Growth Phase and pH Influence Peptide Signaling for Competence Development in Streptococcus mutans

Abstract

The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in midand late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.

Effects of Arginine on Streptococcus mutans Growth, Virulence Gene Expression, and Stress Tolerance

“…XIP-dependent signaling through ComRS, a comX-lacZ promoter-reporter fusion strain was grown in the defined medium FMC with or without 1.5% arginine; XIP does not activate comX in complex medium (35). In this case, cells grown in the presence of arginine showed a 3-fold decrease in ␤-galactosidase activity compared with that of cells grown in FMC without arginine supplementation (Fig.…”

“…Optical densities were measured at OD 420 and OD 550 from the supernates after the cells were removed by centrifugation. ␤-Galactosidase was expressed in Miller units (35,53). Transformation assays.…”

“…Streptococcus mutans was routinely cultured using mBHI broth that was constituted at 90% (33 g/liter) of the concentration recommended (37 g/liter) by the supplier (Difco) in a 5% CO 2 aerobic atmosphere at 37°C. Cells for CAT assays, ␤-galactosidase (LacZ) assays, and for the extraction of RNA were obtained by growing S. mutans overnight in BHI and diluting the overnight cultures 50-fold in mBHI, TY (12) constituted with 0.5% glucose, or the chemically defined medium, FMC (35). A version of each medium further supplemented with 1.5% L-arginine (Arg) was included, and cultures were incubated in a 5% CO 2 aerobic atmosphere at 37°C until mid-exponential phase (OD 600 of 0.5).…”

Discovery of Novel Peptides Regulating Competence Development in Streptococcus mutans

“…Activated ComR enhances transcription of comX by binding directly to the promoter region of comX, as well as to the comS promoter region, to create a positive-feedback loop. In S. mutans, XIP signaling and CSP signaling are highly dependent on growth phase and environment (12,13). Depending on the conditions, XIP activation of ComX occurs uniformly throughout the population, whereas CSP activates comX in only a subpopulation of cells (9,14).…”

Control of Natural Transformation in Salivarius Streptococci through Specific Degradation of σ X by the MecA-ClpCP Protease Complex

“…ComR is a transcriptional regulator of the Rgg family and ComS is the precursor of the secreted competence X /ComX/SigX-inducing peptide (XIP) (19-21, 23, 24). The activation mechanism of the ComRS system was deeply investigated in Streptococcus thermophilus (19)(20)(21)27) and Streptococcus mutans (22)(23)(24)28). The extracellular pheromone XIP is not sensed by a phosphorelay system but is imported in the cytoplasm by the oligopeptide transporter Opp (Ami) (20,24,27), where it directly interacts with ComR (20).…”