Intracellular Signaling by the <i>comRS</i> System in <i>Streptococcus mutans</i> Genetic Competence

Abstract

Entry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In <i>Streptococcus mutans</i>, the immediate activator of <i>comX</i> is the ComRS quorum system. ComS is the precursor of XIP, a seven-residue peptide that is imported into the cell and interacts with the cytosolic receptor ComR to form a transcriptional activator for both <i>comX</i> and <i>comS</i> Although intercellular quorum signaling by ComRS has been demonstrated, observations of bimodal expression of <i>comX</i> suggest that <i>comRS</i> may also function as an intracellular feedback loop, activating <i>comX</i> without export or detection of extracellular XIP. Here we used microfluidic and single-cell methods to test whether ComRS induction of <i>comX</i> requires extracellular XIP or ComS. We found that individual <i>comS</i>-overexpressing cells activate their own <i>comX</i>, independently of the rate at which their growth medium is replaced. However, in the absence of lysis they do not activate <i>comS</i>-deficient mutants growing in coculture. We also found that induction of <i>comR</i> and <i>comS</i> genes introduced into <i>Escherichia coli</i> cells leads to activation of a <i>comX</i> reporter. Therefore, ComRS control of <i>comX</i> does not require either the import or extracellular accumulation of ComS or XIP or specific processing of ComS to XIP. We also found that endogenously and exogenously produced ComS and XIP have inequivalent effects on <i>comX</i> activation. These data are fully consistent with identification of intracellular positive feedback in <i>comS</i> transcription as the origin of bimodal <i>comX</i> expression in <i>S. mutans</i><b>IMPORTANCE</b> The ComRS system can function as a quorum sensing trigger for genetic competence in <i>S. mutans</i> The signal peptide XIP, which is derived from the precursor ComS, enters the cell and interacts with the Rgg-type cytosolic receptor ComR to activate <i>comX</i>, which encodes the alternative sigma factor for the late competence genes. Previous studies have demonstrated intercellular signaling via ComRS, although release of the ComS or XIP peptide to the extracellular medium appears to require lysis of the producing cells. Here we tested the complementary hypothesis that ComRS can drive <i>comX</i> through a purely intracellular mechanism that does not depend on extracellular accumulation or import of ComS or XIP. By combining single-cell, coculture, and microfluidic approaches, we demonstrated that endogenously produced ComS can enable ComRS to activate <i>comX</i> without requiring processing, export, or import. These data provide insight into intracellular mechanisms that generate noise and heterogeneity in <i>S. mutans</i> competence.