title: 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, 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 H2O2-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 H2O2, thereby enhancing disulfide bond formation in the cytoplasm, and modifies a regulatory network of disulfide-bonded proteins to increase intracellular O2(•-) levels.
authors.affiliation: Department of Microbiology, Catholic University of Daegu School of Medicine , Daegu705-718, Republic of Korea.
authors.forename: Yong-Hak
authors.initials: YH
authors.lastname: Kim
authors.forename: Myeong-Hee
authors.initials: MH
authors.lastname: Yu
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003553:Cystine / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D008826:Microbial Sensitivity Tests; D009601:Nitroreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D060066:Protein Interaction Maps; D011549:Pseudomonas / Q000201:enzymology; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D003553:Cystine; C029492:aromatic NADH-dependent nitroreductase; D009601:Nitroreductases