We tested the hypothesis that during metabolism of naphthalene and other substrates by Pseudomonas sp. strain As1 oxidative stress arises and can be reduced by antioxidant enzymes. Our approach was to prepare plasmid constructs that conferred expression of two single antioxidant enzymes [Fpr (ferredoxin-NADP+ reductase) and SOD (superoxide dismutase)] and the pair of enzymes SOD plus AhpC (alkyl hydroperoxide reductase). The fpr, sodA and ahpC genes were placed under the transcriptional control of both the constitutive lac promoter and their respective native promoters. Both HPLC and growth-rate analyses showed that naphthalene metabolism was enhanced in the recombinant strains. All antioxidant-overexpressing recombinant strains, with the exception of one with an upregulated sodA gene due to the lac promoter [strain As1 (sodA)], exhibited resistance to the superoxide generating agent paraquat (PO). The growth of strain As1 (sodA) was inhibited by PQ, but this growth defect was rapidly overcome by the simultaneous overproduction of AhpC, which is a known hydrogen peroxide scavenger. After PQ-induced oxidative damage of the [Fe-S] enzyme aconitase, recovery of enzyme activity was enhanced in the recombinant strains. Reporter strains to monitor oxidative stress in strain As1 were prepared by fusing gfp (encoding green fluorescent protein, GFIP) to the fpr promoter. Growth on salicylate and naphthalene boosted the GFP fluorescent signal 21 - and 14-fold, respectively. Using these same oxidative stress reporters, overexpression of fpr and sodA was found to considerably reduce PO-induced stress. Taken together, these data demonstrate that the overproduction of Fpr or SodA contributes to oxidative tolerance during naphthalene degradation; however, elevated SOD activity may trigger the generation of excess hydrogen peroxide, resulting in cell death.
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