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Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX.

ABSTRACT: 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.

SUBMITTER: Son M

PROVIDER: S-EPMC3468698 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX.

Son Minjun M Ahn Sang-Joon SJ Guo Qiang Q Burne Robert A RA Hagen Stephen J SJ

Molecular microbiology 20120816 2

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 ...[more]

PMID: 22845615

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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.

Project description:An eye tracking experiment explored the gaze behavior of deaf individuals when perceiving language in spoken and sign language only, and in sign-supported speech (SSS). Participants were deaf (n = 25) and hearing (n = 25) Spanish adolescents. Deaf students were prelingually profoundly deaf individuals with cochlear implants (CIs) used by age 5 or earlier, or prelingually profoundly deaf native signers with deaf parents. The effectiveness of SSS has rarely been tested within the same group of children for discourse-level comprehension. Here, video-recorded texts, including spatial descriptions, were alternately transmitted in spoken language, sign language and SSS. The capacity of these communicative systems to equalize comprehension in deaf participants with that of spoken language in hearing participants was tested. Within-group analyses of deaf participants tested if the bimodal linguistic input of SSS favored discourse comprehension compared to unimodal languages. Deaf participants with CIs achieved equal comprehension to hearing controls in all communicative systems while deaf native signers with no CIs achieved equal comprehension to hearing participants if tested in their native sign language. Comprehension of SSS was not increased compared to spoken language, even when spatial information was communicated. Eye movements of deaf and hearing participants were tracked and data of dwell times spent looking at the face or body area of the sign model were analyzed. Within-group analyses focused on differences between native and non-native signers. Dwell times of hearing participants were equally distributed across upper and lower areas of the face while deaf participants mainly looked at the mouth area; this could enable information to be obtained from mouthings in sign language and from lip-reading in SSS and spoken language. Few fixations were directed toward the signs, although these were more frequent when spatial language was transmitted. Both native and non-native signers looked mainly at the face when perceiving sign language, although non-native signers looked significantly more at the body than native signers. This distribution of gaze fixations suggested that deaf individuals - particularly native signers - mainly perceived signs through peripheral vision.

Project description:UnlabelledThe 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. mutansImportanceThe 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.

Dataset Information

Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX.

Publications

Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX.

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