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 mid- and 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.

FIG 1

Competence gene expression and transformation efficiency in response to exogenously added sXIP as a function of growth phase. (A) LacZ activity measured from a reporter fusion with the comX promoter. The P_comX-lacZ strain was grown to an OD₆₀₀ of 0.2 (early-exponential phase), 0.4 (mid-exponential phase), or 0.8 (late-exponential phase) in FMC medium and was then incubated with 1% DMSO (to match the concentration of the solvent for XIP) or 2 μM sXIP for 1 h. Then LacZ assays were performed. (B) Transformation efficiency of UA159 as a function of growth phase. A plasmid (pDL278) was added to growing cultures in FMC medium at OD₆₀₀ values of 0.2, 0.4, and 0.8 after the addition of 2 μM sXIP, as described in Materials and Methods. After 2.5 h, cultures were plated onto BHI agar plates, and CFU were enumerated after 48 h of incubation at 37°C under a 5% CO₂ aerobic atmosphere. Transformation efficiency was calculated by dividing the number of transformants by the total number of viable bacteria. Data are means ± standard deviations (error bars) for three biological replicates conducted in triplicate. Statistical analyses were performed using Student's t test. *, P < 0.05; **, P < 0.01.

FIG 2

Effects of supernatants from cultures at different growth phases on comX induction by exogenous sXIP. The P_comX-lacZ strain was grown to optical densities of 0.2 (early-exponential phase), 0.4 (mid-exponential phase), and 0.8 (late-exponential phase) in FMC medium and was then centrifuged. (A) Cell pellets from cultures at an OD₆₀₀ of 0.2 were resuspended in supernatants derived from cultures at different growth phases and were incubated with 1% DMSO or 2 μM sXIP for 1 h. Then LacZ assays were performed. (B) Cell pellets from cultures at an OD₆₀₀ of 0.4 or 0.8 were resuspended in supernatants from cultures at an OD₆₀₀ of 0.2 or in fresh FMC medium. The supernatants were also supplemented with 25 mM glucose. The resuspended cultures were incubated with 1% DMSO or 2 μM sXIP for 1 h, and LacZ assays were performed. Data are means ± standard deviations (error bars) for three biological replicates conducted in triplicate. Statistical analyses were performed using Student's t test. *, P < 0.05.

FIG 3

pH effects on induction of comX by exogenous sXIP. (A) The P_comX-lacZ strain was grown to an optical density of 0.4 (pH 6.5) or 0.8 (pH 5.8) in FMC medium and was then centrifuged. The supernatant pH was adjusted to the same value (pH 6.9) as that of the culture at an OD₆₀₀ of 0.2 by use of 1 M NaOH and was incubated with 1% DMSO or 2 μM sXIP for 1 h. Then LacZ assays were performed. Nonneutralized supernatants were used as controls. (B) The response of comX to exogenous XIP was analyzed in cultures at an optical density of 0.2 (early-exponential phase), 0.4 (mid-exponential phase), or 0.8 (late-exponential phase) in FMC medium that had been buffered at pH 7.0 with 100 mM K-phosphate. (C) Cells from cultures at an OD₆₀₀ of 0.2 were centrifuged to separate pellets from supernatants. The supernatants were then adjusted to pH values ranging from 5.0 to 9.0 by using 6 M HCl or 1 M NaOH. The pellets were resuspended in the pH-adjusted supernatants and were incubated with 2 μM sXIP for 1 h. The pellets were also resuspended in supernatants without pH adjustment, incubated with or without sXIP, and then used as positive or negative controls, respectively. The expression of comX was measured by LacZ assays. Data are means ± standard deviations (error bars) for three biological replicates conducted in triplicate. Statistical analyses were performed using Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Growth inhibition by addition of sXIP to cells at different growth phases. Overnight cultures of S. mutans UA159 were diluted 1:20 into fresh FMC medium, and growth was monitored by using a Bioscreen C growth monitor. sXIP at 0.2, 2, or 20 μM was added either at the beginning of culture (A) or when the OD₆₀₀ reached 0.2 (B), 0.4 (C), or 0.8 (D). DMSO (1%) was added to the culture as a control. The optical density was measured every 30 min for 48 h, with shaking for 15 s before each reading. Data points are averages for triplicate samples.

FIG 5

Expression of comX in cells engineered to endogenously overproduce ComS or mXIP and grown in FMC medium (A) or BHI broth (B). Strains pIB184ComS/P_comX-lacZ (overexpressing ComS), pIB184mXIP/P_comX-lacZ (overexpressing mXIP), and pIB184/P_comX-lacZ (vehicle control) were grown to optical densities of 0.2 (early-exponential phase), 0.4 (mid-exponential phase), or 0.8 (late-exponential phase) in FMC medium or BHI broth, and the expression of comX was then measured by LacZ assays. Data are means ± standard deviations (error bars) for three biological replicates conducted in triplicate. Statistical analyses were performed using Student's t test. *, P < 0.05; **, P < 0.01.

FIG 6

Responses of comX to exogenously added CSP at different growth phases in BHI medium. (A) The P_comX-lacZ strain was grown to an optical density of 0.2 (early-exponential phase), 0.4 (mid-exponential phase), or 0.8 (late-exponential phase) in BHI medium and was incubated with or without 0.32 μM sCSP for 1 h. Then LacZ assays were performed. (B) Effects of pH on comX expression in response to exogenous CSP. The P_comX-lacZ strain was grown to an optical density of 0.4 (pH 6.4) or 0.8 (pH 5.3) in BHI medium and was then centrifuged. The supernatants were either neutralized to the pH value (6.8) of cultures at an OD₆₀₀ of 0.2 or supplemented with 11.1 mM glucose. The resuspended cultures were then incubated with or without 0.32 μM sCSP for 1 h. Nonneutralized supernatants were used as controls. Then LacZ assays were performed. (C) Effects of culture supernatants on comX induction by CSP. Cell pellets from cultures at an OD₆₀₀ of 0.4 or 0.8 were resuspended in supernatants from cultures at an OD₆₀₀ of 0.2 or in fresh BHI medium and were incubated with or without 0.32 μM sCSP for 1 h. The expression of comX was measured by LacZ assays. Data are means ± standard deviations (error bars) for three biological replicates conducted in triplicate. Statistical analyses were performed using Student's t test. *, P < 0.05; **, P < 0.01.