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Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

ABSTRACT: Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (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, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (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, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity.

SUBMITTER: Lemme A

PROVIDER: S-EPMC3133041 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

Lemme André A Gröbe Lothar L Reck Michael M Tomasch Jürgen J Wagner-Döbler Irene I

Journal of bacteriology 20110211 8

Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompete ...[more]

PMID: 21317319

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Project description:Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm.IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

Project description:In Streptococcus mutans, an oral colonizer associated with dental caries, development of competence for natural genetic transformation is triggered by either of two types of peptide pheromones, competence-stimulating peptides (CSPs) (18 amino acids [aa]) or SigX-inducing peptides (XIPs) (7 aa). Competence induced by CSP is a late response to the pheromone that requires the response regulator ComE and the XIP-encoding gene comS. XIP binds to ComR to allow expression of the alternative sigma factor SigX and the effector genes it controls. While these regulatory links are established, the precise set of effectors controlled by each regulator is poorly defined. To improve the definition of all three regulons, we used a high-resolution tiling array to map global changes in gene expression in the early and late phases of the CSP response. The early phase of the CSP response was limited to increased gene expression at four loci associated with bacteriocin production and immunity. In the late phase, upregulated regions expanded to a total of 29 loci, including comS and genes required for DNA uptake and recombination. The 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 four bacteriocin-related loci. Comparison of the new data with published transcriptome data permitted the identification of all of the operons in each regulon: 4 for ComE, 2 for ComR, and 21 for SigX. Finally, a core set of 27 panstreptococcal competence genes was identified within the SigX regulon by comparison of transcriptome data from diverse streptococcal species. IMPORTANCES. mutans has the hard surfaces of the oral cavity as its natural habitat, where it depends on its ability to form biofilms in order to survive. The comprehensive identification of S. mutans regulons activated in response to peptide pheromones provides an important basis for understanding how S. mutans can transition from individual to social behavior. Our study placed 27 of the 29 transcripts activated during competence within three major regulons and revealed a core set of 27 panstreptococcal competence-activated genes within the SigX regulon.

Project description:Streptococcus gallolyticus subsp. gallolyticus, a member of the group D streptococci, is normally found in the bovine rumen and human gut. It is an opportunistic pathogen that was recently determined to be a bacterial driver of colorectal cancer, in addition to causing other diseases, such as infective endocarditis, bacteremia, neonatal meningitis, and septicemia. As an emerging pathogen, not much is known about this bacterium, its virulence mechanisms, or its virulence regulatory pathways. Previous studies suggest that S. gallolyticus subsp. gallolyticus uses a ComRS pathway, one of many Streptococcus quorum-sensing circuitries, for competence. However, thus far, the ubiquitous ComABCDE pathway has not been studied, nor has its regulatory role in S. gallolyticus subsp. gallolyticus We therefore sought to study the S. gallolyticus subsp. gallolyticus ComABCDE quorum-sensing pathway and have identified its peptide pheromone, which is termed the competence-stimulating peptide (CSP). We further determined that this peptide regulates the production of bacteriocin-like inhibitory substances (BLISs), a phenotype that has been linked with the ComABCDE pathway in both Streptococcus pneumoniae and Streptococcus mutans Our data show that S. gallolyticus subsp. gallolyticus TX20005 produces a 21-mer CSP signal, which differs from CSP signals of other Streptococcus species in that its active form begins three residues after the double-glycine leader signal of the ComC precursor peptide. Additionally, our data suggest that this peptide might not be related to competence induction, as opposed to CSP signaling peptides in other Streptococcus species. This study provides the first evidence that S. gallolyticus subsp. gallolyticus utilizes quorum sensing to eliminate competitors, presenting a potential pathway to target this emerging human pathogen.IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus is an emerging human pathogen known as a causative agent of infective endocarditis, and recently, of colorectal cancer. In this work, we revealed a functional quorum-sensing circuitry in S. gallolyticus subsp. gallolyticus, including the identification of the central signaling peptide pheromone, competence-stimulating peptide (CSP), and the regulatory role of this circuitry in the production of bacteriocin-like inhibitory substances (BLISs). This work uncovered a mechanism by which this bacterium outcompetes other bacterial species and thus provides a potential tool to study this opportunistic pathogen.

Dataset Information

Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

Publications

Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

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