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. 2015 Sep 11;362(19):fnv159. doi: 10.1093/femsle/fnv159

Bidirectional signaling in the competence regulatory pathway of Streptococcus mutans

Minjun Son 1, Robert C Shields 2, Sang-Joon Ahn 2, Robert A Burne 2, Stephen J Hagen 1,*
Editor: Jan-Ingmar Flock
PMCID: PMC4809993  PMID: 26363019

Abstract

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.

Keywords: transformation, single cell, bistability, fluorescence, feedback, bimodality, quorum sensing

Single-cell studies show that signaling is bidirectional between the two quorum-sensing systems that regulate genetic competence in Streptococcus mutans.

Graphical Abstract Figure.

Graphical Abstract Figure.

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Single-cell studies show that signaling is bidirectional between the two quorum-sensing systems that regulate genetic competence in Streptococcus mutans.

INTRODUCTION

As Streptococcus mutans is a primary causative agent of human dental caries, the mechanisms that control its virulence are of great interest. Multiple interacting regulatory systems in S. mutans control the development of natural genetic competence as well as critical virulence attributes that include biofilm formation, acid tolerance, carbohydrate catabolism and bacteriocin production. Consequently, S. mutans competence and its regulation by both endogenous factors and environmental inputs have been the subject of intensive study (Ahn, Wen and Burne 2006; Fontaine et al. 2015).

At the center of competence regulation in S. mutans is ComX (also called SigX), an alternative sigma factor for genes that control uptake of exogenous DNA. ComX is absolutely required for transformation (Aspiras, Ellen and Cvitkovitch 2004). Its expression can be activated by the quorum-sensing peptides CSP (competence stimulating peptide) and XIP (sigX or comX inducing peptide). Activation of comX is also exquisitely sensitive to environmental parameters, such as media composition and pH (Son et al. 2012, 2015; Guo et al. 2014) and to many intracellular and envelope-associated factors that are still being unraveled (Merritt et al. 2007; Senadheera et al. 2012; Ahn et al. 2014; Kaspar et al. 2015). This work focuses on the interaction between the two signaling systems that detect CSP and XIP, respectively. It aims to clarify the direction of information flow between them and to determine which system is the origin of bimodality or heterogeneity in the expression of comX.

CSP induces competence in only a subpopulation of S. mutans cells, largely because CSP activation of comX is bimodal: when cells are supplied with CSP in complex growth medium, only about 1–50% of cells induce comX strongly. The remainder of the population does not activate comX (Aspiras, Ellen and Cvitkovitch 2004; Lemme et al. 2011; Son et al. 2012). Such bimodal gene expression in a population generally arises from a positive feedback regulatory loop (or equivalently a double negative feedback loop) (Dubnau and Losick 2006; Smits, Kuipers and Veening 2006). Positive feedback regulation can maintain a gene in either its activated or inactivated state, so that two different phenotypes are found within one population of cells. However, the mechanism that creates such bimodality in comX expression has been questioned, as the full pathway by which CSP stimulates comX in S. mutans is not known (Federle and Morrison 2012).

While CSP induces comX in only a subpopulation of cells (i.e. bimodally), XIP induces comX in the entire population (i.e. unimodally). Extracellular XIP is imported by an oligopeptide permease and is detected by the ComRS system: ComR is an Rgg regulator that interacts with XIP to form the transcriptional activator of both comX and comS (Mashburn-Warren, Morrison and Federle 2010). Because comS encodes the precursor for XIP, the ComRS system both produces and detects the peptide signal that activates comX and comS. ComRS can therefore be interpreted as a positive feedback loop (Son et al. 2012).

Extracellular CSP is processed by SepM (Hossain and Biswas 2012) to form CSP-18, which is detected by the ComDE two-component system. The CSP receptor ComD phosphorylates the response regulator ComE, which in turn activates multiple bacteriocin genes, including cipB (Perry et al. 2009). CSP stimulation of the ComDE system leads to stimulation of the ComRS system, which leads to bimodal activation of comX (Dufour et al. 2011). Bimodal and unimodal behavior of comX is highly sensitive to growth medium. XIP elicits comX activity only in a chemically defined (peptide-free) medium, while CSP stimulates comX only in complex media, containing small peptides (Son et al. 2012).

Figure 1 presents two possible models for the origin of bimodality in comX. In Fig. 1A, bimodality originates from an as-yet-unidentified feedback loop involving ComDE, which detects CSP but not XIP (Federle and Morrison 2012). If an unknown feedback mechanism induces bimodality in comE expression, the resulting bimodal activation of the pathway from ComDE to comX could result in bimodal comX expression. The XIP pathway bypasses comDE and would presumably induce comX unimodally. Evidence that bimodality originates in ComDE comes from transcriptome data showing that cells that activated comX in response to CSP expressed comDE at roughly twice the level of cells that did not (Lemme et al. 2011).

Figure 1.

Figure 1.

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Two models for induction of a bimodal distribution in the expression of both comE and comX in a population of S. mutans by exogenous CSP. (A) An unidentified positive feedback mechanism is posited to activate comDE bimodally, leading to bimodal activation of downstream bacteriocin genes such as cipB that stimulate the ComRS system and comX. (B) Positive feedback in the ComRS system allows it to become activated bimodally when stimulated by upstream bacteriocin genes, leading to bimodal expression of comX. ComX regulation of comE expression then leads to bimodal activation of comE.

The model of Fig. 1B attributes comX bimodality to autofeedback behavior of the ComRS system. We have proposed that the small peptides present in complex media strengthen intracellular feedback of the ComS/XIP signal in the ComRS system, enabling bistable expression of comS and consequently also comX (Son et al. 2012). In this model, the presence or absence of bimodality in comX is a feature of the environmental response of the ComRS system. It does not arise in the ComDE CSP-sensing pathway (Son et al. 2012). Bimodal behavior of comE would then be a consequence of (possibly indirect) regulation of PcomE by ComRS and comX.

Here we aim to determine whether (a) comE exhibits its own bimodal behavior that leads to bimodal expression in the ComRS system (Fig. 1A), (b) the ComRS system drives bimodal comX behavior which in turn modulates comE expression (Fig. 1B) or (c) comE and comX are regulated by two independent bimodal mechanisms that are stimulated by CSP, but not XIP. We used fluorescent protein reporters to study the cell-to-cell heterogeneity of com bacteriocin gene activity and the coupling between comX and comE expression in populations of S. mutans. We find that comE responds to the activation of comX and that its bimodality is dependent on the ComRS system. These results indicate two-way communication between the XIP- and CSP-sensing pathways that regulate competence and bacteriocin production in S. mutans.

MATERIALS AND METHODS

Preparation of reporter strains

Table S1 (Supporting Information) lists the S. mutans deletion and gfp fluorescent reporter strains used in this study. The supporting information describes the preparation of these strains.

Experimental procedures

Overnight cultures were washed and diluted 20-fold in fresh medium, which was either defined medium (DM) or complex medium (CM). The DM was FMC (Terleckyj, Willett and Shockman 1975), while the CM was a mixture of equal parts by volume of BHI (Becton Dickinson) and FMC. Prior to inoculation, the pH of fresh medium was adjusted to 7 by adding small amounts of NaOH or HCl. Cultures were then incubated at 37°C in a 5% CO2 aerobic atmosphere. When OD600 reached 0.1, exogenous synthetic CSP or XIP was added (Son et al. 2012). For the kinetics studies of Fig S1 (Supporting Information), CSP-18 (the 18-residue truncated CSP) was also tested, in addition to the 21-residue CSP (Hossain and Biswas 2012). Except as indicated for the kinetics studies, each sample was incubated for 2–3 h and then sonicated at 30% amplitude for 10 s (Fisher FB120 ultrasonicator) and aliquoted onto glass coverslips. Cells were imaged by phase contrast and green fluorescence using an inverted microscope (Nikon TE2000U) equipped with a computer controlled motorized stage and cooled CCD camera. The GFP concentration within each cell was quantified by analyzing phase and green fluorescence microscopy images as previously described (Kwak et al. 2012; Son et al. 2012, 2015).

Quantitative real-time PCR (qRT-PCR)

The supporting information describes the methods for qRT-PCR of comX and comE expression in UA159 and in a strain carrying a mutation in the cin-box of PcomE.

RESULTS

Bimodal and unimodal comE induction by CSP and XIP

Figure 2 shows the population distribution of expression levels for genes in the CSP detection pathway after 2 h incubation with peptide signals in DM or CM. Exogenous CSP activated PcomE bimodally in a population growing in complex medium, such that roughly half of the population was upregulated relative to the control. However, CSP elicited no PcomE response in a DM. In DM, XIP induced population-wide upregulation of PcomE, with most cells showing some activation above baseline. In CM, XIP elicited no PcomE response. This response of PcomE to XIP, CSP and growth media is closely similar to that of PcomX (Son et al. 2012): PcomX, which is directly stimulated by the ComRS system, exhibited bimodal response to CSP in CM and unimodal response to XIP in DM, while showing no response in the other combinations. The similar pattern of expression of comE and comX suggests that PcomE receives direct or indirect stimulation from the comRS system.

Figure 2.

Figure 2.

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Histograms showing individual cell activation of (A) PcomE, and (B) PcipB in response to exogenous XIP and CSP in CM or DM. For each indicated promoter and condition, the histograms show the gfp reporter activity from single-cell measurements in a population of S. mutans. Each histogram bar indicates the percentage of cells that were activated at that level. Red (blue) histograms indicate results with (without) CSP or XIP peptide. Heavy red and blue bars indicate the mean expression levels in the corresponding histograms.

Unimodal cipB induction by CSP in both complex and defined media

In the model of Fig. 1A, bimodal expression of comE drives bimodal activation of the competence regulon including comX. Consequently, genes directly controlled by the ComDE two-component system would be expected to exhibit bimodal activation in response to CSP in CM. Figure 2B shows the effect of CSP and growth media on cipB, which is directly activated by the CSP-mediated ComDE system and encodes a self-acting bacteriocin (mutacin V). Prior studies showed that in a ΔcipB strain, comX response to CSP is decreased 13-fold compared to wild type, and transformation is not induced by CSP (Perry, Cvitkovitch and Levesque 2009; Dufour et al. 2011). We found PcipB responded unimodally to CSP in both complex and defined media: CSP induced a population-wide 25- to 40-fold increase in PcipB activity. Unimodal PcipB response was observed at all CSP concentrations studied (Fig. S1, Supporting Information). A unimodal response to CSP was also observed in two other bacteriocin genes studied, smu.423 (nlmD or mutacin VI) and smu.1906 (Fig. S2, Supporting Information). Therefore, unlike comE, which showed bimodal response to CSP, the ComDE two-component system that detects CSP does not stimulate bacteriocins like cipB in a bimodal or media-dependent fashion. Even though the pattern of expression of comE is similar to that of comX, the ComDE system does not transmit that pattern downstream in its stimulation of cipB.

comX and comE respond with similar kinetics

Figure S3 shows the kinetics of the comX, comE and cipB responses to full-length CSP, CSP-18 and XIP. XIP and CSP-18 generally produced a more rapid response from comE and comX than did full-length CSP. However, in all cases comX was activated either simultaneously with, or slightly faster than, comE. The data are therefore consistent with activation of comE by ComX, as in Fig. 1B.

cipB upregulation by the ComRS system

Figure S3 also shows that CSP elicited a strong and immediate unimodal upregulation of cipB, indicating rapid transduction of the CSP signal, regardless of growth medium. Since cipB is activated by ComE-P under CSP stimulation (Perry et al. 2009), we expected that upregulation of comE by XIP would enhance cipB activity, even in the absence of exogenous CSP. Figure S3 shows that PcipB did respond to XIP, although more slowly than did PcomE. However, PcipB did not respond to XIP or CSP in a comE mutant (Fig. 3A). The finding that stimulation of PcipB by XIP is comE dependent is consistent with a regulatory link from ComRS (and ComX), through ComE, to cipB, as in Fig. 1B.

Figure 3.

Figure 3.

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PcipB induction in comE and cin-box mutants. GFP fluorescence was measured 2.5 h after adding CSP or XIP. (A) A ΔcomE strain harboring a PcipB-gfp reporter was supplied with 1 μM of synthetic CSP or 500 nM of synthetic XIP in CM and DM. (B) A strain in which the cin box of PcomE was mutated, and (C) wild type were supplied with 100 nM or 500 nM XIP in DM. Red and blue histograms show response with and without peptide, respectively. Heavy lines indicate mean response. Control data for wild type ± CSP are shown in Fig. 2.

The ComRS circuit is essential for comE regulation by CSP or XIP

Figure 4 tests the hypothesis that ComRS and ComX drive upregulation of comE. It shows comE behavior in ΔcomR and ΔcomS strains, in which the comR and comS genes were deleted and replaced with a non-polar antibiotic resistance determinant (Son et al. 2012; Kaspar et al. 2015), disrupting ComRS autofeedback behavior. In the ΔcomR strain, neither CSP nor XIP elicited a response from comE. In the ΔcomS strain, comE was unresponsive to CSP but was activated unimodally by XIP in DM. Identical behavior has been observed for comX, which could not be activated in the ΔcomR mutant but could be activated unimodally (but not bimodally) in the ΔcomS mutant (Mashburn-Warren, Morrison and Federle 2010; Son et al. 2012). Therefore, the upregulation of comE by CSP or XIP requires a functional ComRS system.

Figure 4.

Figure 4.

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PcomE activation by XIP and CSP in ΔcomR and ΔcomS mutants. GFP fluorescence was measured at 2 h after adding CSP or XIP. Red and blue histograms show response with and without peptide, respectively. Heavy lines indicate mean response.

ComX-binding site in PcomE

Genes under the control of ComR contain a 20-bp palindromic ComR-box with the inverted repeat ‘GACA-N12-TGTC’ (Fontaine et al. 2013). As no ComR-binding site exists in the comE promoter region, our data suggest direct activation of PcomE by ComX. Although the cin-box motif recognized by ComX is generally regarded to be ‘TACGAATA’, analysis of 12 late competence gene promoters suggests some heterogeneity in the first three DNA bases of the motif (Fig. 5A). Figure 5B shows that a cin-box ComX-binding region TGCGAATA can be identified in PcomE, 109 bp upstream of the start codon of comE. To confirm the functionality of this region, we mutated four bases of the cin-box (Δcin-box or PcomE::CTTA strain) and then compared comX and comE expression using qRT-PCR.

Figure 5.

Figure 5.

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Role of a cin-box regulatory region in the promoter sequence of comE. (A) Sequence logo for a consensus comX binding site (cin-box) sequence logo, based on 12 late competence genes, (Crooks et al. 2004). (B) The cin-box DNA motif is apparent in PcomE. The Δcin-box strain in which PcomE carries a mutation at base 4 of this motif (GAAT to CTTA) was prepared. (C) At 1 h after adding 1% DMSO to WT and Δcin–box (PcomE::CTTA) strains (at OD600 ∼ 0.2 in DM, no XIP), mRNA levels for comE were measured using qRT-PCR. (D) Similarly, 1 h after adding 1% DMSO (control), 100 nM and 500 nM XIP in DM, mRNA levels for comX and comE genes were measured. comX and comE copy numbers were normalized to the 16S housekeeping gene, and the relative expression in +XIP conditions compared to UA159 or PcomE::CTTA in the presence of 1% DMSO.

Because the cin-box is adjacent to the –10 site of PcomE, we first confirmed (Fig. 5C) that mutation of the cin-box did not suppress comE expression in the absence of XIP. However, in the presence of XIP the Δcin-box strain showed significantly reduced expression of comE in comparison to the wild type, even though both showed similar upregulation of comX (Fig. 5D).

We also tested the effect of the PcomE cin-box on activation of the mutacin system by the ComRS system. Figures 3B and 3C show that 500 nM XIP induced a robust 10-fold increase in the median PcipB activity in UA159, but had a far weaker effect (roughly 1.5-fold) in the Δcin-box strain. Figure 3 also shows that while higher XIP concentrations led to higher cipB expression in UA159, cipB expression in the Δcin-box strain was insensitive to higher XIP levels. These data demonstrate that the cin-box of PcomE is functionally important to signaling from the ComR/S system to comE and cipB.

DISCUSSION

The activation of comX by the CSP-ComDE pathway is heterogeneous, such that a population of S. mutans exhibits a bimodal distribution in the expression of comX. As comE itself shows bimodal expression in response to CSP, we have investigated whether comE bimodality is the origin (Fig. 1A) or the consequence (Fig. 1B) of bimodality in comX. The lack of bimodal expression in cipB and other bacteriocins under CSP stimulation implies that comE does not induce bimodal activation of the signaling pathway to comX. Moreover, the observation that comE exhibits the same sensitivity to XIP, CSP and growth media as does comX strongly indicates that comE receives input from ComRS and comX. We have further shown that the ComRS system is required for comE activation by CSP and XIP (Fig. 4). Overall, our data indicate that bimodality originates in ComRS and propagates to comE, as in Fig. 1B. The sensitivity of these effects to the cin-box ComX-binding site in the comE promoter region further supports direct regulation of comE by ComX.

Consequently, ComX and ComDE may participate in a complex feedback loop in which ComX enhances the activity of the ComDE system, which then feeds back to the ComRS system and to PcomX. Interestingly, this scheme positions the ComRS feedback circuit as an inner, environment-sensitive feedback loop within the larger comX-comE loop. A key question is whether flow of information from comX to comE in the larger loop has any measurable effect on the function of the competence regulon. If comX-induced upregulation of comE does enhance the influence of ComDE on the ComRS system, cells in which comX is activated could express cipB at a higher level. Higher cipB expression in comX-activated cells could enhance or stabilize the existing bimodal distribution of comX activity under CSP stimulation. In this way, the outer feedback loop may enhance the heterogeneity of late competence gene expression in a population and inhibit stochastic transitions between the competent and non-competent states.

It is curious that bimodal expression of comE leads to unimodal expression of cipB. However, Fig. S1 (Supporting Information) shows that the response of cipB to CSP saturates near 100 nM CSP, a concentration that is below the CSP level required for strong comX induction. Therefore, the unimodal behavior of cipB may be due to the fact that even modest CSP concentrations with baseline ComE levels are sufficient to saturate the expression of cipB. Upregulation of comE may then have no effect on signaling through cipB. By contrast, the fraction of subpopulation inducing comX at 1 μM CSP is roughly twice as large as at 100 nM CSP (Son et al. 2012). This finding raises the question of how increasing CSP concentrations can provide greater stimulation of comX if the activity of cipB is already saturated. One may speculate that the highest CSP concentrations activate an additional, unidentified signaling pathway in parallel to cipB that further stimulates the ComRS system and comX. If this pathway is responsive to upregulation of comE, then the larger feedback loop could enhance heterogeneity as proposed above.

Figure 1B represents the CSP and XIP signaling pathways as more nearly parallel than sequential. XIP is detected by ComRS and activates comX efficiently, while CSP is detected by ComDE and activates cipB (and other bacteriocins) efficiently; meanwhile each of the two pathways stimulates the other, weakly or heterogeneously. An interesting question is why S. mutans employs two parallel quorum-sensing systems with bidirectional crosstalk. CSP sensing appears much less sensitive to certain environmental parameters than is XIP sensing. Activation of cipB by the CSP signal is unimodal and is insensitive to media composition (Fig. 2B) or extracellular pH. By contrast, ComRS and comX show complex response to both parameters (Son et al. 2012, 2015; Guo et al. 2014). Thus, the two quorum-sensing systems of S. mutans may collect different information about the environment for different purposes. Bacteriocin production depends on the simpler CSP system, which is more nearly a sensor of population density, whereas competence development under XIP control is sensitive to fine changes in media and pH, but produces less phenotypic diversity. The bidirectional flow of information between the two systems may therefore allow regulation of additional behaviors in response to combinations of these two inputs.

Supplementary Material

Supplementary data are available at FEMSLE online
femsle_fnv159_index.html (635B, html)

Acknowledgments

Experimental assistance from Delaram Ghoreishi is gratefully acknowledged.

SUPPLEMENTARY DATA

Supplementary data are available at FEMSLE online.

FUNDING

This work was supported by the National Institute of Dental and Craniofacial Research under awards 1R01DE023339 and 1R01DE13239.

Conflict of interest. None declared.

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Supplementary Materials

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