The GlnR Regulon in Streptococcus mutans Is Differentially Regulated by GlnR and PmrA

Abstract

GlnR-mediated repression of the GlnR regulon at acidic pH is required for optimal acid tolerance in Streptococcus mutans, the etiologic agent for dental caries. Unlike most streptococci, the GlnR regulon is also regulated by newly identified PmrA (SMUGS5_RS05810) at the transcriptional level in S. mutans GS5. Results from gel mobility shift assays confirmed that both GlnR and PmrA recognized the putative GlnR box in the promoter regions of the GlnR regulon genes. By using a chemostat culture system, we found that PmrA activated the expression of the GlnR regulon at pH 7, and that this activation was enhanced by excess glucose. Deletion of pmrA (strain ΔPmrA) reduced the survival rate of S. mutans GS5 at pH 3 moderately, whereas the GlnR mutant (strain ΔGlnR) exhibited an acid-sensitive phenotype in the acid killing experiments. Elevated biofilm formation in both ΔGlnR and ΔPmrA mutant strains is likely a result of indirect regulation of the GlnR regulon since GlnR and PmrA regulate the regulon differently. Taken together, it is suggested that activation of the GlnR regulon by PmrA at pH 7 ensures adequate biosynthesis of amino acid precursor, whereas repression by GlnR at acidic pH allows greater ATP generation for acid tolerance. The tight regulation of the GlnR regulon in response to pH provides an advantage for S. mutans to better survive in its primary niche, the oral cavity.

Fig 1. Schematic diagram of S. mutans GS5 SMUGS5_RS05810 (pmrA) and glnRA, and the growth kinetics of GS5 and its derivatives.

(A) The relative locations of the predicated GlnR box in the 5’ flanking region of pmrA and glnR are indicated by a vertical arrow. The putative terminators of pmrA and glnRA are indicated by a lollypop-shaped symbol. The regions within pmrA and glnR that are replaced by Ωkan in strain ΔPmrA and erm in strain ΔGlnR, respectively, are indicated by a horizontal bracket. (B) The growth kinetics of wild-type S. mutans GS5, ΔGlnR, ΔPmrA, and the double knockout strain (ΔGlnR_PmrA) in BHI are shown. A representative graph of at least three experiments is shown.

Fig 2. Alignment of the GlnR box of the S. mutans GS5 GlnR regulon genes.

The distance between the GlnR box and the translational start site of each gene is listed.

Fig 3. EMSA demonstrating the interaction of GlnR and PmrA to the promoters of genes in the GlnR regulon.

The p_glnR-, p_glnQ-, p_gdhA-, p_citB-, p_nrgA-1-, p_nrgA-2-, p_glnP- and p_pmrA-specific DNA probes were mixed with various amounts of protein extracts. All reactions were performed with 0.025 pmol of biotin-labeled probes. (A) Lane 1, probe only. Lanes 2 to 7, use of 8 to 256 nM of MBP-GlnR in the reactions in twofold increments, respectively. Lane 8, use of 256 nM of MBP-GlnR with a non-specific competitor. In the reactions containing the p_pmrA probe, 16 nM to 512 nM of MBP-GlnR was used instead. (B) Lane 1, probe only. Lanes 2 to 8, use of 8 nM to 512 nM of MBP-PmrA in the reactions in twofold increments, respectively. Lane 9, use of 512 nM of MBP-PmrA with a non-specific competitor.

Fig 4. The impact of growth pH and carbohydrate concentrations on the regulatory activity of PmrA.

Wild-type GS5 and strain ΔPmrA were grown in chemostat in TY containing 20 mM (A) or 100 mM glucose (B) at pH 7.2 or pH 5.5. The relative quantity of mRNA of the GlnR regulon gene was measured by qPCR. The ΔCq of GS5 grown at pH 7.2 was used as the reference. Numbers are the means and standard deviations of three independent experiments. The significant difference between wild-type GS5 and strain ΔPmrA under each growth condition was determined by Student’s t test. **, P < 0.001; *, P < 0.01.

Fig 5. Acid killing assay demonstrating the impact of GlnR and PmrA on the viability of S. mutans at pH 3.

The viable counts were determined by serial dilution and plating on BHI agar plates. Numbers are the mean and standard deviation of three independent experiments. Significant differences between strains were determined by Student’s t test. **, P < 0.005; *, P < 0.05.

Fig 6. The biofilm formation of S. mutans GS5 and its derivatives.

(A) The static biofilm formation. Cells were grown in BM containing 10 mM glucose. The amount of the crystal violet retained by the biofilm cells was measured by spectrophotometry. Values shown are the mean and standard deviation of three samples. Significant differences between GS5 and the mutant strains at each time point were determined by Student’s t test. **, P < 0.001; *, P < 0.01. (B) Structures of biofilms formed by wild-type S. mutans GS5 and its derivatives in the flow cell system. The biofilms were grown in BHI for 16 h. The biofilms were treated with the SYTO 9/PI fluorochrome reagents and visualized by CLSM. The live cells (green fluorescence) and dead cells (red fluorescence) are shown. Both the top view and side view of the biofilms are shown.

Fig 7. Model for the regulation of the GlnR regulon in S. mutans GS5.

The putative GlnR box in the promoter is indicated by an open rectangle. The model suggests that PmrA binds to the GlnR box and activates the expression at pH 7, whereas GlnR represses the expression at acidic pH. Such regulation allows a quick switch between nitrogen metabolism and acid tolerance.