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
Abstract
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 (O2(•-))-generating paraquat and 10-100-fold more resistant to H2O2. A significant increase in intracellular levels of O2(•-), but not H2O2, 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 O2(•-) levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, wit...Continue Reading
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