# Quorum Sensing Regulation of Competence and Bacteriocins in _Streptococcus pneumoniae_ and _mutans_
## 7 The Multiple Layers of Competence Regulation in _Streptococcus mutans_
pneumoniae_ ComE homolog, Table 1) [25]. Notably, while _cipB_ transcription was observed, [PERSON] et al. did not detect bacteriocin activity by _S. mutans_ cells when grown in CDM and stimulated with exogenous MIP. The reason for this apparent lack of activity is not known, but was proposed that media-dependent post-transcriptional and/or post-translational mechanisms might account for this phenomenon [25]. Most perplexing to studies that have strived to describe MIP and XIP stimulatory responses is that the MIP-induced SigX response requires an intact _comS_, even though exogenous addition of XIP appears ineffective in peptide-rich conditions [79]. Numerous past studies are also consistent with the perceived linkage between competence and bacteriocin pathways [22; 24; 64; 68; 71; 80; 81]. Among these was a confounding observation, where in using a P_sigX_:_sff_ (green fluorescent protein) reporter, [PERSON] et al. [82] described a bimodal pattern of _sigX_ induction in cells growing in a biofilm, suggesting an apparent bimodal regulation of SigX by MIP. With these obvious overlaps between MIP- and XIP-mediated effects, there has been much interest in dissecting the relationship between the bacteriocin and competence quorum sensing pathways in _S. mutans_. One of the first studies aimed at untangling these pathways combined GFP reporter activity with fluorescence-activated cell sorting (FACS) to allow for analyzing the response of a population to MIP at the individual cell level [59]. Briefly, an _S. mutans_ culture expressing a P_sigX_:_sff_ reporter was grown in the presence of 0.2 uM MIP in peptide-rich medium for ~2-2.5 h before the population was observed to display two modes of GFP expression and were accordingly sorted into low and high _sigX_-expressing populations. The GFP transcriptional reporter was fused directly downstream of the promoter of interest, in this case P_sigX_, to monitor _sigX_ induction. As predicted by previous results indicating bimodal expression patterns, this method yielded two distinct populations of SigX-expressing cell where 30%-50% of cells expressed P_sigX_:_sff_ when stimulated with MIP when compared to undetectable expression in unstimulated cells [22; 59; 82]. Strikingly, microarray analysis of each subpopulation revealed that genes required for competence (i.e., _comR_, _comS_, _sigX_ and the SigX-regulated transformasome genes) were enriched only within the SigX-expressing population. Moreover, bacteriocin-related genes, including _cipB_, were expressed at similar levels across the two populations [59]. By using FACS, [PERSON] et al. [59] demonstrated population heterogeneity within MIP-stimulated cells for the first time. However, the source(s) of the apparent bimodal expression patterns had yet to be resolved [12]. In another study, [PERSON] et al. investigated the effect of MIP and XIP treatment on _S. mutans_ cells grown in peptide-rich medium, chemically defined medium, or varying ratios in combination under flow conditions and added new insight into the intricacy of SigX regulation while also confirming key findings reported by [PERSON] et al. (2015); [PERSON] et al. (2016). Notably, _S. mutans_ grown in FMC (a variant of CDM) and exposed to MIP did not exhibit detectable levels of SigX expression. However, this effect could be reversed upon addition of non-specific peptides (as little as 0.8% %/\(\
u\) peptide-rich media into FMC), resulting in bimodal expression of SigXX [PERSON] et al. (2016). Bimodality required an intact _comS_ locus but did not require _opp_, which is essential for SigX expression in CDM. This surprising result led the authors to propose that in peptide-rich media, ComS can bypass the need to exist extracellular, and instead can activate SigX without a need to act as an intercellular signal, supporting a model in which bimodality is established through positive feedback of internal ComS on ComR signaling [PERSON] et al. (2016). This proposition questions whether ComRS actually serves as a quorum-sensing system in peptide-rich medium.