A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance.
ABSTRACT: Streptococcus mutans displays complex regulation of genetic competence, with ComX controlling late competence gene transcription. The rcrRPQ operon has been shown to link oxidative stress tolerance, (p)ppGpp metabolism and competence in S.?mutans. Importantly, an rcrR polar (?rcrR-P) mutant is hyper-transformable, but an rcrR non-polar (?rcrR-NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA-Seq and quantitative real-time polymerase chain reaction revealed little expression in the 5' region of comX in ?rcrR-NP, but high level expression in the 3' region. Northern blotting with comX probes revealed two distinct transcripts in the ?rcrR-P and ?rcrR-NP strains, and 5' Rapid Amplification of cDNA Ends mapped the 5' terminus of the shorter transcript to nt +140 of the comX structural gene, where a unique 69-aa open reading frame, termed XrpA, was encoded in a different reading frame than ComX. Two single-nucleotide substitution mutants (comX::T162C; comX::T210A) were introduced to disrupt XrpA without affecting the sequence of ComX. When the mutations were in the ?rcrR-NP genetic background, ComX production and transformation were restored. Overexpression of xrpA led to impaired growth in aerobic conditions and decreased transformability. These results reveal an unprecedented mechanism for competence regulation and stress tolerance by a gene product encoded within the comX gene that appears unique to S.?mutans.
Project description:A MarR-like transcriptional repressor (RcrR) and two predicted ABC efflux pumps (RcrPQ) encoded by a single operon were recently shown to be dominant regulators of stress tolerance and development of genetic competence in the oral pathogen Streptococcus mutans. Here, we focused on polar (?rcrR-P) and nonpolar (?rcrR-NP) rcrR mutants, which are hyper- and nontransformable, respectively, to dissect the mechanisms by which these mutations impact competence. We discovered two open reading frames (ORFs) in the 3' end of the rcrQ gene that encode peptides of 27 and 42 amino acids (aa) which are also dramatically upregulated in the ?rcrR-NP strain. Deletion of, or start codon mutations in, the ORFs for the peptides in the ?rcrR-NP background restored competence and sensitivity to competence-stimulating peptide (CSP) to levels seen in the ?rcrR-P strain. Overexpression of the peptides adversely affected competence development. Importantly, overexpression of mutant derivatives of the ABC exporters that lacked the peptides also resulted in impaired competence. FLAG-tagged versions of the peptides could be detected in S. mutans, and FLAG tagging of the peptides impaired their function. The competence phenotypes associated with the various mutations, and with overexpression of the peptides and ABC transporters, were correlated with the levels of ComX protein in cells. Collectively, these studies revealed multiple novel mechanisms for regulation of competence development by the components of the rcrRPQ operon. Given their intimate role in competence and stress tolerance, the rcrRPQ-encoded peptides may prove to be useful targets for therapeutics to diminish the virulence of S. mutans.
Project description:An intimate linkage between the regulation of biofilm formation, stress tolerance and genetic competence exists in the dental caries pathogen Streptococcus mutans. The rcrRPQ genes encode ABC exporters (RcrPQ) and a MarR-family transcriptional repressor of the rcr operon (RcrR) that play a dominant role in the regulation of the development of genetic competence and connect competence with stress tolerance and (p)ppGpp production in S. mutans. Here we identify the target for efficient RcrR binding in the rcr promoter region using purified recombinant RcrR (rRcrR) protein in electrophoretic mobility shift assays and show that DNA fragments carrying mutations in the binding region were not bound as efficiently by rRcrR in vitro. Mutations in the RcrR binding site impacted expression from the rcrR promoter in vivo and elicited changes in transformation efficiency, competence gene expression, and growth inhibition by competence-stimulating peptide; even when the changes in rcrRPQ transcription were minor. An additional mechanistic linkage of RcrR with competence and (p)ppGpp metabolism was identified by showing that the rRcrR protein could bind to the promoter regions of comX, comYA and relP, although the binding was not as efficient as to the rcrRPQ promoter under the conditions tested. Hence, tightly controlled autogenous regulation of the rcrRPQ operon by RcrR binding to specific target sites is essential for cellular homeostasis, and RcrR contributes to the integration of genetic competence, (p)ppGpp metabolism, and acid and oxidative stress tolerance in S. mutans through both direct and indirect mechanisms.
Project description:In the dental caries pathogen Streptococcus mutans, an MarR-like transcriptional regulator (RcrR), two ABC efflux pumps (RcrPQ) and two effector peptides encoded in the rcrRPQ operon provide molecular connections between stress tolerance, (p)ppGpp metabolism and genetic competence. Here, we examined the role of RcrRPQ in the oral commensal S. gordonii. Unlike in S. mutans, introduction of polar or non-polar rcrR mutations into S. gordonii elicited no significant changes in transformation efficiency. However, S. gordonii rcrR mutants were markedly impaired in their ability to grow in the presence of hydrogen peroxide, paraquat, low pH or elevated temperature. Sensitivity to paraquat could also be conferred by mutation of cysteine residues that are present in the RcrR protein of S. gordonii, but not in S. mutans RcrR. Thus, stress tolerance is a conserved function of RcrRPQ in a commensal and pathogenic streptococcus, but the study reveals additional differences in regulation of genetic competence development between S. mutans and S. gordonii.
Project description:Streptococcus mutans, a primary agent of dental caries, has three (p)ppGpp synthases: RelA, which is required for a mupirocin-induced stringent response; RelP, which produces (p)ppGpp during exponential growth and is regulated by the RelRS two-component system; and RelQ. Transcription of relPRS and a gene cluster (SMu0835 to SMu0837) located immediately upstream was activated in cells grown with aeration and during a stringent response, respectively. Bioinformatic analysis predicted that SMu0836 and SMu0837 encode ABC exporters, which we designated rcrPQ (rel competence-related) genes, respectively. SMu0835 (rcrR) encodes a MarR family transcriptional regulator. Reverse transcriptase PCR (RT-PCR) and quantitative RT-PCR analysis showed that RcrR functions as an autogenous negative regulator of the expression of the rcrRPQ operon. A mutant in which a polar insertion replaced the SMu836 gene (?836polar) grew more slowly and had final yields that were lower than those of the wild-type strain. Likewise, the ?836polar strain had an impaired capacity to form biofilms, grew poorly at pH 5.5, and was more sensitive to oxidative stressors. Optimal expression of rcrPQ required RelP and vice versa. Replacement of rcrR with a nonpolar antibiotic resistance marker (?835np), which leads to overexpression of rcrPQ, yielded a strain that was not transformable with exogenous DNA. Transcriptional analysis revealed that the expression of comYA and comX was dramatically altered in the ?835np and ?836polar mutants. Collectively, the data support the suggestion that the rcrRPQ gene products play a critical role in physiologic homeostasis and stress tolerance by linking (p)ppGpp metabolism, acid and oxidative stress tolerance, and genetic competence.
Project description:The microbes that inhabit the human oral cavity are subjected to constant fluctuations in their environment. To overcome these challenges and gain a competitive advantage, oral streptococci employ numerous adaptive strategies, many of which appear to be intertwined with the development of genetic competence. Here, we demonstrate that the regulatory circuits that control development of competence in Streptococcus mutans, a primary etiological agent of human dental caries, are integrated with key stress tolerance pathways by the molecular alarmone (p)ppGpp. We first observed that the growth of a strain that does not produce (p)ppGpp (?relAPQ, (p)ppGpp(0)) is not sensitive to growth inhibition by comX inducing peptide (XIP), unlike the wild-type strain UA159, even though XIP-dependent activation of the alternative sigma factor comX by the ComRS pathway is not impaired in the (p)ppGpp(0) strain. Overexpression of a (p)ppGpp synthase gene (relP) in the (p)ppGpp(0) mutant restored growth inhibition by XIP. We also demonstrate that exposure to micromolar concentrations of XIP elicited changes in (p)ppGpp accumulation in UA159. Loss of the RelA/SpoT homolog (RSH) enzyme, RelA, lead to higher basal levels of (p)ppGpp accumulation, but to decreased sensitivity to XIP and to decreases in comR promoter activity and ComX protein levels. By introducing single amino acid substitutions into the RelA enzyme, the hydrolase activity of the enzyme was shown to be crucial for full com gene induction and transformation by XIP. Finally, loss of relA resulted in phenotypic changes to ?rcrR mutants, highlighted by restoration of transformation and ComX protein production in the otherwise non-transformable ?rcrR-NP mutant. Thus, RelA activity and its influence on (p)ppGpp pools appears to modulate competence signaling and development through RcrRPQ and the peptide effectors encoded within rcrQ. Collectively, this study provides new insights into the molecular mechanisms that integrate intercellular communication with the physiological status of the cells and the regulation of key virulence-related phenotypes in S. mutans.
Project description:Transcriptional profiling of the Δ835np 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, Δ835np mutant vs the wild-type strain. Biological replicates: 4 per strain, independently grown and harvested. One replicate per array
Project description:Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. 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 reveal through both genomic and proteomic techniques that XrpA is the first described negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ?xrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.
Project description:A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g. comR, comS, comD, comE, cipB, clpX, rcrR, ciaH, but disclosed an additional 20 genes that were not previously competence-associated. The competence phenotypes of mutants were characterized, including using fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes.IMPORTANCEStreptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology and genetics of S. mutans and related pathogens.
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.