Fig 9.

The PA4774::lux mutant is more susceptible to lipid peroxidation than wild-type PAO1. Mid-log-phase cultures were stained with 2 μM C₁₁-BODIPY^581/591 and exposed to various concentrations of H₂O₂, and lipid peroxidation (green fluorescence) was measured every 10 min for 2 h. (A) Lipid peroxidation in PAO1 cultures grown under noninducing conditions with 2 mM Mg²⁺ and treated with increasing concentrations of H₂O₂. (B) Effect of growth under low-Mg²⁺ (0.02 mM) or high-Mg²⁺ (2 mM) conditions on resistance to oxidative membrane damage in PAO1 following 25 mM H₂O₂ treatment. (C) To complement the PA4774::lux mutant lipid peroxidation phenotype, the PA4774::lux_CC strain was challenged with 25 mM H₂O₂ treatment (P < 0.01). (D) The addition of 1 mM exogenous spermidine (Spd) was tested for the ability to restore parental levels of lipid peroxidation to the PA4774::lux mutant following 10 mM H₂O₂ treatment (P < 0.05). (C and D) Green C₁₁-BODIPY^581/591 fluorescence was measured after 100 min of H₂O₂ exposure. Each value shown is the average of three technical replicates with standard deviations, and each experiment was performed three times. The unpaired Student's t test was used to compare the levels of lipid peroxidation among strains.