Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity

Abstract

A Staphylococcus aureus mutant (SPW1) which is unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to the sodA family of manganese-dependent superoxide dismutases (SOD). Whole-cell lysates of the parental 8325-4 strain demonstrated three zones of SOD activity by nondenaturing gel electrophoresis. The activities of two of these zones were dependent on manganese for activity and were absent in SPW1. The levels of SOD activity and sodA expression were growth-phase dependent, occurring most during postexponential phase. This response was also dependent on the level of aeration of the culture, with highest activity and expression occurring only under high aeration. Expression of sodA and, consequently, SOD activity could be induced by methyl viologen but only during the transition from exponential- to postexponential-phase growth. SPW1 was less able to survive amino acid limitation and acid stress but showed no alteration in pathogenicity in a mouse abscess model of infection compared to the parental strain 8325-4.

FIG. 1

Alignment of the S. aureus SodA amino acid sequence with those of E. coli [P00448, P09157], S. typhimurium [P43019], L. pneumophila [P31108], B. subtilis [P54375], P. putida [P77928], L. monocytogenes [P28764], and the putative S. aureus enzyme [S54793] (only partially sequenced). Numbers represent the position of the amino acid sequence. Fe and Mn denote metal iron cofactor of enzyme. Residues in bold are predicted to bind metal iron cofactor. Boxed residues have been predicted to bind Mn²⁺. Asterisks pinpoint deletions, which discriminate Mn- from Fe-SOD.

FIG. 2

SOD activity of whole-cell extracts of S. aureus 8325-4 and SPW1. Extracts were separated on a 12.5% (wt/vol) nondenaturing polyacrylamide gel, and stained for SOD activity. Lane 1, 8325-4; lane 2, SPW1; lane 3, 8325-4 metal ion depleted; lane 4, 8325-4 metal ion depleted, reconstituted with Mn²⁺.

FIG. 3

SOD activity and expression of sodA during growth and the effect of methyl viologen. (A) Total SOD activity. 8325-4 (○,●,▵,▴,□,■) and SPW1 (⧫,◊) were grown under high (▵,▴,○,●,⧫,◊) or low (□,■) aeration in BHI or in BHI with 10 μM methyl viologen (▵,▴). Closed symbols represent OD₆₀₀, and open symbols represent SOD activity as described in Materials and Methods. (B) Expression of sodA. SPW1 was grown in BHI with high aeration with (▵,▴) or without (○,●) 10 μM methyl viologen. Growth was measured by OD₆₀₀ (closed symbols) and β-galactosidase activity (open symbols) as described in Materials and Methods. (C) Effect of growth phase on methyl viologen induction of sodA. Expression of sodA was measured during growth of SPW1 in BHI (●) and in BHI with 10 μM methyl viologen added 0 h (○), 2.75 h (▴), 4.5 h (▵), and 7 h (■) after inoculation. Cells were incubated with high aeration, and expression was measured as described in Materials and Methods. Growth as measured by OD₆₀₀ is shown for one representative culture (□).

FIG. 3

SOD activity and expression of sodA during growth and the effect of methyl viologen. (A) Total SOD activity. 8325-4 (○,●,▵,▴,□,■) and SPW1 (⧫,◊) were grown under high (▵,▴,○,●,⧫,◊) or low (□,■) aeration in BHI or in BHI with 10 μM methyl viologen (▵,▴). Closed symbols represent OD₆₀₀, and open symbols represent SOD activity as described in Materials and Methods. (B) Expression of sodA. SPW1 was grown in BHI with high aeration with (▵,▴) or without (○,●) 10 μM methyl viologen. Growth was measured by OD₆₀₀ (closed symbols) and β-galactosidase activity (open symbols) as described in Materials and Methods. (C) Effect of growth phase on methyl viologen induction of sodA. Expression of sodA was measured during growth of SPW1 in BHI (●) and in BHI with 10 μM methyl viologen added 0 h (○), 2.75 h (▴), 4.5 h (▵), and 7 h (■) after inoculation. Cells were incubated with high aeration, and expression was measured as described in Materials and Methods. Growth as measured by OD₆₀₀ is shown for one representative culture (□).

FIG. 4

Effect of methyl viologen on cell survival. Cells (8 h postexponential phase) of SPW1 were resuspended in PBS (●) or in PBS containing 10 μM methyl viologen (○). Cell viability was determined by plating on CDM agar.

FIG. 5

Role of SodA in acid tolerance and adaptation. (A) S. aureus 8325-4 (●,○) and SPW1 (▴,▵) were grown and treated with acid as described in Materials and Methods. Exponential-phase cells were either incubated at pH 4 (37°C) for 1 h (○,▵) or untreated (●,▴) prior to pH 2 assault. Viability was determined by plating on CDM agar. Results are representative of three independent experiments showing less than fivefold variability. (B) Induction of sodA expression and SOD activity during acid adaptation. Exponential-phase cells (OD₆₀₀ = 0.8) were resuspended in CDM pH 7 (●,▴) or pH 4 (○,▵). Expression of sodA (SPW1, ▴,▵) and total SOD activity (8325-4, ●,○) were determined as described in Materials and Methods. Error bars represent the standard deviations of three independent samples.