Fig. 6.

NOS-mediated protection from oxidative stress in B. subtilis. (A) Effect of nos deletion on H₂O₂ sensitivity. wt and Δnos cells were grown aerobically in LB to late log phase (OD₆₀₀ ∼ 0.8–0.9) at 30°C. An aliquot from each culture was diluted with an equal amount of fresh prewarmed LB for 2 min (diluted). Both diluted and undiluted aliquots were treated with 1 mM or 10 mM H₂O₂ for 30 min. The percentage of surviving cells was determined by colony formation. Values shown are the means and SD (error bars) from four independent experiments. (B) Exogenous Cys sensitizes B. subtilis to oxidative stress and induces NOS-mediated protection. Cells were grown as in A and diluted with an equal volume of saline or saline plus Cys (100 μM). Arg (100 μM) was added to all samples. After 2 min of incubation, cells were treated with 10 mM H₂O₂ for 30 min. Values shown are the means ± SD from three experiments. (C) Effect of fresh medium dilution on nitrite levels in wt and Δnos cell cultures. Conditions were as in A. Samples for nitrite measurements were taken 5 min after dilution. LB, nitrite level in LB. Values shown are the means ± SD from three experiments. (D) Effect of dilution on KatA activity. Conditions were as in A. Cells were collected 1 min after dilution and lysed immediately, and catalase activity was measured as described in Experimental Procedures. Values shown are the means ± SE from three experiments.