Project description:Recently, we described the function of a novel two-gene regulatory system consisting of a LytTR family transcription regulator and a membrane protein, which we referred to as the hdrRM operon. We determined that the HdrRM system controls the expression of another similar regulatory system annotated as SMU.2080 and SMU.2081. Like hdrRM, the SMU.2080 – 2081 operon encodes a LytTR family transcription regulator and membrane protein, which we now refer to as BrsR and BrsM, respectively. In this study, we used microarray to examine the regulatory role of BrsR and found that it functions as a transcription activator for a variety of bacteriocins and bacteriocin-related genes. Streptococcus mutans UA140 derivatives, lacA ( genotype: ΔbrsRM /pFW5::PlacA ) and lacA2080 (genotype:ΔbrsRM /pFW5::PlacA-brsR), were cultivated overnight at 37°C in a chemically-defined medium containing 0.5% (wt/vol) glucose as carbon source . The overnight cultures were diluted 1:30 in CDM with 1% (wt/vol) lactose in a total volume of 30 ml. The cells were allowed to grow to an optical density at 600 nm of 0.3 and RNA was extracted. The transcriptional profile of the whole genome was examined with microarray.
Project description:Transcriptional profiling of early logarithmic phase culture (O.D=0.2-0.3) of Streptococcus mutans UA159 comparing control of untreated Streptococcus mutans UA159 bacteria with Streptococcus mutans UA159 bacteria spplemented with 20µM synthetic DPD (pre-AI-2) which regulates gene expression via AI-2 quorum sensing system.Three compairisons were performed at pHs of 7,6 and 5.
Project description:In the human pathogen Streptococcus mutans, the canonical peptide-based quorum sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work showed that the CSP signaling peptide is a stress-signaling alarmone that controls different stress-induced phenotypes. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions. Transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary phase cells and the CSP-induced stationary phase cells. The data obtained contribute to the understanding of the CSP-induced phenotypes in S. mutans.
Project description:In Streptococcus mutans, an oral colonizer associated with dental caries, competence for natural transformation can be triggered by both CSP and XIP pheromones. Competence induced by CSP is a late response that requires induction of the XIP encoding gene comS, but the mechanism(s) linking the two systems remains unknown. To learn how the CSP and XIP pheromone regulatory pathways are temporally linked, we mapped the global changes in gene expression at early and late phases of the CSP response and investigated the effect of deletion of comS on the S. mutans transcriptional profile. The early phase of the CSP response was characterized by an increase in gene expression at five loci associated with bacteriocin production and immunity. In the late phase, the up-regulated regions expanded to include a total of 27 loci, including comS and genes required for DNA uptake and recombination. In the absence of comS, no increase in expression of the genes up-regulated as a late response was observed in response to CSP, whereas expression of those regulated as an early response was maintained. These results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just five bacteriocin-related loci
Project description:RNA-Seq was used to compare the transcriptome of Streptococcus mutans UA159 during growth alone in monoculture, in coculture with Streptococcus gordonii DL1, Streptococcus sanguinis SK36 or Streptococcus oralis 34, and in a quadculture containing all four species. Individual cultures of commensal species Streptococcus gordonii DL1, Streptococcus sanguinis SK36 and Streptococcus oralis 34 were sequenced as well. This revealed a common transcriptome pattern in S. mutans when grown in mixed-species culture, indepenedent of the species identity that S. mutans was cultured with. Additionally, transcriptome changes in the commensal species could also be determined when undergoing competition from S. mutans. RNA-Seq was used to compare the transcriptome of Streptococcus mutans UA159 during growth alone in monoculture or in coculture with Streptococcus sobrinus NIDR 6715, Lactobacillus casei ATCC 4646 or Corynebacterium matruchotii ATCC 14266. These data were compared to previous coculture and quadculture RNA-Seq data with commensal streptococci (GSE209925). These data confirmed a common transcriptome pattern in S. mutans when grown in mixed-species culture with commensal streptococci that is not present with non-commensal streptococci, indepenedent of the species identity that S. mutans was cultured with.
Project description:Streptococcus mutans, an oral pathogen associated with dental caries, colonizes tooth surfaces as polymicrobial biofilms known as dental plaque. S. mutans expresses several virulence factors that allow the organism to tolerate environmental fluctuations and compete with other microorganisms. We recently identified a small hypothetical protein (90 amino acids) essential for the normal growth of the bacterium. Inactivation of the gene, SMU.2137, encoding this protein caused a significant growth defect and loss of various virulence-associated functions. An S. mutans strain lacking this gene was more sensitive to acid, temperature, osmotic, oxidative, and DNA damage-inducing stresses. In addition, we observed an altered protein profile and defects in biofilm formation, bacteriocin production, and natural competence development, possibly due to the fitness defect associated with SMU.2137 deletion. Transcriptome sequencing revealed that nearly 20% of the S. mutans genes were differentially expressed upon SMU.2137 deletion, thereby suggesting a pleiotropic effect. Therefore, we have renamed this hitherto uncharacterized gene as sprV (streptococcal pleiotropic regulator of virulence). The transcript levels of several relevant genes in the sprV mutant corroborated the phenotypes observed upon sprV deletion. Owing to its highly conserved nature, inactivation of the sprV ortholog in Streptococcus gordonii also resulted in poor growth and defective UV tolerance and competence development as in the case of S. mutans Our experiments suggest that SprV is functionally distinct from its homologs identified by structure and sequence homology. Nonetheless, our current work is aimed at understanding the importance of SprV in the S. mutans biology.
Project description:In Streptococcus mutans, an oral colonizer associated with dental caries, competence for natural transformation can be triggered by both CSP and XIP pheromones. Competence induced by CSP is a late response that requires induction of the XIP encoding gene comS, but the mechanism(s) linking the two systems remains unknown. To learn how the CSP and XIP pheromone regulatory pathways are temporally linked, we mapped the global changes in gene expression at early and late phases of the CSP response and investigated the effect of deletion of comS on the S. mutans transcriptional profile. The early phase of the CSP response was characterized by an increase in gene expression at five loci associated with bacteriocin production and immunity. In the late phase, the up-regulated regions expanded to include a total of 27 loci, including comS and genes required for DNA uptake and recombination. In the absence of comS, no increase in expression of the genes up-regulated as a late response was observed in response to CSP, whereas expression of those regulated as an early response was maintained. These results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just five bacteriocin-related loci To distinguish the immediate, and presumably direct, regulatory response to CSP from later, and presumably indirect, effects of exposure to this pheromone, we have assembled a comprehensive strand-specific microarray census of mRNA in strain UA159 at both early and late times in the CSP response as well as in a comS mutant.
Project description:Streptococcus mutans, the organism most frequently associated with the development of dental caries, is able to utilize a diverse array of carbohydrates for energy metabolism. One such molecule is trehalose, a disaccharide common in human foods, which has recently been implicated in enhancing the virulence of epidemic strains of the pathogen, Clostridium difficile. In this study, deletion mutants of all three genes in the putative S. mutans trehalose utilization operon were characterized and shown to be required for wild-type levels of growth when trehalose was the only carbohydrate source provided. Interestingly, the TreR transcriptional regulator appeared to be critical for responding to oxidative stress, and for mounting a protective stress tolerance response following growth at moderately acidic pH. RNAseq of a treR deletion mutant suggested that in S. mutans, TreR acts as a trehalose-sensing activator of transcription of the tre operon, rather than a repressor, as described in other species. In addition, deletion of treR caused the down-regulation of a number of genes involved in genetic competence and bacteriocin production, supporting results of a recent study linking trehalose and the S. mutans competence pathways. Finally, deletion of treR compromised the ability of S. mutans to inhibit the growth of the competing species, Streptococcus gordonii and Lactococcus lactis. Taken together, this study solidifies the role of the S. mutans tre operon in trehalose utilization and suggests novel functions for the TreR regulator, including roles in stress response and competitive fitness.
Project description:Transcriptional profiling of the competence-stimulating peptide (CSP) response in Streptococcus mutans of FACS-separated subpopulations and mixed cells of a clonal culture. CSP-mediated competence development in S. mutans is a transient and biphasic process since only a subpopulation induces expression of ComX in the presence of CSP and activation of the DNA uptake machinery in this fraction shuts down ~3-4 hours post induction. Here we combine, for the first time in bacteria to our knowledge, flow cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and non-competent fractions of CSP-treated S. mutans cells. Sorting was guided by a ComX-GFP reporter and the transcriptome analysis demonstrated the successful combination of both methods because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, among them ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. By contrast, expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and mutacin V confirmed this expression pattern on the single cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, uptake of DNA could be shown on the single cell level. This study demonstrates that all cells in the population respond to CSP through activation of bacteriocin-related genes but that two subpopulations segregate, one becoming competent and another one that lyses, resulting in intrapopulation diversity of the clonal culture. Two conditions: induced and not induced with CSP. Three induced cell populations: competent subpopulation, incompetent subpopulation, all cells. Two biological replicates each population.
Project description:The two-component system (TCS) is a specific regulatory system in bacteria and plays an important role in sensing and adapting to the environment. In this study, we evaluated the roles of TCSs in the major cariogenic pathogen Streptococcus mutans in resistance to several types of bacteriocin. In a comprehensive analysis using individual TCS mutants, we found that two novel TCSs were associated with resistance against distinct lantibiotics nisin A (class I type A[I]) and nukacin ISK-1(class I type A[II]). One TCS, SMU.659-660 (designated as NsrRS), was related to resistance against nisin A produced by Lactococcus lactis ATCC 11454. The other TCS, SMU.1146-1145 (designated as LcrRS), was related to resistance against nukacin ISK-1 produced by Staphylococcus warneri ISK-1. NsrRS induced the expression of SMU.658 (designated as NsrX), which constitutes an operon with nsrRS, in response to nisin A. Inactivation of nsrX increased susceptibility to nisin A. Additionally, NsrX expression in S. mutans increased the binding affinity to nisin A compared to a no-expression strain. LcrRS induced the expression of SMU.1148-50 (lctFEG), which encodes an ABC transporter and is located upstream of lcrRS, in response to nukacin ISK-1. Inactivation of lctFEG significantly increased susceptibility to nukacin ISK-1. Electrophoretic mobility shift assay analysis revealed that NsrR and LcrR bound directly to regions upstream of nsrX and lctFEG, respectively. This is the first report that two distinct TCSs, NsrRS and LcrRS, are independently involved in resistance to nisin A and nukacin ISK-1 in S. mutans.