Kinetic analysis of carolacton regulated genes
Ontology highlight
ABSTRACT:
ORGANISM(S): Streptococcus mutans UA159
PROVIDER: GSE26441 | GEO | 2011/08/01
SECONDARY ACCESSION(S): PRJNA136787
REPOSITORIES: GEO
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Project description:Transcriptional profiling of carolacton-treated Streptococcus mutans biofilm cells in comparison to untreated biofilms in a time series approach.
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Project description:Carolacton is a novel biofilm inhibitor that kills biofilm cells of Streptococcus mutans and inhibits growth of the clinically relevant and human pathogenic bacterium Streptococcus pneumoniae TIGR4 in nanomolar concentrations. Interestingly, Carolacton also inhibits Escherichia coli with a defective outer membrane protein TolC . The cellular target of Carolacton is still unknown. Here, we adressed the differential transcription of cellular RNAs when E.coli TolC was grown in the presence of Carolacton. This was done to identify transcriptional regulatory networks that are directly affected by treatment of the strain with Carolacton. In order to gain insights into the primary transcriptional response, early time-points were chosen for sampling, which should not reflect secondary responses (e.g. due to differences in growth phase, nutrient depletion etc.).
2017-02-15 | GSE93125 | GEO
Project description:Carolacton is a novel biofilm inhibitor that kills biofilm cells of Streptococcus mutans in nanomolar concentrations. Interestingly, Carolacton also inhibits growth of the clinically relevant and human pathogenic bacterium Streptococcus pneumoniae TIGR4. The cellular target of Carolacton is still unknown. Here, we adressed the differential transcription of cellular RNAs when S. pneumoniae TIGR4 was grown in the presence of Carolacton. This was done to identify transcriptional regulatory networks that are directly affected by treatment of the pneumococcus with Carolacton. In order to gain insights into the primary transcriptional response, early time-points were chosen for sampling, which should not reflect secondary responses (e.g. due to differences in growth phase, drop in pH etc.). To achieve a thorough overview over all affected cellular RNA species, such as mRNAs, small regulatory RNAs and tRNAs, and not to lose small transcripts during library preparation, RNAs were separated according to size and used to construct two separate libraries for sequencing.
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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).
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Project description:Streptococcus mutans was grown for 48 h in a biofilm in the absence (single species) and in the presence (dual species) of Veillonella parvula. In addition V. parvula single species 48 h biofilms were grown, to be used as a control. RNA was harvested from all types of biofilms and the transcript levels of the two types of biofilms containing S. mutans were compared with the use of S. mutans microarrays. V. parvula RNA was hybridized to S. mutans microarrays as a control for possible cross-hybridisation.
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