Project description:Transcriptional profiling of the M-NM-^T835np mutant vs the wild-type strain grown in BHI to OD600=0.5. An intimate linkage between the regulation of biofilm formation, stress tolerance and genetic competence exists in the dental caries pathogen Streptococcus mutans. We previously reported that the rcrRPQ genes, which encode ABC exporters (RcrPQ) and a MarR-family transcriptional regulator (RcrR) that represses the expression of the rcr operon, play a dominant role in regulation of development of genetic competence and connect competence with stress tolerance and (p)ppGpp production in S. mutans. Here we identify the region required for efficient RcrR binding to the rcr promoter region using purified recombinant RcrR (rRcrR) protein in electrophoretic mobility shift and fluorescence polarization assays. DNA fragments carrying mutations in the binding region resulted in decreased binding of rRcrR. Direct binding of RcrR to the comX gene promoter in vitro revealed that RcrR has the potential to directly regulate competence development. Likewise, direct interactions of rRcrR with the promoter region of relP, encoding a (p)ppGpp synthase, show the potential for RcrR-dependent modulation of (p)ppGpp pools. Introduction of mutations into the RcrR binding sites in the rcrR promoter region in vivo demonstrated that optimal RcrR binding is essential for proper expression of competence genes and optimal transformation efficiency. Transcription profiling by RNA-seq and mircroarrays of strains with mutations in rcrR showed that genes in the competence pathway were those most differentially expressed. Thus, here and in a accompanying paper, we establish mechanisms by which the products of the rcrRPQ operon integrate com gene expression, genetic transformation, ppGpp metabolism and stress tolerance in S. mutans Two-condition experiment, M-NM-^T835np mutant vs the wild-type strain. Biological replicates: 4 per strain, independently grown and harvested. One replicate per array

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.

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).

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.

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835np mutant vs wild-type

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