Mutacin Production Research Articles

Nattokinase, a Subtilisin-like Alkaline-Serine Protease, Reduces Mutacin Activity by Inactivating the Competence-Stimulating Peptide in Streptococcus mutans.

Streptococcus mutans is a major cariogenic organism because of its ability to form biofilms on tooth surfaces. Bacteriocins produced by S. mutans (known as mutacins) are indirect pathogenic factors that play a role in the persistence of this microbe in the oral environment. Nattokinase, a subtilisin-like alkaline serine protease, potently inhibits biofilm formation without affecting S. mutans growth. However, effective strategies utilizing nattokinase to control mutacin production by S. mutans are lacking. In this study, we evaluated the effect of nattokinase on mutacin activity in 46 strains of S. mutans with different mutacin genotypes isolated from the dental plaques of pediatric patients with caries. Nattokinase reduced the activity of mutacin against oral streptococci at a concentration of 1 mg/mL in all clinical isolates. Furthermore, nattokinase reduced the expression of non-lantibiotic mutacin structural genes (nlmABCD) and inactivated the extracellular competence-stimulating peptide involved in comDE activation, which regulates non-lantibiotic mutacin gene expression. These results suggest that nattokinase may reduce the virulence of S. mutans and could potentially be used as a new caries-preventive agent as an alternative to conventional drug treatments.

VicR overexpression in Streptococcus mutans causes aggregation and affects interspecies competition.

Streptococcus mutans is considered to be a major causative agent of dental caries. VicRK is a two-component signal transduction system (TCSTS) of S. mutans, which can regulate the virulence of S. mutans, such as biofilm formation, exopolysaccharide production, acid production, and acid resistance. Meanwhile, it can also regulate the production of mutacins (nlmC) through the TCSTS ComDE. In this study, we found that the vicR-overexpressing strain was more likely to aggregate to form cell clusters, leading to the formation of abnormal biofilm; the overexpression of vicR increased the length of the chain of S. mutans. Furthermore, the expression of the mutacins in the vicR overexpression strain was increased under aerobic conditions. Compared with the control strain and the parental strain, the vicR overexpression strain was more competitive against Streptococcus gordonii. But there was no significant difference against Streptococcus sanguinis. In clinical strains, the expression level of vicR was positively correlated with their competitive ability against S. gordonii. Transcriptional profiling revealed 24 significantly upregulated genes in the vicR-overexpressing strain, including nlmA, nlmB, nlmC, and nlmD encoding mutacins. Electrophoretic mobility shift assays and DNase I footprinting assays confirmed that VicR can directly bind to the promoter sequence of nlmD. Taken together, our findings further demonstrate that VicRK, an important TCSTS of S. mutans, is involved in S. mutans cell morphology and biofilm formation. VicRK regulates the production of more mutacins in S. mutans in response to oxygen stimulation. VicR can bind to the promoter sequence of nlmD, thereby directly regulating the production of mutacins NlmD.

Analysis of the Streptococcus mutans Proteome during Acid and Oxidative Stress Reveals Modules of Protein Coexpression and an Expanded Role for the TreR Transcriptional Regulator.

ABSTRACTStreptococcus mutans promotes a tooth-damaging dysbiosis in the oral microbiota because it can form biofilms and survive acid stress better than most of its ecological competitors, which are typically health associated. Many of these commensals produce hydrogen peroxide; therefore, S. mutans must manage both oxidative stress and acid stress with coordinated and complex physiological responses. In this study, the proteome of S. mutans was examined during regulated growth in acid and oxidative stresses as well as in deletion mutants with impaired oxidative stress phenotypes, Δnox and ΔtreR. A total of 607 proteins exhibited significantly different abundances across the conditions tested, and correlation network analysis identified modules of coexpressed proteins that were responsive to the deletion of nox and/or treR as well as acid and oxidative stress. The data explained the reactive oxygen species (ROS)-sensitive and mutacin-deficient phenotypes exhibited by the ΔtreR strain. SMU.1069-1070, a poorly understood LytTR system, had an elevated abundance in the ΔtreR strain. S. mutans LytTR systems regulate mutacin production and competence, which may explain how TreR affects mutacin production. Furthermore, the protein cluster that produces mutanobactin, a lipopeptide important in ROS tolerance, displayed a reduced abundance in the ΔtreR strain. The role of Nox as a keystone in the oxidative stress response was also emphasized. Crucially, this data set provides oral health researchers with a proteome atlas that will enable a more complete understanding of the S. mutans stress responses that are required for pathogenesis, and will facilitate the development of new and improved therapeutic approaches for dental caries.IMPORTANCE Dental caries is the most common chronic infectious disease worldwide and disproportionately affects marginalized socioeconomic groups. Streptococcus mutans is considered a primary etiological agent of caries, with its pathogenicity being dependent on coordinated physiological stress responses that mitigate the damage caused by the oxidative and acid stress common within dental plaque. In this study, the proteome of S. mutans was examined during growth in acidic and oxidative stresses as well in nox and treR deletion mutants. A total of 607 proteins were differentially expressed across the strains/growth conditions, and modules of coexpressed proteins were identified, which enabled mapping the acid and oxidative stress responses across S. mutans metabolism. The presence of TreR was linked to mutacin production via LytTR system signaling and to oxidative stress via mutanobactin production. The data provided by this study will guide future research elucidating S. mutans pathogenesis and developing improved preventative and treatment modalities for dental caries.

Complete Genome Sequences of Two Mutacin-Producing Streptococcus mutans Strains, T8 and UA140.

Streptococcus mutans is known to produce various antimicrobial peptides called mutacins. Two clinical isolates, T8 and UA140, are well characterized regarding their mutacin production, but genome sequence information was previously unavailable. Complete genome sequences of these two mutacin-producing strains are reported here.

Insight into the Effect of Small RNA srn225147 on Mutacin IV in Streptococcus mutans.

Streptococcus mutans (S. mutans) is a serious microbe causing dental caries. Mutacin IV is an effective bacteriocin produced by S. mutans to antagonize numerous non-mutans streptococcal species. However, the posttranscriptional regulation of mutacin IV remains unclear. This study aimed to analyze the effect of small RNA srn225147 on mutacin IV. The functional prediction suggested that srn225147 is involved in the production of mutacin IV, an important secondary metabolite. According to RNAhybrid and RNAPredator prediction, the mutacin IV formation-associated gene comD is a target of srn225147. We further analyzed the roles of srn225147 and comD in 20 S. mutans clinical strains with high production of mutacin IV (High-IV group) and lacking mutacin IV (None-IV group). Levels of comD expression were significantly higher in the High-IV group, whereas the Non-IV group showed relatively higher expression of srn225147, with a negative correlation observed between srn225147 and comD. Moreover, compared to the mimic negative control (NC) group, comD expression was decreased at 400-fold srn225147 overexpression but increased at approximately 1400-fold overexpression. Although the production of mutacin IV in the 1400-fold change srn225147 mimic group was larger than that in the 400-fold change mimic group, there was no significant difference in the production of mutacin IV between the srn225147 mimic group and mimic NC group. These results indicate that srn225147 has a two-way regulation effect on the expression of comD but that its regulation in the production of mutacin IV is weak.

Dissecting structure/function relationship of Streptococcus mutans membrane protein chaperones/insertases, YidC1 and YidC2

BackgroundApproximately, one third of most bacterial open reading frames encode membrane proteins; therefore, understanding the machinery that translocates these proteins to their functional location is of great interest. Most membrane proteins are translocated co‐translationally by the signal recognition particle (SRP) pathway via the SecYEG translocon. Additionally the Oxa/Alb/YidC (mitochondria/chloroplast/bacteria) family of membrane‐localized protein chaperone/insertases assists in membrane protein translocation in a Sec‐dependent manner, or independently if the proteins are small with two or fewer transmembrane (TM) domains. Most gram‐negative bacteria contain a single yidC whose deletion is lethal. Most gram‐positive bacteria contain two or more YidCs. The cariogenic bacterium S. mutans encodes YidC1 and YidC2, each with similar 5 TM domain topologies. Deletion of yidC2 results in a severe phenotype similar to disruption of the SRP pathway including impaired growth, sensitivity to acid, osmotic, and oxidative stresses, and loss of competence and mutacin production. Deletion of DyidC1 leads to increased localization of surface proteins and hyperadherence. The goal of this study is to better understand the structure and function of YidC1 and YidC2 as related to their known phenotypes.MethodsThe significance of the two cytoplasmic loops, C1 and C2, of YidC1 and YidC2 were evaluated in domain swapping experiments in which DNA segments encoding one or both C1 and C2 domains of yidC1 were incorporated into yidC2 and vice versa. Chimeric genes were integrated into the non‐essential gtfA locus of the DyidC1 and DyidC2 strains. Next the residual endogenous yidC was deleted, thus generating strains with a sole chimeric YidC for phenotypic analyses. In addition, a naturally‐acquired W138R point mutation within yidC1 was identified in a phenotypically‐reverted DyidC2 mutant; therefore, the significance of this residue was evaluated as well.Results and ConclusionsAn S. mutans strain producing a chimeric YidC2 harboring the C1 loop of YidC1 was similar to the DyidC2 strain with respect to sensitivity to acid and osmotic stress suggesting a functional contribution of YidC2's C1 loop to stress tolerance. Transfer of YidC2 C1 into YidC1 enabled YidC1 to rescue acid and osmotic stress sensitivity. Also, a deliberately engineered W138R point mutation within YidC1 enabled it to rescue growth and stress phenotypes of the DyidC2 mutant. Sequence comparison revealed that the YidC1 tryptophan 138 residue corresponds to serine 152 in YidC2, the only polar residue within its TM3. Additionally, engineered W138K/S mutations within YidC1 also enabled rescue of DyidC2‐associated phenotypes. These data reveal that functional activities unique to YidC2 reside within both its C1 domain and TM3 that can be transferred to YidC1.Support or Funding InformationNIH grant: R01 DE08007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning

Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as to the role of backbone amide protons in the interaction between CSP and the ComD receptor. We then utilized the reverse alanine approach to develop CSP-based analogs with enhanced activities. The two most potent analogs were found to induce bacteriocin production at sub-nanomolar concentration using an interspecies inhibition assay. Overall, our analysis revealed that the 18-CSP sequence is not optimized and can be improved by replacement of multiple positions with alanine. Our results further suggest that the hydrophobic residues in S. mutans 18-CSP are involved in both receptor binding and activation.

Efflux inhibitor suppresses Streptococcus mutans virulence properties.

It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans.

Mutacins and bacteriocins like genes in Streptococcus mutans isolated from participants with high, moderate, and low salivary count

ObjectiveTo detect S. mutans producers of mutacins and bacteriocins like substances (BLIS) from saliva of participants with low, moderate, and high salivary counts. Material and methods123 strains of S. mutans were obtained from participants with low, moderate, and high salivary counts (age 18 and 20 years old) and their antibacterial capacity analyzed. By using PCR amplification, the expression levels of mutacins and BLIS genes were studied (expressed in arbitrary units/ml) in all three levels. ResultsS. mutans strains from participants with low salivary counts show high production of mutacins (63%). In contrast, participants with moderate and high salivary counts depict relatively low levels of mutacins (22 and 15%, respectively). Moreover, participants with low salivary counts showed high expression levels of genes encoding mutacins, a result that correlates with the strong antimicrobial activity of the group. Participants with moderate and high salivary counts however depict low expression levels of mutacin related genes, and little antimicrobial activity. No BLIS were detected in any of the groups studied. ConclusionS. mutans isolated from the saliva of participants with low bacterial counts have significant antibacterial capacity compared to that of participants with moderate and high salivary counts. The superior lethality of S. mutans in participants with low salivary counts is likely due to the augmented expression of mutacin- related genes.

Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production.

The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcusmutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence.

Evaluation of Antimicrobial susceptibility pattern of Streptococcus mutans isolated from dental plaques to chlorhexidine, nanosil and common antibiotics

Streptococcus mutans is one of the most important leading causes of dental diseases worldwide and is considered as one of the main causative agent of dental caries. Increasing resistance of oral pathogens to conventional antibacterial agents has resulted to find alternative therapies to overcome resistance development problems. The aim of this study was to examine susceptibility of Streptococcus mutans isolates to some antibiotics, chlorhexidine and nanosil. The study subjects comprised of caries active individual volunteers attending the outpatient department of different dental college hospitals of Kerman. The saliva sample of 2-5 ml was collected from each individual in sterile capped bottles and were immediately transported to the laboratory and processed for the screening of S. mutans. The specific selective media, Blood agar and Tryptone yeast cysteine media agar were used for the screening and isolation of Streptococcus mutans and incubated anaerobically at 37?C for 48 hrs. Colonies of mutans streptococci were examined under a dissecting microscope and identified by their distinctive colony morphology and biochemical tests .Mutacin production of all isolates were investigated.The isolates were tested for susceptibility to some antibiotics (penicillin, gentamycin, vancomycin, cephalotin), chlorhexidine and nanosil mouthwashes by disc diffusion method. The results showed that the majority of mutacin and non mutacin-producing isolates were more sensitive to the nanosil than antibiotics and chlorhexidine. More extensive research on the use of mouthwashes containing silver nanoparticles is suggested.DOI: http://dx.doi.org/10.3126/ijls.v9i2.12040 International Journal of Life Sciences 9 (2) : 2015; 18-21

ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans

The ComED two-component signal transduction system (TCSTS) and the exogenous CSP can provoke the competence development and mutacin production of Streptococcus mutans. More binding fashions of ComE with DNA are the molecular basis for the ComED TCSTS to play pleiotropic roles. The interaction of nlmC and comC in regulation makes the expression of nlmC different from that of nlmAB and the ComE molecular feature is shown in the regulation pattern of nlmC. By electrophoretic mobility shift assay, ComE binding sites were discoveried in PnlmC314 and PnlmC419 for the first time. In the wild-type background, the promoter activity of nlmC is not only stronger than that of nlmAB but also rushes to a peak value of 3,116.0 MU at the point of 100 min in liquid culture with no supply of exogenous CSP; the data indicate that the regulation of nlmC expression is charged in an known coordination mechanism. In the mutant background, by monitoring the promoter activity of nlmC and nlmAB, it is proved that they are both controlled directly by the ComED TCSTS. But the surprising result is the deletion of comC to make nlmC promoter lose its activity completely. Based on those data, it is deduced that Pnlm419, PnlmC314 and PnlmC, which comprise the nlmC regulating region, coordinate to incite nlmC expression and to make nlmC expression different from that of nlmAB because of a synergistic effect, whereas they also could inhibit comC expression by blocking the RNA polymerase moving through the binding of ComE with the comC encoding region.

An extracelluar protease, SepM, generates functional competence-stimulating peptide in Streptococcus mutans UA159.

Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium Streptococcus mutans employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In S. mutans, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for streptococcal extracellular protease required for mutacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in S. mutans.

The mutacins of Streptococcus mutans: regulation and ecology

Streptococcus mutans is generally recognized as a causative agent of human dental caries. The production of mutacins (bacteriocins) by S.mutans is considered to be an important factor in the colonization and establishment of S.mutans in the dental biofilm. Two types of mutacins have been characterized: the lantibiotics and the non-lantibiotics. The lantibiotics generally have a wider spectrum of activity than the non-lantibiotics, which make them attractive targets for development into new antimicrobial modalities. The non-lantibiotics are much more prevalent among strains of S.mutans and play a significant role in both community-level and population-level interactions in the dental biofilm. These interactions are directly mediated through the ComCDE two-component system and the newly characterized LytTR Regulation Systems HdrRM and BrsRM. These systems coordinate natural competence development and mutacin production as a means to acquire transforming DNA either by killing closely related streptococcal species in the vicinity of S.mutans, or through an altruistic suicide mechanism among a subpopulation of competent cells within the S.mutans community. As more S.mutans strains are sequenced, it is anticipated that additional mutacins with novel functions will be discovered, which may yield further insights into the ecological role of mutacins within the oral biofilm.

Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as expression of virulence properties. However, with the exception of the Spx global regulator, the molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans are not well understood. Here, microarray analysis was used to provide novel insights into the scope of ClpXP proteolysis in S. mutans. In a ΔclpP strain, 288 genes showed significant changes in relative transcript amounts (P≤0.001, twofold cut-off) as compared with the parent. Similarly, 242 genes were differentially expressed by a ΔclpX strain, 113 (47 %) of which also appeared in the ΔclpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the Δclp strains. Among the upregulated genes were those encoding enzymes required for the biosynthesis of intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced expression of glg and mle genes in ΔclpP and ΔclpX strains correlated with increased storage of intracellular polysaccharide and enhanced malolactic fermentation activity, respectively. Expression of several genes known or predicted to be involved in competence and mutacin production was downregulated in the Δclp strains. Follow-up transformation efficiency and deferred antagonism assays validated the microarray data by showing that competence and mutacin production were dramatically impaired in the Δclp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulence-related traits that are influenced by ClpXP proteolysis.

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

BackgroundThe increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances.ResultsMutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested.ConclusionMutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

Optimization of the production of the lantibiotic mutacin 1140 in minimal media

Mutacin 1140 is produced by Streptococcus mutans and belongs to the type A lantibiotic family. Experiments were done to optimize production of mutacin 1140 in minimal media enabling a more cost efficient downstream purification method. The development of a small volume fermentation method enabled a rapid screen of several variables in a standard shaking incubator. This method provided a fast approach for determining components that promote mutacin 1140 production in minimal media broth. Lactose was determined to be the optimal carbon source for mutacin 1140 production. High concentrations of CaCl(2) (0.3% w/v) and MgSO(4) (0.77% w/v) promoted an increase in mutacin 1140 production, while ZnCl(2) and FeCl(3) appeared to impair production. Optimization of mutacin 1140 production in minimal media resulted in more than a 100-fold increase in production compared to the base medium used to begin our optimizations. The yield has been estimated by RP-HPLC to be ~10 mg/L.

ClpP of Streptococcus mutans Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance

Streptococcus mutans is the primary etiological agent of human dental caries and, at times, of infective endocarditis. Within the oral cavity, the pathogen is subjected to conditions of stress. A well-conserved protein complex named ClpP (caseinolytic protease) plays a vital role in adaptation under stress conditions. To gain a better understanding of the global role of the ClpP protease in cellular homeostasis, a transcriptome analysis was performed using a DeltaclpP mutant strain. The expression levels of more than 100 genes were up- or downregulated in the DeltaclpP mutant compared to the wild type. Notably, the expression of genes in several genomic islands, such as TnSmu1 and TnSmu2, was differentially modulated in the DeltaclpP mutant strain. ClpP deficiency also increased the expression of genes associated with a putative CRISPR locus. Furthermore, several stress-related genes and genes encoding bacteriocin-related peptides and many transcription factors were also found to be altered in the DeltaclpP mutant strain. A comparative analysis of the two-dimensional protein profile of the wild type and the DeltaclpP mutant strains showed altered protein profiles. Comparison of the transcriptome data with the proteomic data identified four common gene products, suggesting that the observed altered protein expression of these genes could be due to altered transcription. The results presented here indicate that ClpP-mediated proteolysis plays an important global role in the regulation of several important traits in this pathogen.

Genes involved in the repression of mutacin I production in Streptococcus mutans

Streptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibiotic mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high-phosphate brain heart infusion agar medium that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones for which we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups, including central metabolism, surface binding and sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when the gene is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.

A phenotypic microarray analysis of a Streptococcus mutans liaS mutant

Streptococcus mutans, a biofilm-forming Gram-positive bacterium that resides in the human oral cavity, is considered to be the primary aetiological agent of human dental caries. A cell-envelope stress-sensing histidine kinase, LiaS, is considered to be important for expression of virulence factors such as glucan-binding protein C and mutacin production. In this study, a liaS mutant was subjected to phenotypic microarray (PM) analysis of about 2000 phenotypes, including utilization of various carbon, nitrogen, phosphate and sulfur sources; osmolytes; metabolic inhibitors; and susceptibility to toxic compounds, including several types of antibiotics. Compared to the parental strain UA159, the liaS mutant strain (IBS148) was more tolerant to various inhibitors that target protein synthesis, DNA synthesis and cell-wall biosynthesis. Some of the key findings of the PM analysis were confirmed in independent growth studies and by using antibiotic discs and E-test strips for susceptibility testing.