation tests, ribotyping, colony hybridisation with DNA probes and polymerase chain reaction (PCR) were used to investigate the relatedness of these atypical isolates. The PCR and colony hybridisation procedures were based on amplification and detection of two genes: the wapA gene which encodes a surface protein found in all S. mutans strains and the gtfA gene which lies within the msm operon. The colony hybridisation and PCR results confirmed loss of the gtfA gene in the melibiose-negative isolates. Three new melibiose-negative isolates were also identified, but in only 2 of these was the gtfA gene absent, the third did not appear to have lost this region of the chromosome. Biotyping, as well as ribotyping based on an EcoRI digest of chromosomal DNA, revealed that the melibiose-negative isolates fell into a number of distinct groups.(ABSTRACT TRUNCATED AT 250 WORDS)

A glucosyltransferase (GTF) gene, designated gtfL, from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.

The range of substrates transported by the sugar-binding protein-dependent msm (multiple sugar metabolism) system of S. mutans was investigated. By determining the ability of unlabeled sugar to compete with radiolabeled melibiose transport, we have demonstrated that the transported sugars included a number of carbohydrates structurally related to raffinose. A model accommodating these results has been devised which accounts for the sugars transported by the msm transport system. Competition with radiolabeled melibiose transport indicated sucrose to be an msm substrate. This was confirmed by examination of uptake of radiolabeled sucrose in scrAB mutants lacking the sucrose-specific phosphotransferase system.

Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

Reports of the isolation of streptococci from dental abscesses have shown an association of the "S. milleri" group with such lesions. There has been considerable confusion regarding the taxonomy of these organisms, but the milleri group has recently been reclassified into three distinct species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. In this study, 45 samples from dental periapical abscesses were examined. Milleri group streptococci were isolated from 16 patients (37%), 15 being identified as S. anginosus and one as S. intermedius. In one patient, S. anginosus was isolated in pure culture, and it would appear that this is the predominant species of milleri group streptococci associated with periapical abscesses.

Metabolic labelling by [14C]palmitic acid showed that growth of Streptococcus mutans LT11 in raffinose, an inducer of the msm operon, resulted in increased production of a 45-kDa lipoprotein corresponding to MsmE, which is believed to be a sugar-binding protein. MsmE was also labelled when an msmE clone was expressed in Escherichia coli. The presence of a lipid anchor on MsmE provides a likely explanation of how the sugar-binding protein component of the msm binding protein-dependent multiple sugar transport system is retained at the cell surface.

Isolates from a collection of phenotypically melibiose-negative (Mel-) Streptococcus mutans from widely-scattered geographical locations were examined and found to lack the activities of the enzymes α-galactosidase and a-glucosidase, in addition to being unable to transport melibiose. Cloned fragments of S. mutans DNA from the region of the chromosome carrying the genes for a-galactosidase ( aga), sucrose phosphorylase ( gtfA), and dextranglucosidase ( dexB), as well as the genes encoding components of the binding-protein-dependent uptake system for raffinose and melibiose, were used in hybridization studies for investigation of the genetic basis of the Mel- phenotype. A region of at least 12 kilobases, containing all the above genes, was found to be deleted from the chromosome of the Mel- strains. It appears that this region of the chromosome is not essential for survival of S. mutans in the oral cavity. The reason for the frequent occurrence of deletions, as opposed to other forms of mutational events, is unknown.