Regulation of gbpC expression in Streptococcus mutans

Abstract

Streptococcus mutans, the principal causative agent of dental caries, produces four glucan-binding proteins (Gbp) that play major roles in bacterial adherence and pathogenesis. One of these proteins, GbpC, is an important cell surface protein involved in biofilm formation. GbpC is also important for cariogenesis, bacteremia, and infective endocarditis. In this study, we examined the regulation of gbpC expression in S. mutans strain UA159. We found that gbpC expression attains the maximum level at mid-exponential growth phase, and the half-life of the transcript is less than 2 min. Expression from PgbpC was measured using a PgbpC-gusA transcriptional fusion reporter and was analyzed under various stress conditions, including thermal, osmotic, and acid stresses. Expression of gbpC is induced under conditions of thermal stress but is repressed during growth at low pH, whereas osmotic stress had no effect on expression from PgbpC. The results from the expression analyses were further confirmed using semiquantitative reverse transcription-PCR analysis. Our results also reveal that CovR, a global response regulator in many Streptococcus spp., represses gbpC expression at the transcriptional level. We demonstrated that purified CovR protein binds directly to the promoter region of PgbpC to repress gbpC expression. Using a DNase I protection assay, we showed that CovR binds to DNA sequences surrounding PgbpC from bases -68 to 28 (where base 1 is the start of transcription). In summary, our results indicate that various stress conditions modulate the expression of gbpC and that CovR negatively regulates the expression of the gbpC gene by directly binding to the promoter region.

FIG. 1.

Mapping of the transcriptional initiation site of gbpC. (A) Schematic representation of the gbpC locus. Open reading frames are represented by block arrows, and their orientations indicate the transcriptional direction. The upstream sequence of the gbpC open reading frame is shown below the diagram. (B) Transcription initiation site as determined using 5′ RACE-PCR. (C) Transcription initiation site as determined using primer extension. (D) Northern blot analysis of total RNA isolated from S. mutans UA159 after growth in THY medium. The predicted −35 and −10 sites are underlined, and the ribosome-binding site (RBS) is indicated. The bent arrow indicates the transcription start site, and the lollipop symbols indicate the location of putative transcriptional termination.

FIG. 2.

Expression of gbpC at different stages of growth. (A) The total RNA was extracted at the indicated time points from IBS131 cells grown in THY broth. (B) RNA samples (50 ng) were subjected to semiquantitative RT-PCR analysis with primers specific for the gbpC gene or the gyrA gene as described in Materials and Methods. The experiments were repeated at least twice with independent RNA isolations. The growth points are as follows: 1, 42 KU; 2, 59 KU; 3, 79 KU; 4, 100 KU; 5, 123 KU; and 6, 136 KU. The gyrA gene was included to ensure that equal amounts of RNA were used for all reactions.

FIG. 3.

Stability of gbpC transcript. The stability of the gbpC transcript was measured using cultures at mid-exponential growth phase (70 KU). RNA was extracted from IBS131 at the times (in minutes) indicated above the lanes following the addition of rifampin to block the synthesis of new mRNA. Northern blotting (A) was performed with 4 μg of total RNA, using the gbpC sequence as a probe. The decay curve calculation (B) was performed as described in the text. Experiments were repeated at least twice, and a representative Northern blot and representative graph are shown.

FIG. 4.

Expression of PgbpC under different stress conditions. The reporter strain IBS131 contains the promoter region of gbpC (PgbpC) along with the sequence encoding the first 18 amino acids of GbpC fused to the gusA gene. IBS131 was grown in THY medium under different stress conditions, as indicated. Expression from PgbpC at mid-exponential phase was quantified by determining the Gus activity as described in the text. The values were normalized with the Gus activity obtained with IBS131 grown in THY medium at 37°C. Experiments were performed at least three times, and the means and standard deviations are shown.

FIG. 5.

GbpC expression is regulated by CovR. (A) Expression of PgbpC in the wild-type and covR mutant strains. Strains were grown in THY broth at 37°C and harvested at mid-exponential phase, and Gus activity was measured as described in the text. The data are the means and standard deviations of at least three experiments that were normalized with wild-type (IBS131) values. (B) Semiquantitative RT-PCR analysis of gbpC transcription. RNA was harvested at mid-exponential (ME) and stationary (ST) phases of growth and subjected to RT-PCR analysis using primer pairs specific for gbpC or gyrA, as indicated. The data are representative of an RT-PCR analysis resulting from at least two independent RNA isolations.

FIG. 6.

Binding of CovR to the PgbpC promoter. A 204-bp PgbpC DNA fragment was radiolabeled with [γ-³²P]ATP using T4 polynucleotide kinase, and 0.52 pmol of labeled DNA was used for binding with various amounts of CovR. CovR-PgbpC DNA reaction mixtures were incubated at room temperature for 40 min, which was followed by electrophoresis in a 50 mM NaPO₄-buffered 5% acrylamide gel (pH 6.5). Gels were electrophoresed at 100 V for 90 min at room temperature, dried, and exposed to a phosphorimager screen. Lane 1 contained no CovR; lanes 2 to 8 contained 1.15, 2.3, 4.6, 9.2, 18.4, 23.0, and 27.6 pmol of CovR, respectively; and lanes 9 to 12 contained 18.4 pmol of CovR along with 1.0 or 5.4 pmol of unlabeled PgbpC DNA (lanes 9 and 10, respectively) or 1.0 or 5.1 pmol of unlabeled PrpsL (lanes 11 and 12, respectively). F, free probe; B, bound probe.

FIG. 7.

DNase I protection assay of the gbpC promoter. EMSA was performed with CovR and a 204-bp PgbpC DNA sequence, as described in the legend to Fig. 6. The amounts of CovR added to 0.52 pmol of the PgbpC were 0, 4.6, 9.2, 18.4, and 27.6 pmol (lanes 1 to 5, respectively). DNase I protection assays were carried out as described in Materials and Methods. Samples were electrophoresed on an 8.0% sequencing gel adjacent to PgbpC sequencing reaction mixtures (lanes G, A, T, and C). Binding regions are indicated by vertical lines, and the transcription start site is indicated by a bent arrow. The region of PgbpC protected from DNase I digestion is distinguished by gray shading. RBS, ribosome-binding site.

FIG. 8.

Stress-induced gbpC expression is CovR independent. RNA was isolated from wild-type (IBS131) (lanes 1) and covR mutant (IBS132) (lanes 2) strains grown under the indicated growth conditions. RNA was subjected to semiquantitative RT-PCR analysis using either gbpC- or gyrA-specific primers as previously described. The data are representative of an RT-PCR analysis resulting from at least two independent experiments. −BUF, no buffer.