Levels of Escherichia coli thioredoxin 1 (Trx1), Trx2, glutaredoxin 1 (Grx1), Grx2, and Grx3 have been determined by novel sensitive sandwich enzyme-linked immunosorbent assay. In a wild type strain, levels of Trx1 increased from the exponential to the stationary phase of growth (1.5-fold to 3400 ng/mg), as did levels of Grx2 (from ϳ2500 to ϳ8000 ng/mg). Grx3 and Trx2 levels were quite stable during growth (ϳ4500 and ϳ200 ng/mg, respectively). Grx1 levels decreased from ϳ600 ng/mg at the exponential phase to ϳ285 ng/mg at the stationary phase. A large elevation of Grx1 (20 -30-fold), was observed in null mutants for the thioredoxin system whereas levels of the other redoxins in all combinations of examined null mutants barely exceeded a 2-3-fold increase. Measurements of thymidine incorporation in newly synthesized DNA suggested that mainly Grx1 and, to a lesser extent, Trx1 contribute to the reduction of ribonucleotides. All glutaredoxin species were elevated in catalase-deficient strains, implying an antioxidant role for the glutaredoxins. Trx1, Trx2, and Grx1 levels increased after exposure to hydrogen peroxide and decreased after exposure to mercaptoethanol. The levels of Grx2 and Grx3 behaved exactly the opposite, suggesting that the transcription factor OxyR does not regulate their expression.Escherichia coli employs two separate pathways that use NADPH to reduce cytosolic disulfides: the thioredoxin and the glutaredoxin systems. The thioredoxin system consists of thioredoxin reductase and thioredoxins 1 and 2 (Trx1 and Trx2).

Three Escherichia coli glutaredoxins catalyze GSHdisulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB ؊ ) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB ؊ cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.

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