Regulation of superoxide dismutase (sod) genes by SarA in Staphylococcus aureus

Abstract

The scavenging of reactive oxygen species (ROS) within cells is regulated by several interacting factors, including transcriptional regulators. Involvement of sarA family genes in the regulation of proteins involved in the scavenging of ROS is largely unknown. In this report, we show that under aerobic conditions, the levels of sodM and sodA transcription, in particular the sodM transcript, are markedly enhanced in the sarA mutant among the tested sarA family mutants. Increased levels of sod expression returned to near the parental level in a single-copy sarA complemented strain. Under microaerophilc conditions, transcription of both sodM and sodA was considerably enhanced in the sarA mutant compared to the wild-type strain. Various genotypic, phenotypic, and DNA binding studies confirmed the involvement of SarA in the regulation of sod transcripts in different strains of Staphylococcus aureus. The sodA mutant was sensitive to an oxidative stress-inducing agent, methyl viologen, but the sarA sodA double mutant was more resistant to the same stressor than the single sodA mutant. These results suggest that overexpression of SodM, which occurs in the sarA background, can rescue the methyl viologen-sensitive phenotype observed in the absence of the sodA gene. Analysis with various oxidative stress-inducing agents indicates that SarA may play a greater role in modulating oxidative stress resistance in S. aureus. This is the first report that demonstrates the direct involvement of a regulatory protein (SarA) in control of sod expression in S. aureus.

FIG. 1.

Transcription of the sodA and sodM genes in the wild type (Wt) and its isogenic sarA mutant (A⁻) in different S. aureus strains. (A and B) Northern analysis of the sodM (A) and sodA (B) transcripts in different strains (as indicated on top of each panel) at various phases of growth. The blots were probed with 600 bp and 650 bp of sodM and sodA DNA fragments containing the entire open reading frames of the sodM and sodA genes, respectively. (C) SOD activity of cell extracts from different S. aureus strains. Extracts from cells grown to the exponential growth phase were separated on a 12% (wt/vol) nondenaturing polyacrylamide gel and stained for SOD activity. Each lane contains approximately 20 μg of protein. Three bands corresponding to SodA, SodM, and a hybrid SodA/SodM activity (54) are indicated. The gel was scanned, and the inverse image generated is shown. (D) Northern analysis of sodM and sodA transcripts in the SH1000 wild-type and sarA mutant strains grown under microaerophilic conditions. The two bands of sodA transcripts are due to transcription from two closely spaced promoters (26). The region of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting is shown as a loading control in panels A, B, and D. The intensity of different transcripts was quantified with the ImageQuant software (Amersham).

FIG. 2.

Northern analysis of the sodM, sodA, and sarA transcripts and SOD activity in the wild-type (Wt) SH1000, sarA mutant, and complemented strains. The different strains were grown to an OD₆₀₀ of ∼1.1 and total RNA isolated. (A to C) The blots were probed with 600-bp sodM (A), 650-bp sodA (B), and 450-bp sarA (C) DNA fragments containing their respective open reading frames. In sarAcpssarB, a 1.5-kb sarB DNA fragment of the sarA locus (1, 7) was integrated into the lipase locus (geh) of the sarA mutant. In sarAcpmsarA, the P1 sarA region was cloned into multicopy shuttle vector pSK236 (29). The sarB region of the sarA locus produces three overlapping transcripts, which are required for optimal expression of SarA (1, 7). The region of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting is shown as loading control. (D) SOD activities of intracellular cell extracts of different S. aureus strains. The intensity of different bands was quantified with the Gene Tool software from Syngene. Other details are as described for Fig. 1C.

FIG. 3.

DNA binding activity of SarA protein. (A) Autoradiograms of 8.0% polyacrylamide gels showing binding of SarA protein to 253-bp and 273-bp promoter fragments of sodA and sodM, respectively. For the sodM promoter, lanes 1 to 3 contain 0 ng, 100 ng, and 200 ng of purified SarA protein, respectively, while lanes 4 to 8 each contain 400 ng of SarA protein. For the sodA promoter, lanes 1 to 3 contain 0 ng, 200 ng, and 400 ng of purified SarA protein, respectively, while lanes 4 to 8 each contain 600 ng of purified SarA protein. Approximately, 0.02 pM of radiolabeled DNA fragments was used in all lanes for both promoters. For competition assays, 25- and 75-fold molar excesses of nonspecific competitor DNA (a 185-bp internal fragment of the sarX gene) were added in lanes 5 and 6, respectively, whereas 25- and 75-fold molar excesses of the respective unlabeled promoter DNA were used in lanes 7 and 8 for each panel. In lanes 9, 1,000 ng of purified mutated SarAR90A protein was used. The arrows indicate the free labeled DNA or the DNA-protein complex. (B) Transcription of sodA and sodM in the SH1000 sarA mutant carrying different P1 sarA gene constructs. The strains were grown microaerophically for 16 h, and total RNA was isolated and hybridized with the sodA or sodM gene probe as indicated. Lane 1, sarA mutant; lane 2, sarA mutant complemented with wild-type P1 sarA construct; lane 3, sarA mutant complemented with P1 sarAR90A construct. The gene constructs used in lanes 2 and 3 were cloned into multicopy shuttle vector pSK236. (C) Nucleotide sequence of the 253-bp DNA fragment upstream of the sodM gene. The −10 and −35 regions of the sodM gene (26) are underlined. The transcriptional start site (+1), ribosome binding site (SD), and translational start (ATG) of the sodM gene are indicated and shown in bold. The consensus SarA binding sites are shown with arrows; solid arrows are for the coding strand of DNA, and broken arrows are for the complementary strand of DNA in the upstream region of translational start site. The consensus regions are deduced from the analysis of the depicted DNA region with the 26-bp SarA consensus binding site (9, 10) using a DNA alignment (LALIGN) program. The deduced six SarA consensus binding regions are located at the different sites on DNA, but four of them are confined within a 47-bp region (shown in bold) on both strands of DNA.

FIG. 4.

MV-induced expression of the sodM and sodA genes. (A) Northern blots of total RNA extracted from exponential-phase cultures of S. aureus parental strain SH1000 (Wt) and its isogenic sarA mutant grown in the absence (−) or presence (+) of 1 mM MV. The blots were hybridized with 600-bp sodM and 650-bp sodA DNA fragments. (B) Northern blots of the sarA transcripts in the wild-type strain grown to the exponential (OD₆₀₀ of ∼1.1) phase of growth and induced with MV for 2 h. The blot was probed with a 450-bp DNA fragment containing the sarA gene. Lower panel, cell extracts from the above-mentioned MV-treated or control SH1000 cells were immunoblotted and probed with anti-SarA polyclonal antibodies at a 1:10,000 dilution. The region of 23S and 16S rRNA of the ethidium bromide stained gel used for blotting is also shown as a loading control in panels A and B. (C) Growth analysis of the wild-type SH1000 and sarA mutant strains in presence or absence of 1.0 mM MV. The different cells depicted are the wild type without MV (○), wild type with MV (□), sarA mutant without MV(▴), and sarA mutant with MV (×). The experiments were repeated at least three independent times. Error bars indicate standard deviations.

FIG. 5.

Genotypic and phenotypic characterization of the sodA single mutant and the sarA sodA double mutant in response to MV. (A) Northern blot analysis of exponential-phase cultures of S. aureus parental strain SH1000 (Wt) and various isogenic single and double mutants (as indicated above each panel) are shown. The blots were hybridized with 600-bp and 650-bp DNA fragments containing open reading frames of the sodM and sodA genes, respectively. The region of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting is also shown as a loading control. (B) SOD activity of intracellular cell extracts of different S. aureus strains as indicated above the panel. Details of loading and conditions are as described for Fig. 1C. (C) Growth of sodA and sodA sarA mutants in the presence or absence of 1.0 mM MV. The different cells depicted are the sodA mutant without MV (○); sodA mutant with MV (□), sodA sarA mutant without MV (▴), and sodA sarA mutant with MV (×). Error bars indicate standard deviations. (D) Sensitivities of the wild type and various sodA and sarA mutant strains to MV as determined by disc diffusion assays. The zone of clearance was measured as an indicator of sensitivity to MV.

FIG. 6.

Transcriptional analysis of various target genes under different growth conditions in the wild-type (Wt) SH1000 and its isogenic sarA mutant. The different cells were grown to the exponential phase of growth (OD₆₀₀ of ∼1.0) and induced with various oxidative stress-inducing agents. After 15 min of exposure, total RNA was isolated and subjected to Northern analysis with sodM and sodA gene probes. The different oxidative stress agents employed are H₂O₂ (2 mM), t-BOOH (0.5 mM), CuOOH (0.5 mM), and diamide (2 mM). The region of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting is also shown as a loading control.