Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159

Abstract

Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.

FIG. 1.

Phenotypic characterization of the ciaH, ciaR, and ciaRH mutants. (A) Growth at pH 6.4 and 5.4. Growth in BHI medium adjusted to different pH values (7.4, 6.4, and 5.4) was monitored in a Bioscreen C system. The results at two different pHs (6.4 and 5.4) showing different growth patterns between the wild type and mutants are presented. Data points are averages of triplicate samples. (B) Biofilm formation. Strains were grown in BM medium supplemented with glucose (BM-glucose) or sucrose (BM-sucrose) at a final concentration of 20 mM for 24 h. Biofilms were assayed in polystyrene microtiter plates by staining with crystal violet and quantified by adding to an ethanol-acetone mix and reading the optical density at 575 nm. (C) Transformation frequency. Plasmid pDL278 (Sp^r) was transformed into wild-type and mutant strains grown in 200 μl BHI medium (OD₆₀₀ = 0.15) with or without CSP. Transformation frequency was determined from the ratio of the number of transformants versus that of the total viable recipients, multiplied by 100. (D) Differential expression of htrA measured by real-time PCR. Data are representative of at least two separate experiments. The data shown in panels B, C, and D are means ± standard deviations (error bars) of at least three replications. *, P < 0.001; Student's t test.

FIG. 2.

Frequency of transformation of the comD, comE, and comED mutants. Data are representative of at least two separate experiments. The data shown are means ± standard deviations (error bars) of at least three replications. See the text for more details.

FIG. 3.

Effects of HS and CSP on the frequency of transformation of UA159 (A) and growth (B). See the text for more details.

FIG. 4.

Induction of the ciaRH, comED, comX, and comYA genes by CSP treatment in the absence or presence of HS (A) and in the presence of BSA (B) by using real-time PCR. S. mutans UA159 was grown in 50 ml BHI medium supplemented with HS (10%, vol/vol), and synthetic CSP was added at a concentration of 0.2 mM when the culture reached an OD₆₀₀ of 0.15. A 12-ml sample was removed at 0, 10, 20, and 40 min after addition of CSP, and RNA was extracted for real-time PCR. BSA was used at the same concentration as the total protein measured in HS using a commercial Bradford reagent. Data shown in panel A are representative of three independent experiments. Data shown in panel B are means ± standard deviations from two independent experiments. *, P < 0.01; Student's t test. See the text for more details.

FIG. 5.

Differential transcriptional profiles of com genes of wild-type and mutant strains of TCS in the presence of horse serum and CSP by real-time PCR. Differential expression is expressed as fold induction at 40 min after adding synthetic CSP, compared to 0 min. The data shown are means ± standard deviations (error bars) of two independent experiments. See the text for more details.

FIG. 6.

Growth of the wild type and SJ233 (ΔcomC) mutant generated with Bioscreen C (A) and the regulation of comC by CiaH as measured by CAT assay (B). The promoter fusions of comC with the cat gene were inserted in a single copy into the chromosome of the wild type (SJ232) and the ciaH mutant (SAB52). Data presented are means ± standard deviations (error bars) of two independent experiments. *, P < 0.001; Student's t test.

FIG. 7.

Working model for competence development in S. mutans UA159. This model presents gene networks controlling competence development, acid tolerance, and biofilm formation through CiaRH and ComE/D TCS driven by exogenous signals. The network is separated into circuits that are CSP dependent and CSP independent. We hypothesize that ComED is the primary conduit for sensing of CSP, while CiaH integrates signals detected in serum proteins, such as HS. We also hypothesize the presence of a third RR which integrates signals derived from CiaH. Possible pathways driven by HS are shown by thick dashed lines. The model includes the possibility that ComE is a target for CiaH and that HtrA is involved in processing of CSP or HS (thin dotted lines). Thus, the model shows that CiaRH and ComED control competence development and com gene expression, as well as various virulence-related functions in a hierarchical and cooperative fashion, responding to CSP and environmental cues.