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Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans.

ABSTRACT:

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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

Importance

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.

SUBMITTER: Moye ZD

PROVIDER: S-EPMC4984281 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans.

Moye Zachary D ZD Son Minjun M Rosa-Alberty Ariana E AE Zeng Lin L Ahn Sang-Joon SJ Hagen Stephen J SJ Burne Robert A RA

Applied and environmental microbiology 20160715 15

<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 ...[more]

PMID: 27260355

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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 the 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, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by 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, the 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.IMPORTANCE Streptococcus 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: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: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.

Dataset Information

Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans.

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Importance

Publications

Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans.

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