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<p>Abstract</p> <p>Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. <it>Escherichia coli </it>strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in <it>E. coli </it>including <it>ahpC, mdaB, nfnB, nfo, sodA, yggX </it>and <it>ygfZ</it>. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, <it>ygfZ </it>and <it>sodA </it>were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of <it>ygfZ</it>. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when <it>sodA </it>was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of <it>E. coli </it>to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing <it>E. coli</it>. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type <it>E. coli </it>but not in the Δ<it>ygfZ </it>mutant. Our results showed that the presence of <it>ygfZ </it>is not only critical for the <it>E coli </it>resistance to plumbagin but also facilitates the plumbagin degradation.</p>