Supporting Information

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Fig. 7. Construction of IS/pM2 and IS/pM5 strains. The 5'-terminal (including the ribosome binding site and start codon) or an internal fragment of nos was amplified by PCR and cloned into the pMutin2 plasmid to yield pM5 and pM2, respectively. wt cells were transformed with these plasmids, and erythromycin (em) resistant colonies were selected on LB agar plates. P_spac, the IPTG-inducible SPO1 bacteriophage promoter; P_nos, the nos gene promoter.

Fig. 8. Functional complementation of the nos deletion. Cells with nos disrupted by either pM2 or pM5 were diluted with fresh LB and treated with H₂O₂ as described in Fig. 6A. Where indicated, 1 mM IPTG was added ≈30 min before dilution. Data are shown as means ± SD from four experiments.

Fig. 9. Effect of exogenous NO on t-butyl hydroperoxide (tBOOH) toxicity. NO (30 mM) was added 5 sec before tBOOH to aerobically growing B. subtilis cells in LB at OD₆₀₀ ~ 0.5. Incubation with tBOOH was for 15 min. Data are shown as means ± SD from three experiments.

Fig. 10. Inhibition of purified KatA by Cys and reactivation by NO or diamide. For inhibition, KatA was mixed with 200 mM Cys for 60 min; for reactivation, either 50 mM NO-donor (MAHMA NONOATE) or 200 mM diamide was added for 5 sec and 20 min, respectively. 100% catalase activity = 0.5 M H₂O₂ min^–1·mg^–1. Data are shown as means ± SE from four experiments.

Fig. 11. Reactivity of Cys and NADPH with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium inner salt (MTS). Indicated amounts of Cys and NADPH were mixed with MTS (0.1 mg/ml) in LB, incubated 10 min at 37°C followed by OD₄₉₀ measurement. Data are shown as means ± SD from three experiments.

Fig. 12. Effect of dilution on the growth rate of wt and Dnos cells. Cells were grown aerobically in LB to late log phase (OD₆₀₀ ~ 0.8-0.9) at 30°C, before 1:1 dilution with fresh prewarmed LB. Data are shown as means ± SD from three experiments.

Fig. 13. NO protection of Staphylococcus aureus from H₂O₂-mediated toxicity. (A) The graph shows the time course of H₂O₂-mediated toxicity. 370 mM H₂O₂ was added at t = 0 to aerobically grown wt cells at OD₆₀₀ ~ 0.5 (in LB at 37°C). Where indicated, 30 mM NO was added 5 sec before H₂O₂. Chloramphenicol (Cm, 100 mg/ml) was added for 5 min before NO/H₂O₂ treatment. The percentage of surviving cells was determined by colony formation. Data are shown as means ± SD from three experiments. (B) A representative agar plate. Cells were treated as in A and plated on LB agar after serial dilution.

Table 1. Transient accumulation of cellular reducing equivalents (RE) upon dilution

RE accumulation was measured by the rate of formazan dye formation. Experimental conditions were as in Fig. 6A. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium inner salt (MTS) (0.1 mg/ml) was added 1 min after dilution. Cultures were incubated for another minute at room temperature, and OD₄₉₀ was measured in clarified supernatants. Data are shown as means ± SD from three experiments.

Supporting Text

We found that NO could not activate purified KatA (Fig. 10); rather, higher concentrations (>100 mM) of NO inhibited it (ref. 1 and data not shown). These results suggested that NO stimulation of catalase in the crude extract was mediated by an additional factor. Dialysis experiments (data not shown) suggested that the putative factor was a small molecule. Because low-molecular-weight (LMW) thiols have been shown to reversibly inhibit various catalases in vitro (2), and NO could readily S-nitrosylate free thiols in vivo (3, 4), we hypothesized that NO could reactivate thiol-inhibited catalase. Cys is the major free thiol in Bacillus subtilis (5) and thus a primary candidate for a KatA inhibitor. Indeed, we found that the presence of Cys at physiological concentration (200 mM) strongly suppressed the activity of purified KatA, whereas NO reversed this effect (Fig. 10). A specific scavenger of free thiols, diamide, also increased the activity of Cys-treated KatA (Fig. 10). Taken together, these data demonstrate that low doses of NO efficiently reactivate catalase that has been inhibited by Cys, and that this mechanism contributes to NO-mediated cytoprotection.

To confirm that the potential polarity effect of nos deletion was not responsible for its failure to alleviate oxidative stress in the experiments using Dnos strain (Fig. 6), we performed a complementation test using the pMutin2 type integrative plasmid to replace the chromosomal copy of nos with either a full copy of nos (pM5) or its truncated variant (pM2) under the control of an IPTG-inducible promoter. In the presence of IPTG, the IS/pM5 strain expresses the whole operon starting with intact nos (Fig. 7 Right), whereas IS/pM2 expresses only downstream gene(s) (Fig. 7 Left). As shown in Fig. 8, addition of IPTG to IS/pM5 cells, but not to IS/pM2, increased their resistance to H₂O₂, indicating that the activity of nos was indeed essential for H₂O₂ resistance.

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