Plumbagin is found in many medicinal plants and has been reported to have antimicrobial activities. We examined the molecular responses of Escherichia coli to plumbagin by using a proteomic approach to search for bacterial genes up-regulated by the drug. The protein profile obtained was compared with that of E. coli without the plumbagin treatment. Subsequent analyses of the induced proteins by mass spectroscopy identified several up-regulated genes, including ygfZ, whose function has not been defined. Analyses of the 5-flanking sequences indicate that most of these genes contain a marbox-like stretch, and several of them are categorized as members of the mar/sox regulon. Representatives of these genes were cloned into plasmids, and the marbox-like sequences were modified by site-directed mutagenesis. It was proven that mutations in these regions substantially repressed the level of proteins encoded by the downstream genes. Furthermore, plumbagin's early effect was demonstrated to robustly induce SoxS rather than MarA, an observation distinctly different from that seen with sodium salicylate.Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a naphthoquinone having antibacterial (6), antifungal (5), anticancer (22), and antimutagenic activities (7). Like other redoxcycling chemicals such as paraquat and menadione, plumbagin has been used as an agent to generate superoxide or reactive oxygen species in order to study oxidative stress (14). Plumbagin has been suggested to activate SoxS by oxidizing the SoxR molecule (10, 21) or inhibiting the repression of MarR (2), the effect of which is exerted on marA, resulting in activation of the mar/sox regulon. In addition, it has been observed that the expression of some members of the mar/sox regulon in Escherichia coli, such as sodA (8), nfo (3), ribA (20), and pqi (21), is up-regulated by treatment with plumbagin.Although plumbagin could induce excessive expression of superoxide dismutase and catalase, overexpression of sodA failed to protect E. coli (17). The toxic effect of plumbagin may not simply result from the production of reactive oxygen species. It has been reported that plumbagin inhibits NADH dehydrogenase, as well as causing respiratory arrest (17). Plumbagin has also been shown to modify the lactose carrier and inhibit its binding with galactoside; the modified carrier then becomes completely inactive (29). The above effects appeared more or less to result directly from the chemical nature of plumbagin. In this report, we focus on the responses of the bacteria to the chemical, in which multiple proteins were simultaneously induced by plumbagin treatment. We report the evaluation of the bacterial regulatory systems after treating E. coli with plumbagin. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. E. coli JM109 was used as the major experimental strain for the chemical treatments and the cloning host. Plasmids pGEM-T-easy (Promega, Madison, WI), pBluescript II SKϩ (Stratagene, La Jolla, CA), and pQE60 (QIAGEN, Valencia, CA) wer...