Control of thioredoxin reductase gene (trxB) transcription by SarA in Staphylococcus aureus

Abstract

Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sarA family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sarA regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus.

FIG. 1.

Transcriptional analysis of the trxB gene in the different S. aureus strains at various phases of growth. A. Northern analysis of RNA isolated from the wild-type RN6390 and its various isogenic sarA family mutants at the postexponential phase of growth (OD₆₀₀ ∼ 1.7). B. Transcription of trxB in the wild-type SH1000 and its isogenic sarA mutant at different phases of growth as indicated. C. Complementation analysis of the sarA mutant with single and multiple copies of the sarA gene at the postexponential phase of growth (OD₆₀₀ ∼ 1.7). In sarA cps sarB, a 1.5-kb sarB DNA region of the sarA locus (11) was integrated into the lipase locus (geh) of a sarA mutant. In sarA cpm sarA, the P1sarA region was cloned into multicopy shuttle vector pSK236 (30). D. Northern analysis of the trxB transcript in the different wild-type and sarA mutant strains of S. aureus grown under microaerophilic conditions for 16 h. E. Northern analysis of the perR transcript in the wild-type and sarA mutant strains of S. aureus grown under microaerophilic conditions for 16 h. All the strains were grown to postexponential phase (OD₆₀₀ ∼ 1.7) and analyzed by Northern hybridization. In all panels, 10 μg of total cellular RNA was loaded onto each lane and the blots were probed with a 950-bp DNA probe containing the open reading frame of the trxB gene or a 440-bp DNA fragment containing the open reading frame of the perR gene. The regions of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting are also shown as loading controls in all panels. The OD₆₀₀ was determined by using a spectrophotometer (Spectronic 20D).

FIG. 2.

Analysis of DNA binding of SarA both in vitro and in vivo. A. Autoradiogram of a nondenaturing 8.0% polyacrylamide gel showing gel shift assay results for purified SarA protein with a γ-³²P-labeled 258-bp trxB promoter fragment (approximately 0.02 pM per lane). Lanes 1 to 5 correspond to 0 ng, 200 ng, 400 ng, 600 ng, and 800 ng of wild-type SarA, while lanes 6 to 8 correspond to 1,000 ng of SarA protein. For competition assays, a 50-fold molar excess of the respective unlabeled trxB promoter DNA was used in lane 7, whereas a 50-fold molar excess of nonspecific competitor DNA (a 185-bp internal fragment of the sarX gene) was added in lane 8. B. Northern blot analyses showing the effects of sarA mutations at various positions on in vivo expression of trxB and sarA genes. Total cellular RNA was isolated from a sarA mutant containing shuttle plasmid or plasmid with wild-type or mutated sarA fragments at the postexponential phase of growth (OD₆₀₀ ∼ 1.7). A total of 10 μg of cellular RNA was loaded onto each lane. The blots were probed with 950-bp trxB and 375-bp sarA gene fragments. The regions of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting are shown as loading controls. C. Autoradiogram of a nondenaturing 8.0% polyacrylamide gel, showing the binding of the mutant forms of SarA to a 258-bp trxB promoter fragment. The various concentrations of mutant SarA proteins used for binding assays are shown at the top of the panels.

FIG. 3.

Analysis of trxB gene expression in response to different oxidative stress-inducing agents and the effect of a cysteine mutation at position 9 of the sarA gene. A. Transcription of the trxB gene in response to oxidative stress-inducing agents under microaerophilic growth conditions. The wild-type strain SH1000 and isogenic sarA mutant strains were grown under microaerophilic conditions for 16 h and treated with oxidizing agents for 1 h. The concentrations of different oxidative stress agents used were as follows: H₂O₂, 2 mM; t-butyl-OOH, 0.5 mM; CuOOH, 0.5 mM; diamide, 2 mM. After exposure, total cellular RNA was isolated and hybridized. B. Northern blot of RNA isolated from isogenic sarA mutant and sarA mutant strains containing shuttle plasmid with the wild-type and mutated sarA fragments grown under microaerophilic conditions for 16 h. In panels A and B, a total of 10 μg of cellular RNA was loaded onto each lane and the blots were hybridized with a 950-bp trxB gene fragment. The regions of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting are also shown as loading controls in all panels. C. Gel shift assay showing the effects of oxidizing agents on DNA binding of purified wild-type SarA or mutant SarAC9G. Purified proteins (500 ng) were incubated with H₂O₂ (15 mM) or diamide (15 mM) for 30 min. Where indicated, 50 mM DTT was added to the oxidized wild-type SarA protein, and incubation continued for further 30 min. All the samples were subsequently employed for gel shift assays with the radiolabeled trxB promoter fragment as described in Materials and Methods.

FIG. 4.

Effects of sarA mutations at various codon positions on in vivo expression of target genes. A. Northern blots of RNA isolated from isogenic sarA mutant and sarA mutant strains containing shuttle plasmid with the wild-type and mutated sarA fragments grown under microaerophilic conditions for 16 h. B. Expression of trxB in the sarA mutant carrying various sarA gene constructs in response to diamide. Northern blot analyses were carried out with RNA isolated from SH1000 sarA mutant or the sarA mutant carrying the wild-type or mutated sarA gene as indicated. The cells were grown under microaerophilic conditions for 16 h and treated with diamide (2 mM) for 1 h, and subsequently total RNA was isolated. In all panels, a total of 10 μg of cellular RNA was loaded onto each lane, and the blots were hybridized with 950-bp trxB and 600-bp sodM gene fragments. The regions of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting are also shown as loading controls.

FIG. 5.

Binding of the wild-type and mutant forms of SarA to the upstream promoter DNA fragments of the trxB and sodM genes. Autoradiograms of nondenaturing 8% polyacrylamide gels show binding of various mutant forms of purified SarA proteins to the trxB promoter (upper panel) or sodM promoter (lower panel). The amount of protein employed in each assay is shown on the top of each lane. Approximately 0.02 pM γ-³²P-labeled DNA fragments was used per lane for both of the promoters.