A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance

Abstract

Streptococcus mutans displays complex regulation of genetic competence, with ComX controlling late competence gene transcription. The rcrRPQ operon has been shown to link oxidative stress tolerance, (p)ppGpp metabolism and competence in S. mutans. Importantly, an rcrR polar (ΔrcrR-P) mutant is hyper-transformable, but an rcrR non-polar (ΔrcrR-NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA-Seq and quantitative real-time polymerase chain reaction revealed little expression in the 5' region of comX in ΔrcrR-NP, but high level expression in the 3' region. Northern blotting with comX probes revealed two distinct transcripts in the ΔrcrR-P and ΔrcrR-NP strains, and 5' Rapid Amplification of cDNA Ends mapped the 5' terminus of the shorter transcript to nt +140 of the comX structural gene, where a unique 69-aa open reading frame, termed XrpA, was encoded in a different reading frame than ComX. Two single-nucleotide substitution mutants (comX::T162C; comX::T210A) were introduced to disrupt XrpA without affecting the sequence of ComX. When the mutations were in the ΔrcrR-NP genetic background, ComX production and transformation were restored. Overexpression of xrpA led to impaired growth in aerobic conditions and decreased transformability. These results reveal an unprecedented mechanism for competence regulation and stress tolerance by a gene product encoded within the comX gene that appears unique to S. mutans.

Fig. 1. qRT-PCR of the comX coding region using 5′ and 3′ primer sets

A. Diagram of 5′ and 3′ primer sets by location within the comX coding region (SMU.1997) and relation to RNA-Seq reads mapped in the ΔrcrR-NP and ΔrcrR-P strains. B. qRT-PCR results of the UA159, ΔrcrR-P and ΔrcrR-NP strains using 5′ (white bars) and 3′ (black bars) comX primer sets. Strains were grown in BHI medium to OD₆₀₀ = 0.5 before cells were harvested and RNA extracted. Data represent averages of three biological replicates measured as triplicate samples. Differences in copies per microgram of RNA in all strains were evaluated for statistical significance between the 5′ and 3′ primer sets by Student’s t-test. **P value 0.009.

Fig. 2

Immunoblotting of ComX in the ΔrcrR-P and ΔrcrR-NP strains. Strains UA159, ΔcomX, ΔrcrR-P and ΔrcrR-NP were grown in BHI medium to an OD₆₀₀ = 0.5 before cells were harvested and whole cell lysates collected. One micromolar sCSP was added at OD₆₀₀ = 0.2 to a subset of the growing cultures. Ten micrograms of protein was loaded and separated on a 12.5% SDS-PAGE gel before transfer onto a polyvinylidene difluoride (PDVF) membrane. ComX was detected by a 1:1000 dilution of primary antisera against full-length recombinant ComX from Streptococcus mutans. Molecular mass standards (in kDa) are shown to the left. The calculated molecular mass of ComX is 19 kDa.

Fig. 3

Northern blot of comX transcripts. Strains UA159, ΔrcrR-P (ΔP) and ΔrcrR-NP (ΔNP) were grown in BHI medium to an OD₆₀₀ = 0.2, at which point 1 μM sCSP was added, if desired. Cells were grown for an additional hour after sCSP addition before harvest by centrifugation. Five microgram of purified RNA was separated on a 1% agarose-formaldehyde gel and transferred to a BrightStar-Plus membrane. A biotin-labeled, 352 bp comX-specific probe was utilized for hybridization and detection. Transcript sizes (right) were estimated by comparison with an RNA Millennium Marker. An image of 23S and 16S rRNA (bottom) from each sample serves as the loading control.

Fig. 4. comX transcripts evaluated by northern blot

A. Strains UA159, ΔrcrR-P (ΔP), ΔrcrR-NP (ΔNP) and ΔcomSΔrcrR-NP (ΔcomS/ΔNP) were grown in FMC medium to an OD₆₀₀ = 0.2, at which point 1% dimethyl sulfoxide (vehicle) or 2 μM sXIP was added. Cells were grown for an additional hour after addition of sXIP, then cells were harvested by centrifugation and RNA prepared. Asterisk (*) in lane ΔcomSΔNP + sXIP denotes the predicted point to which a full-length comX mRNA would migrate. B. Strains UA159 (WT), ΔrcrR-P (ΔP), ΔrcrR-NP (ΔNP), ΔcomS/ΔrcrR-NP (ΔS), ΔcomRΔrcrR-NP (ΔR) and Δ PcomXΔrcrR-NP (ΔPx) were grown in FMC medium to an OD₆₀₀ = 0.5, at which point cells were harvested. Five microgram of purified RNA was separated on a 1% agarose-formaldehyde gel and transferred to a BrightStar-Plus membrane. A biotin-labeled, 352 bp comX-specific probe was utilized for hybridization and detection. Transcript sizes (right) were estimated by comparison with an RNA Millennium Marker. An image of 23S and 16S rRNA (bottom) from each sample serves as the loading control.

Fig. 5. Mapping of 5′ termini within the comX coding region

A. 5′ termini of the comX coding region transcripts were mapped using 5′ Rapid Amplification of cDNA Ends (5′-RACE) and are bolded, underlined and labeled (5′ Terminus) within the DNA sequence. Elements regulating comX transcription are labeled by underlining (ComRS box) and bolding (-35, -10). Shine-Dalgarno sequences are underlined (RBS) and start (ATG) and stop (TGA; TAG) codons are bolded for the ComX and XrpA protein coding sequences. Sequence shown is chromosomal position 1 872 020–1 872 623 of the Streptococcus mutans UA159 genome. B. Illustration of the organization of xrpA within the comX coding region. comX is 483 bp in length with the 210 bp xrpA ORF beginning at the 161st bp of comX.

Fig. 6. Mutation of xrpA in the ΔrcrR-NP background

A. Sequence of the comX::T162C and comX::T210A mutations. A thymine is mutated to cytosine at the 162nd bp of comX (bolded), changing the xrpA start codon from ATG to ACG (underlined) to prevent translation initiation. In comX::T210A, a thymine at the 210th bp of comX is mutated to adenosine to create a premature stop codon. In both mutations, the ComX protein coding sequence is unchanged. B. Transformation efficiency between strains UA159, ΔrcrR-NP, comX::T162C/ΔrcrR-NP and comX::T210A/ΔrcrR-NP in BHI without sCSP (white bars) or BHI with addition of 1 μM sCSP (black bars). Transformation efficiency is determined by dividing the number of transformants by the total number of viable bacteria and is expressed as a percentage. Differences in transformation efficiency between strains were evaluated for statistical significance by Student’s t-test. *P value < 0.05. C. Growth curves of strains ΔrcrR-NP (circles), comX::T162C/ΔrcrR-NP (triangles) and comX::T210A/ΔrcrR-NP (squares) in BHI with addition of 1 μM sCSP. Growth was monitored with a Bioscreen C lab system every 30 min for 24 h. Growth is displayed as an average of three biological replicates monitored in three individual wells. D. Immunoblot against ComX in the UA159, ΔrcrR-NP, comX::T162C/ΔrcrR-NP and comX::T210A/ΔrcrR-NP strains. One micromolar sCSP was added at OD₆₀₀ = 0.2 to a subset of the cultures, and cells were harvested at OD₆₀₀ = 0.5. Ten microgram of protein from extracted whole cell lysates was loaded and separated on a 12.5% SDS-PAGE gel before transfer onto a polyvinylidene difluoride (PDVF) membrane. ComX was detected by a 1:1000 dilution of primary antiserum against full-length recombinant ComX from Streptococcus mutans. Molecular mass standards (in kDa) are shown to the left. The calculated molecular mass of ComX is 19 kDa.

Fig. 7. xrpA mRNA and start codon drive GFP production in vivo

A. Diagram of translational fusion constructs used to drive GFP production in vivo. XrpA::gfp contains the 5′ comX coding sequence (bases 1–169) fused to GFP. PcomXXrpA::gfp is the same construct as XrpA::gfp, but the 5′ end is extended to include the comX promoter. B. Relative expression of the XrpA::gfp and PcomXXrpA::gfp strains in either the UA159 or ΔrcrR-NP genetic backgrounds, grown in FMC with addition of either 1% dimethyl sulfoxide (vehicle) or 2 μM sXIP. The strains indicated in the figure, from left to right, are UA159, XrpA::gfp/UA159, PcomXXrpA::gfp/UA159, XrpA::gfp/ΔrcrR-NP, and PcomXXrpA::gfp/ΔrcrR-NP. Fluorescence of GFP was measured using a Synergy 2 multimode microplate reader (filters: excitation 485/20 nm, emission 528/20 nm). Differences in relative expression between strains were evaluated for statistical significance by Student’s t-test. **P value < 0.01. Relative expression (RFU/OD₆₀₀) is calculated as fluorescent units minus background, divided by OD₆₀₀ (~OD₆₀₀ 0.5). An immunoblot of detecting the expressed GFP, which contains a 6-His tag, using an anti 6-His antibody (0.5 ng μl⁻¹ Anti-6X His tag^®) against extracted whole cell lysates (5 μg of protein loaded on 12.5% SDS-PAGE gel) is shown below for each referenced strain. The calculated molecular mass of XrpA-GFP fusion is 28.5 kDa.

Fig. 8. XrpA can decrease competence and ComX levels

A. Comparison of transformation efficiency between UA159 and comX::T162C (ΔxrpA). Strains were grown in either BHI with or without 1 μM sCSP or in FMC with either 1% dimethyl sulfoxide (vehicle) or 2 μM sXIP. Differences in transformation efficiency between strains were evaluated for statistical significance in all medium conditions by Student’s t-test. *P value 0.015. B. Comparison of transformation efficiency between pIB184 (vector only) and pIB184xrpA (xrpA overexpressing), grown in either BHI with or without 1 μM sCSP or in FMC with either 1% dimethyl sulfoxide (vehicle) or 2 μM sXIP. Differences in transformation efficiency between strains were evaluated for statistical significance in all medium conditions by Student’s t-test. *P value 0.035 and 0.014 respectively. C. Immunoblot against ComX in either pIB184 (V) or pIB184xrpA (XrpA). One micromolar sCSP or 2 μM sXIP was added at OD₆₀₀ = 0.2, and cells were harvested at OD₆₀₀ = 0.5. Ten microgram of protein from clarified whole cell lysates was loaded and separated on a 12.5% SDS-PAGE gel before transfer onto a polyvinylidene difluoride (PDVF) membrane. ComX was detected by a 1:1000 dilution of primary antisera against full-length recombinant ComX from Streptococcus mutans. Molecular mass standards (in kDa) are shown to the left. The calculated molecular mass of ComX is 19 kDa.

Fig. 9. Growth of xrpA-overexpressing strain in aerobic conditions

A. Growth curves of the pIB184 (circles), pIB184comX (squares) and pIB184xrpA (triangles) strains in BHI without a 50 μl sterile mineral oil overlay to examine growth inhibitory effects of air. B. Growth curves of the pIB184 (circles), pIB184comX (squares) and pIB184xrpA (triangles) strains in BHI containing 1 μM sCSP. Strains were grown without a mineral oil overlay. Growth was monitored using a Bioscreen C lab system every 30 min for 24 h. Growth is displayed as an average of three biological replicates monitored in three individual wells.