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. 2011 Feb;193(4):862-74.
doi: 10.1128/JB.01257-10. Epub 2010 Dec 10.

A transcriptional regulator and ABC transporters link stress tolerance, (p)ppGpp, and genetic competence in Streptococcus mutans

Kinda Seaton  1 , Sang-Joon Ahn, Ann M Sagstetter, Robert A Burne
Affiliations

A transcriptional regulator and ABC transporters link stress tolerance, (p)ppGpp, and genetic competence in Streptococcus mutans

Kinda Seaton et al. J Bacteriol. 2011 Feb.

Abstract

Streptococcus mutans, a primary agent of dental caries, has three (p)ppGpp synthases: RelA, which is required for a mupirocin-induced stringent response; RelP, which produces (p)ppGpp during exponential growth and is regulated by the RelRS two-component system; and RelQ. Transcription of relPRS and a gene cluster (SMu0835 to SMu0837) located immediately upstream was activated in cells grown with aeration and during a stringent response, respectively. Bioinformatic analysis predicted that SMu0836 and SMu0837 encode ABC exporters, which we designated rcrPQ (rel competence-related) genes, respectively. SMu0835 (rcrR) encodes a MarR family transcriptional regulator. Reverse transcriptase PCR (RT-PCR) and quantitative RT-PCR analysis showed that RcrR functions as an autogenous negative regulator of the expression of the rcrRPQ operon. A mutant in which a polar insertion replaced the SMu836 gene (Δ836polar) grew more slowly and had final yields that were lower than those of the wild-type strain. Likewise, the Δ836polar strain had an impaired capacity to form biofilms, grew poorly at pH 5.5, and was more sensitive to oxidative stressors. Optimal expression of rcrPQ required RelP and vice versa. Replacement of rcrR with a nonpolar antibiotic resistance marker (Δ835np), which leads to overexpression of rcrPQ, yielded a strain that was not transformable with exogenous DNA. Transcriptional analysis revealed that the expression of comYA and comX was dramatically altered in the Δ835np and Δ836polar mutants. Collectively, the data support the suggestion that the rcrRPQ gene products play a critical role in physiologic homeostasis and stress tolerance by linking (p)ppGpp metabolism, acid and oxidative stress tolerance, and genetic competence.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of the SMu0835-SMu0836-SMu0837-SMu0838-SMu0839 (rcrRPQ) gene cluster and the relPRS operon in S. mutans UA159. SMu0835 (rcrR) encodes a predicted transcriptional regulator of the MarR family, SMu0836 (rcrP) and SMu0837 (rcrQ) are annotated as an ABC-type multidrug/protein/lipid transport system, SMu0838 encodes a thiol peroxidase, SMu0839 encodes a predicted bacteriocin immunity protein, SMu0840 (relP) encodes a GTP pyrophosphokinase, SMu0841 (relR) encodes a response regulator, and SMu0842 (relS) encodes a sensor histidine kinase of a classic two-component system.
FIG. 2.
FIG. 2.
Real-time RT-PCR. SMu0836 (A), SMu0837 (B), and nonpolar kanamycin resistance cassette (C) mRNA levels are shown. In all cases, cells were grown to mid-exponential phase (OD600 = 0.5), total RNA was extracted, and reverse transcription was done using gene-specific primers followed by quantitative real-time PCR. The data are presented as the copy number of each gene per μg of input RNA. *, differs from the wild type at P < 0.05 (Student's t test). WT, wild type.
FIG. 3.
FIG. 3.
Comparison of wild-type and mutant strain growth. (A) Growth of nonpolar strains versus that of the wild type. The strains were grown in triplicate to mid-exponential phase in BHI broth, diluted 1:100, transferred to fresh BHI broth, overlaid with sterile mineral oil, and placed in a Bioscreen C apparatus at 37°C to monitor growth. Diamonds, wild type; squares, Δ835np; ×, Δ837np; triangles, Δ836np. (B) Growth of the Δ836p versus the wild type. Strains were grown as described for panel A. Diamonds, wild type; squares, Δ836p. (C) Growth of the nonpolar strains versus that of the wild type at pH 5.5. Cells were grown to mid-exponential phase in BHI broth, diluted 1:100 in BHI broth acidified to pH 5.5, covered with sterile mineral oil, and placed in a Bioscreen C apparatus at 37°C to monitor growth. Diamonds, wild type; squares, Δ835np; ×, Δ837np; triangles, Δ836np. (D) Growth of Δ836p versus the wild type at pH 5.5. Strains were grown as described for panel C. Diamonds, wild type; squares, Δ836p. The results are representative of those from three independent experiments performed in triplicate.
FIG. 4.
FIG. 4.
Growth with oxidative stress. (A) Growth of mutant strains versus wild-type strain without oil overlay. The different strains were grown to mid-exponential phase in BHI broth and then diluted 1:100 into fresh BHI broth and placed in a Bioscreen C apparatus at 37°C to monitor growth. Diamonds, wild type; squares, Δ835np; triangles, Δ836np; asterisks, Δ837np; ×, Δ836p. (B) Growth of the mutants versus the wild-type strain in 25 mM paraquat. Cells were grown to mid-exponential phase in BHI broth and then diluted 1:100 in BHI that contained 25 mM paraquat. Diamonds, wild type; squares, Δ836p; triangles, ΔrelP. The results are representative of those from three independent experiments performed at least in triplicate.
FIG. 5.
FIG. 5.
Biofilm formation. The differences in biofilm formation of the mutants compared with that of the wild-type (WT) strain in glucose. Cells were grown to an OD600 of 0.5 in BHI broth and then diluted 1:100 into BM supplemented with 20 mM glucose in microtiter plates. Biofilm formation was quantified after 48 h, as described in Materials and Methods. The results are representative of those from three independent experiments performed at least in triplicate.
FIG. 6.
FIG. 6.
Quantitative real-time PCR. comYA (A) and comX (B) mRNA levels are shown. In both cases, cells were grown to mid-exponential phase (OD600 = 0.5), total RNA was extracted, and reverse transcription was done using gene-specific primers, followed by quantitative real-time PCR. The copy number of each mRNA per μg of input RNA was quantified. The data represent the difference in fold change in copy number of the mutant strains compared to the copy number for the wild-type (WT) strain, where the wild-type level was set at 1.0. *, differs from the wild type at P < 0.05 (Student's t test).
FIG. 7.
FIG. 7.
Quantitative real-time PCR. relP (A), relRS (B), SMu0835 (C), and SMu0836 (D) mRNA levels are shown. In all cases cells were grown to an OD600 of 0.5, total RNA was extracted, and reverse transcription was done using gene-specific primers, followed by quantitative real-time PCR. The copy number per μg of input RNA was calculated. The data represent the fold change in copy numbers of the mutant strains compared to the copy number for the wild-type (WT) strain, where the wild-type value was set to 1.0. *, differs from the wild type at P < 0.05 (Student's t test).
FIG. 8.
FIG. 8.
CAT assays. (A) CAT activity from the relP promoter. The relP promoter was fused to a promoterless cat gene in the pJL105 integration vector. The cat-promoter fusion was transformed into the wild-type and Δ836p strains. (B) The SMu0835 promoter was fused to a promoterless cat gene in the pJL105 integration vector. The cat-promoter fusion was transformed into the wild-type and ΔrelP mutant strains. In all cases cells were grown to mid-exponential phase (OD600 = 0.5) and CAT specific activity was measured as described in Materials and Methods. The results are from three independent experiments performed at least in triplicate. *, differs from the wild type at P < 0.05 (Student's t test).
FIG. 9.
FIG. 9.
Accumulation of (p)ppGpp accumulation in mutant versus wild-type (WT) strains with and without 0.003% hydrogen peroxide. Cells were grown to an OD600 of 0.2 in FMC and labeled with [32P]orthophosphate. H2O2 (0.003%) was added where noted during the labeling. The cells were incubated for 1 h, and (p)ppGpp was extracted using 13 M formic acid. A total of 2 × 105 cpm of each sample was spotted onto PEI-cellulose plates for TLC in 1.5 M KH2PO4 buffer.
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References

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