Expression of Four Methionine Sulfoxide Reductases in Staphylococcus aureus

Abstract

Staphylococcus aureus possesses three MsrA enzymes (MsrA1, MsrA2, MsrA3) that reduce the S-epimer of methionine sulfoxide (MetO) and an MsrB enzyme that reduces R-MetO. The four msr genes are expressed from three different promoters. The msrA1/msrB genes are coexpressed. To determine the expression pattern of msr genes, three independent reporter strains were constructed where msr promoter was cloned in front of a promoterless lacZ and the resulting construct was integrated in the chromosome. Using these strains, it was determined that the msrA1/B expression is significantly higher in S. aureus compared to msrA2 or msrA3. Expression of msrA1/B was highest during stationary phase growth, but the expression of msrA2 and msrA3 was highest during the early to midexponential growth phase. Expression of msrA1/B was induced by oxacillin and the expression of msrA3 was upregulated by salt. Expression of msrA2 remained unchanged under all tested conditions.

Figure 1

Growth comparison of the msr(A1/B)P-lacZ, msrA2P-lacZ, and msrA3P-lacZ reporter constructs in S. aureus strain SH1000. Growth was measured by recording OD₆₀₀ periodically. Values indicate averages of data from three independent experiments ± standard deviation (SD). The msr(A1/B)P-lacZ, msrA2P-lacZ, and msrA3P-lacZ reporter strains are represented by closed circles, open circles, and closed triangles, respectively.

Figure 2

β-galactosidase activity levels in msr(A1/B)P-lacZ (a), msrA2P-lacZ (b), and msrA3P-lacZ (c) reporter strains during different stages of growth under standard growth conditions. Growth and β-galactosidase activity were measured at different time points spectrophotometrically. For precise OD₆₀₀ determination, the late-stage cultures were diluted appropriately to bring cell density in measurable range of the spectrophotometer. OD₆₀₀ is indicated by open circles, and β-galactosidase activity (OD₄₂₀) is indicated by closed circles. Values indicate averages of data from three independent experiments ± standard deviation (SD).

Figure 3

Expression of the msr(A1/B), msrA2, and msrA3 loci in S. aureus SH1000 under different environmental stress conditions. Cultures of S. aureus SH1000 msr(A1/B)P-lacZ, msrA2P-lacZ, and msrA3P-lacZ reporter strains were grown to OD₆₀₀ of 0.3 at 37°C and treated separately with H₂O₂ (15 mM) (2), oxacillin (1.2 μg/mL) (3), pH 5.0 (4), pH 9.0 (5), and TSB with 1.5 M added NaCl (6) for 1 h. β-galactosidase activity (lighter bar) and growth (OD₆₀₀) (darker bar) were subsequently determined. β-galactosidase activity and growth of cells in TSB control are represented in bars 1. Values indicate averages of data from three independent experiments ± standard deviation (SD).

Figure 4

Expression of the msr(A1/B), msrA2, and msrA3 loci in S. aureus SH1000 in the presence of different oxidizing chemical agents. Cultures grown to OD₆₀₀ of 0.3 at 37°C were treated separately with the following stresses for 1 h: diamide (5 mM) (2), N-ethylmaleimide (NEM) (0.05 mM) (3), methyl viologen (MV) (20 mM) (4), menadione (MD) (0.05 mM) (5), cumene peroxide (CuOOH) (0.0125%) (6), and sodium nitroprusside (SNP) (5 mM) (7). β-galactosidase activity (lighter bar) and growth (OD₆₀₀) (darker bar) were subsequently determined. β-galactosidase activity and growth of cells in TSB control are represented in bars 1. Values indicate averages of data from three independent experiments ± standard deviation (SD).