Oxygen and nitrate-dependent regulation of dmsABC operon expression in Escherichia coli: sites for Fnr and NarL protein interactions

The regions of dmsA regulatory DNA required for Fnr and NarL interactions in response to anaerobiosis and nitrate, respectively, were examined. Mutations within the Fnr site that deviated from the wild type sequence, TTGATaccgAACAA, or that removed an entire half-site, either impaired or abolished the anaerobic activation of dmsA-lacZ expression. The region for phosphorylated NarL (NarL-phosphate) binding at the dmsA promoter was identified by DNase I and hydroxyl radical footprinting methods. A large 97 bp region that overlaps the Fnr and RNA polymerase recognition sites was protected by NarL-phosphate but not by the non-phosphorylated form of NarL. Hydroxyl radical footprinting analysis confirmed the NarL-phosphate DNase I protections of both dmsA strands and revealed 8–9 protected sites of 3–5 bp occurring at ten bp intervals that are offset by 3 bp in the 3' direction.These findings suggest that multiple molecules of phosphorylated NarL bind along one face of the DNA and may interfere with Fnr and/or RNA polymerase interactions at the dmsA regulatory region. The interplay of these transcription factors insures a hierarchical expression of the dmsABC genes when respiration of the preferred electron acceptors, oxygen and nitrate, is not possible.Escherichia coli like many enteric and soil bacteria can respire anaerobically by using a variety of amine-N-oxides and methyl-sulfoxides as electron acceptors. This ability depends on the regulated synthesis of a membrane bound DMSO (dimethylsulfoxide) and/or TMAO (trimethylamine N-oxide) reductase enzyme. Use of these compounds in E. coli occurs by a broad substrate enzyme encoded by the dmsABC operon located at 20 minutes on the chromosome [1-3]. The 100-fold activation of dmsABC gene expression in response to anaerobiosis is controlled by the Fnr regulatory protein [4]. Following a re-examination, the dmsABC P1 transcription start site was located 223 nucleotides upstream of the translational start of dmsA[5,6]. This c