Overexpression of reactive cysteine-containing 2-nitrobenzoate nitroreductase (NbaA) and its mutants alters the sensitivity of escherichia coli to reactive oxygen species by reprogramming a regulatory network of disulfide-bonded proteins

摘要

The effects of redox-sensitive proteins on Escherichia coli were investigated by overexpressing Pseudomonas 2-nitrobenzoate nitroreductase (NbaA) and its mutants. Overexpression of wild-type and mutant NbaA proteins significantly altered the sensitivity of E. coli to antibiotics and reactive oxygen species regardless of the enzyme activity for reduction of 2-nitrobenzoic acid. The overexpressed proteins rendered cells 100-10000-fold more sensitive to superoxide anion (O _2 ~-)-generating paraquat and 10-100-fold more resistant to H _2O _2. A significant increase in intracellular levels of O _2 ~-, but not H _2O _2, was observed during expression of wild-type and truncated (δ65-74, δ193-216, and δ65-74δ193-216) NbaA. From two-dimensional nonreducing/reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry analyses, 29 abundant proteins in the cytoplasm were identified to form interchain disulfide bonds, when cells were exposed to polymyxin B. Of them, down-regulation and modifications of SodB, KatE, and KatG were strongly associated with elevated cellular O _2 ~- levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, with ～2-fold up-regulation of heterodimeric integration host factor, Ihf. Activity gel assays revealed significant reduction of glyceraldehyde-3-phosphate dehydrogenase with constant levels of 6-phosphogluconate dehydrogenase. These changes would support a high level of NADPH to reduce H _2O _2-induced disulfide bonds by forced expression of thioredoxin A via thioredoxin reductase. Thus, overexpression of wild-type and truncated NbaA partially compensates for the lack of KatE and KatG to degrade H _2O _2, thereby enhancing disulfide bond formation in the cytoplasm, and modifies a regulatory network of disulfide-bonded proteins to increase intracellular O _2 ~- levels.