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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.

SUBMITTER: Kaspar J

PROVIDER: S-EPMC4414889 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance.

Kaspar Justin J Ahn Sang-Joon SJ Palmer Sara R SR Choi Sang Chul SC Stanhope Michael J MJ Burne Robert A RA

Molecular microbiology 20150304 3

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 (ΔrcrR-P) mutant is hyper-transformable, but an rcrR non-polar (ΔrcrR-NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA-Seq and quantitative real-time polymerase chain reaction revealed ...[more]

PMID: 25620525

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Project description:The dental caries pathogen Streptococcus mutans is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence of S. mutans Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in S. mutans Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of comX in a progressive and cumulative fashion, whereas the response to H2O2 displayed a strong threshold behavior. Low concentrations of H2O2 had little effect on induction of comX or the bacteriocin gene cipB, but expression of these genes declined sharply if extracellular H2O2 exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2 affect the S. mutans competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others.IMPORTANCEStreptococcus mutans inhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of S. mutans and its important virulence-associated behaviors, such as genetic competence. S. mutans competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence S. mutans competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects on S. mutans competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.

Project description:Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses, and ecological competitiveness. One example is activation of natural genetic competence by ComR and the com X -inducing peptide (XIP) in Streptococcus mutans Although the competence pathway can be activated by the addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signaling molecule has not been rigorously tested. Coculture model systems were developed that included a "sender" strain that overexpressed the XIP precursor (ComS) and a "responder" strain harboring a green fluorescent protein (GFP) reporter fused to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using (i) planktonic culture systems, (ii) cells suspended in an agarose matrix, or (iii) cells growing in biofilms. XIP was shown to be freely diffusible, and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting the spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with an endogenously produced peptide(s) in populations in various environments and physiologic states.IMPORTANCE The comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids, and the addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence, and evolution of a ubiquitous human pathogen and related organisms.

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A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance.

Publications

A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance.

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