Identification of the Major Oxidatively Damaged Proteins in Escherichia coli Cells Exposed to Oxidative Stress

In the present study we have analyzed protein oxidation on Escherichia coli when these cells were submitted to different stress conditions such as hydrogen peroxide, superoxide-generating compounds, and iron overloading. Carbonyl groups on oxidized cell proteins were examined by Western blot immunoassay. When anaerobically grown E. coli cells were exposed to hydrogen peroxide stress, alcohol dehydrogenase E, elongation factor G, the heat shock protein DNA K, oligopeptidebinding protein A, enolase, and the outer membrane protein A were identified as the major protein targets. A similar immunostained band pattern was found when cells were shifted from anaerobic to aerobic conditions in the presence of different concentrations of iron; it is relevant to note that oxidation of outer membrane protein C, not observed in peroxide stress conditions, was clearly detected as the concentration of iron was increased in the culture media. The hydrogen peroxide stress performed under aerobic conditions affected the b-subunit of F0F1-ATPase; the rest of the oxidized protein pattern was very similar to that found for anaerobic conditions, with the exception of alcohol dehydrogenase E, a protein not synthesized aerobically. Cells submitted to superoxide stress using menadione showed a more specific pattern in which elongation factor G and the b-subunit of F0F1-ATPase were affected significantly. When paraquat was used, although the degree of oxidative damage was lower, the same two modified proteins were detected, and DNA K was also clearly damaged. Cell viability was affected to different extents depending on the type of stress exerted. The results described in this paper provide data about the in vivo effects of oxidative stress on protein oxidation and give insights into understanding how such modifications can affect cellular functions.This work was supported in part by Grant PB94-0829-C02-02 from the Dirección General de Investigación Científica y Técnica and the Comissionat per Universitats i Recerca de la Generalitat de Catalunya and Ajuntament of Lleida (Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact