Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus

Abstract

The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.

FIG. 1.

Growth analysis of B. subtilis 168 and S. aureus COL cells after NO stress. Cells were grown in synthetic medium, and the cultures were initiated at an OD₅₀₀ of 0.5 (0 min) (○). The growth of parallel cultures exposed to 100 μM (B. subtilis) or 500 μM (S. aureus) of the NO donor MAHMA NONOate is also shown (•).

FIG. 2.

Synthesis of cytoplasmic proteins of B. subtilis after NO stress. The 2D gel images of newly synthesized proteins (labeled with l-[³⁵S]methionine) from exponentially growing cells (shown in green) and cells exposed to 100 μM concentrations of the NO donor MAHMA NONOate for 10 min (shown in red) were overlaid. Identified proteins with an increased synthesis rate 1 to 30 min after stress are labeled and color coded for their membership to specific regulons as indicated.

FIG. 3.

Synthesis of cytoplasmic proteins of S. aureus after NO stress. The 2D gel images of newly synthesized proteins (labeled with l-[³⁵S]methionine) from exponentially growing cells (shown in green) and cells exposed to 500 μM concentrations of the NO donor MAHMA NONOate for 5 min (shown in red) were overlaid. Identified proteins with increased synthesis after 1 to 60 min after stress are labeled. Proteins whose synthesis was also induced after a shift from aerobic to anaerobic growth conditions are colored blue (28).

FIG. 4.

Proteins with changed synthesis after NO stress in S. aureus COL involved in glycolysis (A), the tricarboxylic acid cycle (B), and fermentation (C). Details of the 2D dual-channel images generated from radioactively labeled cytoplasmic protein extracts of exponentially growing cells (colored in green) and of cells exposed to 500 μM concentrations of the NO donor MAHMA NONOate (colored in red) for 1, 5, 10, 30, and 60 min are shown.

FIG. 5.

Formation of lactate and butanediol in S. aureus and B. subtilis 20 min after exposition to 100 μM (only for B. subtilis) or 500 μl (for B. subtilis and S. aureus) of the NO donor MAHMA NONOate. Cells were grown in synthetic medium to an OD₅₀₀ of 0.5 and exposed to the NO donor. For metabolite analyses, samples were taken immediately before and 20 min after addition of the NO donor. Cells were separated from the supernatant by filtration, and the obtained supernatants were used for further analyses. Lactate and butanediol were detected and quantified by ¹H-NMR. The graphs show the increase of the concentration per minute and the OD₅₀₀ within 20 min after the addition of the NO donor for lactate and butanediol. The values are given as means ± the standard deviation of three parallel studies of two independently analyzed cell cultures.

FIG. 6.

Details of the 2D dual-channel image generated from the images of Sypro Ruby-stained cytoplasmic proteins of exponentially growing cells exposed to 10 mM H₂O₂ for 10 min (shown in red) and cells incubated with 100 μM (B. subtilis) or 500 μM (S. aureus) of the NO donor MAHMA NONOate prior to H₂O₂ addition (shown in green). Note that a lower amount of the proteins shows an acidic isoelectric point shift in cells preincubated with NO, indicating diminished irreversible thiol oxidation. AhpC, alkyl hydroperoxide reductase subunit C; HchA, chaperone protein HchA (Hsp31); GapA1, glyceraldehyde 3-phosphate dehydrogenase; PurQ, phosphoribosylformylglycinamidine synthase 1.