Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus

Abstract

A gene encoding superoxide dismutase (SOD), sodM, from S. aureus was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the S. aureus SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The sodM gene expressed from a plasmid rescued an Escherichia coli double mutant (sodA sodB) under conditions that are otherwise lethal. SOD activity gels of S. aureus RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in a sodM mutant, while the two lower bands were absent in a sodA mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the sodA and sodM sodA mutants but not the sodM mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the sodM sodA mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an S. aureus sodA mutant are maintained.

FIG. 1

Amino acid sequence alignments of bacterial SODs. The SodM (AF273269) and SodA (7) (AF121672) proteins of S. aureus and the SOD proteins from Bacillus subtilis (Bs-MnSOD, D86856), Listeria monocytogenes (Lm-MnSOD, M80526), E. coli (Ec-MnSOD, AE000465; Ec-FeSOD, AE000261), Pseudomonas putida (Pp-FeSOD, U64798), and Legionella pneumophila (Lp-FeSOD, D12922) are shown. Discriminating amino acids (31) used to differentiate between Mn- and Fe-SODs are in bold. The underlined region represents the partial SodM amino acid sequence identified by Poyart et al. (33).

FIG. 2

Activity gel analysis of E. coli (Ec) and S. aureus SODs. Lane 1, E. coli (sodA sodB) containing pCR2.1sodM; lane 2, E. coli (sodA sodB); lane 3, E. coli (sodA sodB) containing pCR2.1; lane 4, E. coli MG1655; and lane 5, S. aureus RN6390. Stained gels were scanned using the AlphaImager 2000 (Alpha Innotech Corp.) imaging system, and the inverse image was generated using NIH Image software.

FIG. 3

SOD sensitivity to hydrogen peroxide and potassium cyanide. Whole-cell lysates prepared from S. aureus RN6390 (lane 1), E. coli MG1655 (lane 2), and E. coli (sodA sodB) containing plasmid pBA23sodC (lane 3) were resolved by nondenaturing polyacrylamide gel electrophoresis and treated with either hydrogen peroxide (B) or potassium cyanide (C) prior to negative staining for SOD activity. (A) Untreated control. The gel was analyzed and the image generated as described for Fig. 2.

FIG. 4

Growth-phase-dependent SOD activity and sodM expression under conditions of low and high aeration. (A) Representative growth curve of S. aureus RN6390 grown under conditions of low (●) and high (○) aeration. Whole-cell lysates and total RNA were isolated under low- and high-aeration conditions at 3, 6, and 12 h of growth. (B) Northern analysis of total RNA hybridized with a sodM-specific probe. RNA concentrations were standardized according to A₂₆₀ values and loaded as either undiluted (U) or twofold serially diluted (numerical values) samples. (C) Nondenaturing polyacrylamide gel of whole-cell lysates stained for SOD activity. The gel was analyzed and the image was generated as described for Fig. 2. (D) Activity of SodM (●), the hybrid (■), and SodA (▴) under low (left)- and high (right)-aeration conditions. Percentages were determined from values generated by quantitative densitometric analysis as described in Materials and Methods.

FIG. 5

SOD activities from S. aureus sod mutants. Lane 1, RN6390; lane 2, sodM mutant; lane 3, sodA mutant; lane 4, double (sodM sodA) mutant; lane 5, double mutant containing pCL15sodM induced with IPTG; lane 6, double mutant containing pCL15sodM uninduced.

FIG. 6

Viable cell count of S. aureus RN6390 (○, ●), the sodM mutant (□, ■), the sodA mutant (▵, ▴), and the double (sodM sodA) mutant (◊, ⧫) grown in the absence (open symbols) and presence (closed symbols) of MV. MV was added at 1.5 (A), 6 (B), and 12 (C) h of growth. Values are means ×/÷ standard errors.

FIG. 7

Viable cell count of S. aureus RN6390 (○, ●), the sodA mutant (□, ■), the double mutant (▵, ▴), and the double mutant containing pCL15sodM (◊, ⧫) grown in the absence (open symbols) and presence (closed symbols) of MV. Values are means ×/÷ standard errors.