Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escherichia coli.
The NADP(H)-dependent enzymes glucose-6-phosphate dehydrogenase (G6PDH) and ferredoxin(flavodoxin)-NADP(H) reductase (FPR), encoded by the zwf and fpr genes, respectively, are committed members of the soxRS regulatory system involved in superoxide resistance in Escherichia coli. Exposure of E. coli cells to the superoxide propagator methyl viologen (MV) led to rapid accumulation of G6PDH, while FPR was induced after a lag period of several minutes. Bacteria expressing G6PDH from a multicopy plasmid accumulated higher NADPH levels and displayed a protracted soxRS response, whereas FPR build-up had the opposite effects. Inactivation of either of the two genes resulted in enhanced sensitivity to MV killing, while further increases in the cellular content of FPR led to higher survival rates under oxidative conditions. In contrast, G6PDH accumulation over wild-type levels of expression failed to increase MV tolerance. G6PDH and FPR could act concertedly to deliver reducing equivalents from carbohydrates, via NADP(+), to the FPR acceptors ferredoxin and/or flavodoxin. To evaluate whether this electron-transport system could mediate reductive repair reactions, the pathway was reconstituted in vitro from purified components; the reconstituted system was found to be functional in reactivation of oxidatively damaged iron-sulfur clusters of hydro-lyases such as aconitase and 6-phosphogluconate dehydratase. Recovery of these activities after oxidative challenge was faster and more extensive in transformed bacteria overexpressing FPR than in wild-type cells, indicating that the reductase could sustain hydro-lyase repair in vivo. However, FPR-deficient mutants were still able to fix iron-sulfur clusters at significant rates, suggesting that back-up routes for ferredoxin and/or flavodoxin reduction might be called into action to rescue inactivated enzymes when FPR is absent.
Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), División Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
Mariana
M
Giró
Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), División Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
Néstor
N
Carrillo
Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), División Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
Adriana R
AR
Krapp
D000154:Aconitate Hydratase / Q000032:analysis; D000222:Adaptation, Physiological; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000502:physiology*; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000502:physiology*; D005287:Ferredoxin-NADP Reductase / Q000502:physiology*; D005288:Ferredoxins / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005954:Glucosephosphate Dehydrogenase / Q000502:physiology*; D006836:Hydro-Lyases / Q000032:analysis; D016254:Mutagenesis, Insertional; D018384:Oxidative Stress; D010269:Paraquat; D018085:Regulon; D013481:Superoxides / Q000378:metabolism / Q000633:toxicity; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005288:Ferredoxins; D005418:Flavodoxin; D015534:Trans-Activators; D014157:Transcription Factors; D013481:Superoxides; C070724:SoxR protein, Bacteria; C070725:SoxS protein, E coli; D005954:Glucosephosphate Dehydrogenase; D005287:Ferredoxin-NADP Reductase; D001616:beta-Galactosidase; D006836:Hydro-Lyases; C022362:phosphogluconate dehydratase; D000154:Aconitate Hydratase; D010269:Paraquat