The Alternative Sigma Factor SigX Controls Bacteriocin Synthesis and Competence, the Two Quorum Sensing Regulated Traits in Streptococcus mutans

Abstract

Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S. mutans cells can split into a competent and non-competent subpopulation. The origin of this population heterogeneity has not been experimentally determined and it is unknown how the two QS systems are connected. We developed a toolbox of single and dual fluorescent reporter strains and systematically knocked out key genes of the competence signaling cascade in the reporter strain backgrounds. By following signal propagation on the single cell level we discovered that the master regulator of competence, the alternative sigma factor SigX, directly controls expression of the response regulator for bacteriocin synthesis ComE. Consequently, a SigX binding motif (cin-box) was identified in the promoter region of comE. Overexpressing the genetic components involved in competence development demonstrated that ComRS represents the origin of bimodality and determines the modality of the downstream regulators SigX and ComE. Moreover these analysis showed that there is no direct regulatory link between the two QS signaling cascades. Competence is induced through a hierarchical XIP signaling cascade, which has no regulatory input from the CSP cascade. CSP exclusively regulates bacteriocin synthesis. We suggest renaming it mutacin inducing peptide (MIP). Finally, using phosphomimetic comE mutants we show that unimodal bacteriocin production is controlled posttranslationally, thus solving the puzzling observation that in complex media competence is observed in a subpopulation only, while at the same time all cells produce bacteriocins. The control of both bacteriocin synthesis and competence through the alternative sigma-factor SigX suggests that S. mutans increases its genetic repertoire via QS controlled predation on neighboring species in its natural habitat.

Fig 1. Previous model of competence development in S. mutans in complex (A) and defined medium (B).

The different classes of structural components of the competence network are explained below in the legend (grey box in the left). Two quorum sensing pathways regulate competence in S. mutans. Components of the ComDE circuit with the autoinducer CSP are shown in green while components of the ComRS circuit and the autoinducer XIP are illustrated in red color. Inactive elements of the circiuts are shown in grey. The two QS pathways are operating in parallel and converge on the central comRS system. Both circuits are connected via an unknown link between ComDE and ComRS in complex medium. The medium determines which autoinducer is biologically active and whether competence is induced bimodally or unimodally across the population. This is illustrated by the gene expression shemes (grey box in the right). The modality of the expression of involved genes is illustrated in the same coloring as used for the gene symbol. The output of the systems, bacteriocin expression and competence, are symbolized below the bacteriocin encoding genes and the late competence genes in yellow. Bacteriocin expression is exclusively observed in complex medium while competence development in defined medium is not coupled to bacteriocin expression.

Fig 2. Single cell co-expression analysis of comE with the late competence genes lytFsm (A), comS (B) and sigX (C) using dual fluorescent reporter strains.

Strains were grown in complex THBY medium and gene expression induced with 2 μM CSP. 150 minutes post induction cells were collected and imaged using fluorescence microscopy. ComE expressing cells show green fluorescence (middle column), while the blue fluorescence (left column) indicates expression of co-expression partners. An overlay of the green and blue channel is shown in the right column. For better visualization the blue fluorescence is shown in red false color.

Fig 3. Overexpression analysis of comE, comR, comS and comRS in the LytFsm reporter strain background.

Expression of the different genes was under the control of the strong constitutive lactococcal P23 promoter on the replicative plasmid pIB166. Strains were grown in THBY under CSP induced (2μM) and uninduced conditions. 180 min post induction samples were collected and analyzed using a flow cytometer. A sample collected directly before induction (0 min) was used as negative control. The distribution of the gfp fluorescence intensity of 50.000 analysed cells as determined by flow cytometry is shown in the density plots. Density plots derived from uninduced cells at t = 180 min are shown in black while the density plots of CSP induced cells at that timepoint are shown in green. Density plots of the control at t = 0 min are shown in grey.

Fig 4. Combined effect of CSP and XIP peptides on lytFsm expression in complex medium.

The LytFsm pAE03 reporter strain was grown in complex medium until the culture reached an OD₆₀₀ of 0.2. The culture was divided and induced with either CSP or XIP alone or treated with combinations of XIP and CSP. Concentrations between 200 nM and 20 μM of each inducer were tested. 2 h post induction cells were harvested and analyzed using flow cytometry. 50.000 cells were analyzed for each condition. Density plots of the log2 gfp fluorescence intensity for the differently treated reporter cells are shown. Controls are shown in black. Increasing inducer concentrations are visualized by increased intensity of the red color. The vertical black line represents the peak fluorescence intensity of the uninduced control. A Effect of increasing concentrations of XIP on the fluorescence of the LytFsm reporter strain. B Effect of increasing concentrations of CSP on the reporter strain. C LytSsm fluorescenc of cells treated with combinations of XIP and CSP in equimolar ratios. D Effect of different molar ratios of XIP to CSP.

Fig 5. Time resolved transcriptome analysis of XIP and CSP induced S. mutans WT cultures in CDM.

S. mutans WT cells were grown in CDM and induced either with 2 μM CSP or 2 μM XIP. Samples were taken 0, 5, 15 and 30 minutes post induction. Samples from uninduced controls were taken at corresponding timepoints. Differentially expressed genes (log2 fold > 1) were clustered into the categories competence, mutacins, quorum sensing and others. Differential expression of the respective genes is displayed in a heatmap. Genes that belong to the two quorum sensing regulons controlling competence in S. mutans but were no differentially expressed are also shown.

Fig 6. RNAsequencing and bioinformatics analysis of the 5`UTR of the comE gene of S. mutans.

A SigX binding motif (cin-box, highlighted in green) was identified 109 bp upstream of the comE start codon. Directly adjacent to the cin-box a Pribnow box (highlighted in red) was found (red corners). RNA sequencing data revealed the transcriptional start site of the comE gene (black line).

Fig 7. Time resolved analysis of comE and cipB expression using flow cytometry.

CipB pAE03 and ComE pAE03 GFP reporter strains were grown in CDM or THBY and were induced with 2 μM CSP or 2 μM XIP. 0, 30, 60, 90, 120 and 180 minutes post induction samples were taken and analyzed using a flow cytometer. Samples from uninduced controls were taken at corresponding timepoints. Density plots of GFP fluorescence (log2 units) are shown during time. In A the density plots of the XIP-induced ComE pAE03 reporter strain grown in CDM (green plots) are compared with the density plots derived from the same strain grown under CSP-induced conditions in THBY (red plots). The colored vertical lines represent the peak log2 GFP fluorescence intensity of the distribution at timepoint 0h. In part B density plots derived from the XIP induced CipB pAE03 reporter strain grown in CDM (yellow plots) are plotted against the density plots of the same strain grown in the same medium under CSP induced conditions (blue plots). C showed the comparative analysis of the ComE pAE03 reporter strain (orange density plots) and the CipB pAE03 strain (blue density plots) both grown in CDM under CSP (2μM) induced conditions.

Fig 8. Modality of CSP induced comE and cipB expression in THBY in different gene deletion backgrounds.

ComE pMR1 and CipB pMR1 reporter strains were grown in THBY under CSP (2 μM) induced conditions. 3 h post induction images were collected using fluorescence microscopy. GFP fluorescence images (green) of the reporters were overlaid with the phase contrast (grey) images of the corresponding strain. Images of the comE reporter strain are shown in the upper panel, while images of the cipB reporter strain are shown in the lower panel.

Fig 9. Phophorylmimetic study.

A CipB reporter strain in the ΔcomE deletion background was transformed with plasmids carrying the coding sequence of the natural comE (ComE nat.), the coding sequence of comE under the control of the strong lactococcal P23 promoter (P23-ComE), or the mutant genes ComE D60E (mimicking stable phorphorylation) and ComE D60A (no phosphorylation possible). A strain transformed with the empty vector (pIB166) was used as negative control. All strains were grown in THBY and fluorescence microscopy was performed 1 h after addition of CSP. Overlays of phase contrast and green fluorescence microscopic images are shown. The right column shows the un-induced controls.

Fig 10. Model of competence development in S. mutans.

Two quorum sensing pathways are operating in S. mutans. Bacteriocin expression is regulated via CSP signaling and the ComDE two-component system (green box). CSP should be renamed MIP (mutacin inducing peptide). Competence development is regulated via XIP signaling and the ComR regulator (red box). Both systems are connected via the alternative sigma-factor SigX which controls comE transcription (red arrow). Competence regulation proceeds in three successive steps: (1) Instantaneous post-transcriptional activation of the regulator ComR. (2) Early transcriptional response: Transcription of comS, resulting in a positive feedback loop for signal synthesis, and of sigX. (3) Late transcriptional response: Transcription of the SigX regulon, including the competence genes and the comE response regulator. Upon competence development the medium determines via which mechanism XIP is imported into the cell. In defined medium XIP is specifically imported via the Opp permease, leading to unimodal competence development. XIP import in complex medium requires bacteriocin activity and might be mediated via permeabilization of the cells. In complex medium, competence development is bimodal. Active bacteriocins and CSP areexclusively observed in complex medium. Post transcriptional mechanisms must be operating in the defined medium, e.g. down-regulation of bacteriocin translation, lack of secretion, or degradation, since the cells sense externally added CSP and transcription of bacteriocin encoding genes is induced.