Overexpressing antioxidant enzymes enhances naphthalene biodegradation in Pseudomonas sp. strain As1.
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 (PQ). 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, GFP) 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 PQ-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.
Kang
Yoon-Suk
YS
Division of Environmental Science and Ecological Engineering, Korea University, Anam-Dong 5 Ga, Seoul, South Korea.
Lee
Yunho
Y
Division of Environmental Science and Ecological Engineering, Korea University, Anam-Dong 5 Ga, Seoul, South Korea.
Jung
Hyungil
H
Department of Biotechnology, Yonsei University, Sinchon-Dong, Seoul, South Korea.
Jeon
Che Ok
CO
Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, South Korea.
Madsen
Eugene L
EL
Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
Park
Woojun
W
Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, South Korea.|Division of Environmental Science and Ecological Engineering, Korea University, Anam-Dong 5 Ga, Seoul, South Korea.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019677:Artificial Gene Fusion; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis* / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009281:Naphthalenes / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D054464:Peroxiredoxins / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D020156:Salicylic Acid / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics
D000900:Anti-Bacterial Agents; D009281:Naphthalenes; D016877:Oxidants; D049452:Green Fluorescent Proteins; C031721:naphthalene; D054464:Peroxiredoxins; D013482:Superoxide Dismutase; D005287:Ferredoxin-NADP Reductase; D020156:Salicylic Acid; D010269:Paraquat