Growth phase and pH influence peptide signaling for competence development in Streptococcus mutans.
ABSTRACT: The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.
Project description:Streptococcus mutans expresses comX (also known as sigX), which encodes a sigma factor that is required for development of genetic competence, in response to the peptide signals XIP and CSP and environmental factors. XIP (sigX inducing peptide) is derived from ComS and activates comX unimodally in chemically defined media via the ComRS system. CSP (competence stimulating peptide) activates comX bimodally in peptide-rich media through the ComDE two-component system. However, CSP-ComDE activation of comX is indirect and involves ComRS. Therefore, the bimodality of CSP-dependent activation of comX may arise from either ComRS or ComDE. Here we study, at the single-cell level, how genes in the CSP signaling pathway respond to CSP, XIP and media. Our data indicate that activation of comX stimulates expression of comE. In addition, activation of comE requires intact comR and comS genes. Therefore, not only does CSP-ComDE stimulate the ComRS pathway to activate comX expression, but ComRS activation of comX also stimulates expression of the CSP-ComDE pathway and its regulon. The results demonstrate the mutual interconnection of the signaling pathways that control bacteriocin expression (ComDE) and genetic competence (ComRS), both of which are linked to lytic and virulence behaviors.
Project description:Genetic competence in Streptococcus mutans is a transient state that is regulated in response to multiple environmental inputs. These include extracellular pH and the concentrations of two secreted peptides, designated CSP (competence-stimulating peptide) and XIP (comX-inducing peptide). The role of environmental cues in regulating competence can be difficult to disentangle from the effects of the organism's physiological state and its chemical modification of its environment. We used microfluidics to control the extracellular environment and study the activation of the key competence gene comX. We find that the comX promoter (PcomX) responds to XIP or CSP only when the extracellular pH lies within a narrow window, about 1 pH unit wide, near pH 7. Within this pH range, CSP elicits a strong PcomX response from a subpopulation of cells, whereas outside this range the proportion of cells expressing comX declines sharply. Likewise, PcomX is most sensitive to XIP only within a narrow pH window. While previous work suggested that comX may become refractory to CSP or XIP stimulus as cells exit early exponential phase, our microfluidic data show that extracellular pH dominates in determining sensitivity to XIP and CSP. The data are most consistent with an effect of pH on the ComR/ComS system, which has direct control over transcription of comX in S. mutans.
Project description:Streptococcus mutans regulates genetic competence through a complex network that receives inputs from a number of environmental stimuli, including two signalling peptides designated as CSP and XIP. The response of the downstream competence genes to these inputs shows evidence of stochasticity and bistability and has been difficult to interpret. We have used microfluidic, single-cell methods to study how combinations of extracellular signals shape the response of comX, an alternative sigma factor governing expression of the late competence genes. We find that the composition of the medium determines which extracellular signal (XIP or CSP) can elicit a response from comX and whether that response is unimodal or bimodal across a population of cells. In a chemically defined medium, exogenous CSP does not induce comX, whereas exogenous XIP elicits a comX response from all cells. In complex medium, exogenous XIP does not induce comX, whereas CSP elicits a bimodal comX response from the population. Interestingly, bimodal behaviour required an intact copy of comS, which encodes the precursor of XIP. The comS-dependent capability for both unimodal and bimodal response suggests that a constituent - most likely peptides - of complex medium interacts with a positive feedback loop in the competence regulatory network.
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:In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.
Project description:Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore, MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.
Project description:All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX-dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX. We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double-tryptophan-containing sigX-inducing peptide (XIP) pheromone. In all three groups, the XIP gene (comS), and sigX have identical, non-canonical promoters consisting of 9 bp inverted repeats separated from a -10 hexamer by 19 bp. comS is adjacent to a gene encoding a putative transcription factor of the Rgg family and is regulated by its product, which we designate ComR. Deletion of comR or comS in Streptococcus mutans abolished transformability, as did deletion of the oligopeptide permease subunit oppD, suggesting that XIP is imported. Providing S. mutans with synthetic fragments of ComS revealed that seven C-terminal residues, including the WW motif, cause robust induction of both sigX and the competent state. We propose that this circuit is the proximal regulator of sigX in S. mutans, and we infer that it controls competence in a parallel way in all pyogenic and bovis streptococci.
Project description:<h4>Unlabelled</h4>The capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes by Streptococcus mutans, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters for comX, comS, and comYA after exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression of comX, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression of comX and comS, encoding the precursor of comX-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence in S. mutans<h4>Importance</h4>The signaling pathways that regulate development of genetic competence in Streptococcus mutans are intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.
Project description:Here we show that S. suis, a major bacterial pathogen of pigs and emerging pathogen in humans responds to a peptide pheromone by developing competence for DNA transformation. This species does not fall within any of the phylogenetic clusters of streptococci previously shown to regulate competence via peptide pheromones suggesting that more species of streptococci may be naturally competent. Induction of competence was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon, extracellular addition of a comX-inducing peptide (XIP), and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. Different comS alleles are present among strains of S. suis. These comS alleles are not functionally equivalent and appear to operate in conjuction with a cognate ComR to regulate comX through a conserved comR-box promoter. We demonstrate that these 'pherotypes' can be genetically transferred between strains, suggesting that similar approaches might be used to control competence induction in other lactic acid bacteria that lack ComR/ComS homologues but possess comX and the late competence regulon. The approaches described in this paper to identify and optimize peptide-induced competence may also assist other researchers wishing to identify natural competence in other bacteria. Harnessing natural competence is expected to accelerate genetic research on this and other important streptococcal pathogens and to allow high-throughput mutation approaches to be implemented, opening up new avenues for research.
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.