Comprehensive transcriptome profiles distinguish the CSP and XIP signaling pathways in Streptococcus mutans UA159
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ABSTRACT:
ORGANISM(S): Streptococcus mutans UA159
PROVIDER: GSE70067 | GEO | 2015/09/01
SECONDARY ACCESSION(S): PRJNA287561
REPOSITORIES: GEO
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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.
2015-09-01 | E-GEOD-70067 | biostudies-arrayexpress
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.
2011-02-01 | E-GEOD-25284 | biostudies-arrayexpress
Project description:S. mutans UA159 was cultivated in chemically defined medium. Changes in the transcriptome in response to the autoinducing peptides CSP and XIP were monitored for 30 minutes.
2015-06-01 | GSE65982 | GEO
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.
2011-02-01 | GSE25284 | GEO
Project description:The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the ‘death effector’. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.
2010-05-29 | GSE22019 | GEO
Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community. Dual-species biofilms of S. mutans and C. albicans and single-species biofilms of S. mutans were cultivated in 24-well microtitre plates in YNBB medium. Transcriptional profiles of S. mutans in single- and dual-species biofilms were analysed at early (6 h) and late (10 h) logarithmic phase of the biofilm growth, as well as after 24 h when biofilms entered stationary phase. Transcriptional profiles of S. mutans grown in the dual-species biofilms were compared to profiles obtained for single-species biofilm from the same time point. Three biological and one to two technical replicas were used in the microarray study. RNA samples were labeled with Cy3 or Cy5 using the ULS fluorescent labeling kit (Kreatech, Germany). Seven hundred nanograms of Cy3 or Cy5 labeled RNA after fragmentation were hybridized to the microarray at 65M-BM-0C for 17 h using the Agilent hybridization chamber according to the manufacturer's instructions. The arrays were scanned using the Agilent DNA microarray scanner and the raw data were extracted using Agilent Feature Extraction software (v. 10.7).
2014-09-01 | E-GEOD-52543 | biostudies-arrayexpress
Project description:The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the M-bM-^@M-^Xdeath effectorM-bM-^@M-^Y. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm. Streptococcus mutans UA159 were grown with 2 uM CSP or without (uninduced control) to mid-log phase. Total RNA was extracted as described above. The cDNAs were prepared for hybridization using the PFGRC protocol. Microarray chips were scanned using a Gene Pix 4000B (Axon) and analyzed using the TM4 Microarray Software Suite (http://www.tm4.org/). Transcript levels were measured by cDNA hybridized to a fourfold redundant S. mutans microarray and averaged for three replicated hybridizations. Differential gene expression was based on a post-normalization cut-off of M-BM-1> twofold.
2010-05-29 | E-GEOD-22019 | biostudies-arrayexpress
Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community.
2014-09-01 | GSE52543 | GEO
Project description:Streptococcus gordonii is a primary colonizer of the multispecies biofilm on tooth surfaces forming dental plaque, and a potential agent of endocarditis. The recent completion of the genome sequence of the naturally competent strain Challis allowed the design of a spotted oligonucleotide microarray to examine a genome-wide response of this organism to environmental signals. Based on temporal responses to synthetic competence signaling peptide (CSP) as indicated by transformation frequencies, the S. gordonii transcriptome was monitored at increments after CSP exposure. Microarray analysis identified 35 candidate early genes and 127 candidate late genes that were up-regulated at 5 and 15 minutes, respectively; these genes were often grouped in clusters. Findings supported published literature on the S. gordonii competence response, with up-regulation of most, but not all, genes that have been reported to affect this species' transformation frequencies. The CSP-induced transcriptomes of S. gordonii were compared to those of published S. pneumoniae strains. Both conserved and species-specific genes were identified. Putative intergenic regulatory sites such as the conserved combox sequence thought to be a binding site for competence sigma factor, were found preceding S. gordonii late responsive genes. In contrast, S. gordonii early CSP-responsive genes were not preceded by S. pneumoniae conserved direct repeats. These studies provide the first insights into a genome-wide transcriptional response of an oral commensal organism. They offer an extensive analysis of transcriptional changes that accompany competence in S. gordonii and form a basis for future intra- and inter-species comparative analyses of this ecologically important phenotype. Keywords: gene expression design
2007-10-30 | GSE8758 | GEO
Project description:BACKGROUND: Streptococcus mutans, the etiological agent of human dental caries, displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (comX inducing peptide, XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. ComR-XIP controls the expression of both PcomX and PcomS, with the latter creating a positive feedback loop. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we began to dissect how XrpA exerts control over competence development in S. mutans. METHODS: RNA-Seq was utilized to compare the transcriptomes of S. mutans wild-type strain UA159 (3 replicates), UA159 with addition of 2 uM sXIP (3 replicates) and with a mutant of xrpA (comX::T162C; 3 replicates). Strains were grown to OD600 nm = 0.5 in FMC medium before harvest. sXIP was added at OD600 nm = 0.2. Deep sequencing was performed at the University of Florida ICBR facilities (Gainesville, FL). Approximately 20 million short-reads were obtained for each sample. After removing adapter sequences from each short-read and trimming of the 3’-ends by quality scores (59), the resulting sequences were mapped onto the reference genome of strain UA159 (GenBank accession no. AE014133) using the short-read aligner. Mapped short-read alignments were then converted into readable formats using SAMTOOLS. RESULTS: Using an optimzed data analysis workflow, we mapped 14-19 million reads per sample to the genome of UA159. For viewing of the mapped reads aligned to the genome, .bam files were uploaded into the Integrative Genomics Viewer (IGV – version 2.3.55) (61). A .csv file containing raw read counts for each replicate (3) was then uploaded to Degust (http://degust.erc.monash.edu/) and edgeR analysis performed to determine Log2 fold change and a false discovery rate (FDR). Comparison of the transcriptome by RNA-Seq of the wild-type with the mutant lacking XrpA revealed 56 differentially expressed genes, with 34 upregulated in xrpA and 22 downregulated. Many of the upregulated genes were competence-related genes, including comX and genes that are a part of the ComX regulon of S. mutans . Since loss of xrpA caused upregulation of competence genes, we also analyzed the transcriptome of UA159 treated with 2 uM sXIP to induce competence, with UA159 treated with vehicle (DMSO) as a control. Cells treated with sXIP had 137 genes differentially expressed compared to the DMSO control. CONCLUSIONS: Collectively, these data reveal that the novel regulator XrpA exerts its influence over competence development by impacting ComRS functions, resulting in decreased expression of comX and late com gene expression that negatively impact transformability. These results highlight XrpA as a new negative regulator of competence signaling as well as broaden our understanding of the complex regulatory mechanisms that modulate competence and virulence in S. mutans.
2018-02-13 | GSE110167 | GEO