Article Figures & Data
Figures
- 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 (Spr) 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.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Source or reference E. coli strains DH10B F−mcrA (mrr-hsdRMS-mcrBC) Ø80lacZ M15 lacX74 deoR recA1 endA1 araD139 (ara-leu)7697 galU galK-rpsL (Strr) nupG Gibco-BRL S. mutans strains UA159 Wild type SAB4 ΔcomX::Kmr This study SAB7 ΔciaR::NPKmr This study SJ233 ΔcomC::Tcr This study SAB19 ΔcomED::Kmr This study SAB21 ΔcomD::Kmr This study SAB22 ΔciaH::Kmr This study SAB22Em ΔcomC::Emr This study SAB23 ΔciaRH::Kmr This study SAB23Em ΔciaRH::Emr This study SAB39 ΔcomE::NPKmr This study SAB52 SAB22Em::PcomC-cata This study SAB53 SAB23Em::PcomC-cat This study SJ232 UA159::PcomC-cat This study Plasmids pALH124 Vector harboring an NPKmr cassette Y. Y. Chen, Univ. of Florida pBGK2 Streptococcus integration vector, Kmr 63, 64 pBGK-PcomC-cat CAT fusion vector including the putative promoter of comC This study pDL278 E. coli-Streptococcus shuttle vector, Spr 30 pGEM-T Easy PCR cloning vector Promega pGEM3 E. coli cloning vector Promega pGEM3cat pGEM3 harboring a promoterless cat 1 pGEM3cat-PcomC pGEM3 harboring the PcomC-cat gene fusion This study pLN2 Vector harboring a Tcr cassette 6 pUC18Em pUC18 harboring an Emr cassette Y. Y. Chen, Univ. of Florida pVT924 Vector harboring a Kmr cassette Y. Y. Chen, Univ. of Florida ↵ a comC promoter fused to chloramphenicol acetyltransferase gene (cat).
- TABLE 2.
Primers used for construction of deletion mutants in this studya
5′-end amplicon Gene deleted 3′-end amplicon Primer Nucleotide sequence (5′→3′) Primer Nucleotide sequence (5′→3′) ciaR-flanking-RV GCCAATGAGTCTCTTCCATGA ciaRH ciaH-BamHI-C CAAGGTATTGGATCCGAGGATAAGA ciaR-BamHI-B TATTGACATGGATCCTGACATTCTC ciaRH ciaH-D2 TTGCCAGAGACATTTGGAAAG ciaH-A TCGAAATATCCCAAGTCATGC ciaH ciaH-BamHI-C CAAGGTATTGGATCCGAGGATAAGA ciaH-BamHI-B TATTGACATGGATCCTGACATTCTC ciaH ciaH-D1 TGAGAAAGACTTGCCAAATATGTTA ciaR-flanking-RV GCCAATGAGTCTCTTCCATGA ciaR ciaR-BamHI-C TAGATAGACGGATCCCGTCTTCTAC ciaR-BamHI-B AGTGTAGGAGGATCCTTGAAGGAT ciaR ciaR-flanking-FW CCACCCTTTTGTCGTGTTCT comED-A3 GCAGGAGCTGGCTATCTTTTT comED comED-BamHI-C AGTAAAATAGGATCCCCTGAGATGG comED-BamHI-B3 CCTTGTTGTGGATCCTCATCTTCCA comED comED-D CCCCTTCCCCATTTTTAGTT comD-A TTTTTGCGGTTAGCAAAATG comD comED-BamHI-C AGTAAAATAGGATCCCCTGAGATGG comD-BamHI-B GAAAGTATCAGGATCCCTTCATTCA comD comED-D CCCCTTCCCCATTTTTAGTT comED-A3 GCAGGAGCTGGCTATCTTTTT comE comE-BamHI-C AGTAAAATAGGATCCCCTGAGATGG comED-BamHI-B3 CCTTGTTGTGGATCCTCATCTTCCA comE comE-D CCCCTTCCCCATTTTTAGTT Comm55 ACTTATCAGATGAGTTTGTCCATC comC Comm-BamHI-35 TAGGATCCGCTAACATTGGAATAAAACAAG Comm-BamHI-53 AAGGATTCTGTTGATAGGCTTCCGCTTCC comC Comm-33 TGCTGTCAAGGGTATCTTGTCAGC ↵ a In addition, primers used for amplification of the putative promoter of comC were as follows: PcomC RI5′, AAGAATTCAAATGCTTGTGTATTCATATG; PcomC-Bm3′, ATGATAGTGTTTTTTTCATGGATCCTCTCC.
- TABLE 3.
Primers used for real-time PCR in this study
Primer Nucleotide sequence (5′→3′) Primer Nucleotide sequence (5′→3′) Product size (bp) ciaR-sense GAAGCAGAGAGTGGCGTTTATG ciaR-antisense TGTCATCCAAACCTTCCTTAGC 145 comD-sense TATGGTCTGCTGCCTGTTGC comD-antisense TGCTACTGCCCATTACAATTCC 97 comX-sense CGTCAGCAAGAAAGTCAGAAAC comX-antisense ATACCGCCACTTGACAAACAG 89 comYA-sense ATTATCTCTGAGGCATCGTCCG comYA-antisense ACCATTGCCCCTGTAAGACTTG 102 htrA-sense AAGTTGTTAGACCCGCTCTTGG htrA-antisense ACCGCTTGTGACATCACTTGG 101
