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

## TL;DR

This study reveals how Streptococcus mutans uses a regulatory factor called SigX to control both genetic competence and bacteriocin production, which are important for its survival in dental plaque.

## Contribution

The discovery that SigX directly regulates ComE and controls both competence and bacteriocin synthesis, revealing a new regulatory mechanism in S. mutans.

## Key findings

- SigX directly controls ComE, the response regulator for bacteriocin synthesis, via a binding motif in the comE promoter.
- ComRS is the origin of population heterogeneity in competence and determines the modality of downstream regulators.
- CSP regulates only bacteriocin synthesis and does not influence competence signaling.

## 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.

Streptococcus mutans is a bacterium of the human dental plaque that contributes to caries development. It controls two important survival mechanisms via a cell-density dependent communication system (quorum sensing): The synthesis of peptide antibiotics, and of a membrane apparatus for genetic competence, i.e. the ability to take up external DNA and integrate it into its own genome. S. mutans synthesizes two different signalling peptides to this end. It has remained elusive, how exactly these signals are propagated within the cell and why only a fraction of the population becomes competent. To actually observe under the microscope which bacterium in the population is activated, and which genes are required for the activation, we constructed strains of S. mutans that reported on the transcription of a gene by starting to fluoresce green. We even constructed strains that reported on two genes simultaneously, by fluorescing either green or blue or both. With these tools, and by additionally knocking out or modifying key genes as needed, we investigated the complete signaling cascade under various conditions. Thus we discovered a central regulatory switch. S. mutans makes sure that external DNA is available when it becomes genetically competent–by killing cells in the environment.