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.
J Proteome Res
; 11(6): 3219-30, 2012 Jun 01.
Article
em En
| MEDLINE
| ID: mdl-22564194
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(â¢-))-generating paraquat and 10-100-fold more resistant to H2O2. A significant increase in intracellular levels of O2(â¢-), 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(â¢-) levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, with â¼2-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(â¢-) levels.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Nitrorredutases
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Espécies Reativas de Oxigênio
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Proteínas de Escherichia coli
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Escherichia coli
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article