The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide. When SoxR is activated by oxidation of its [2Fe–2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression. How the oxidized SoxR returns to and is maintained in its reduced state has been under question. To identity genes that constitute the SoxR‐reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants. Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE–rseABC operon. In‐frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype. The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR. Electron paramagnetic resonance analysis of SoxR and measurement of re‐reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria that serves as a barrier against harmful molecules, including antibiotics. The waaYZ locus that encodes the LPS core biosynthetic function in Escherichia coli was found to be induced strongly by superoxide generators but not by H 2 O 2 , ethanol, or heat shock. This induction was dependent on SoxRS, a superoxide and nitric oxide sensing system, through a soxbox in the waaY promoter that binds SoxS. A ⌬waaYZ mutant became more sensitive to some superoxide generators, and the activation of SoxR by these drugs became more sensitized in the mutant. Through phenotypic microarray analysis, we found that the mutant became sensitive to a wide variety of chemicals not restricted to oxidizing agents. We found that the mutant is under envelope stress and is altered in LPS composition, as monitored by the level of E activation and changes in the electrophoretic mobility of LPS, respectively. waaY expression was also regulated by MarA (multiple-antibiotic resistance regulator), which shares a binding site (soxbox) with SoxS, and was induced by salicylate, a nonoxidative compound. These results demonstrate a novel way of protecting gram-negative bacteria against various compounds by modifying LPS, possibly through phosphorylation. Since either oxidant or nonoxidant compounds elicit resistance toward themselves and other toxic drugs, this mechanism could serve as an efficient way for pathogenic bacteria to enhance survival during antibiotic treatment within an oxidant-rich host immune environment.

Summary SoxR is a [2Fe-2S]-containing sensor-regulator, which is activated through oxidation by redox-active compounds (RACs

Bacteria in natural habitats are exposed to myriad redox-active compounds (RACs), which include producers of reactive oxygen species (ROS) and reactive electrophile species (RES) that alkylate or oxidize thiols. RACs can induce oxidative stress in cells and activate response pathways by modulating the activity of sensitive regulators. However, the effect of a certain compound on the cell has been investigated primarily with respect to a specific regulatory pathway. Since a single compound can exert multiple chemical effects in the cell, its effect can be better understood by time-course monitoring of multiple sensitive regulatory pathways that the compound induces. We investigated the effect of representative RACs by monitoring the activity of three sensor-regulators in the model actinobacterium Streptomyces coelicolor; SoxR that senses reactive compounds directly through oxidation of its [2Fe–2S] cluster, CatR/PerR that senses peroxides through bound iron, and an anti-sigma factor RsrA that senses RES via disulfide formation. The time course and magnitude of induction of their target transcripts were monitored to predict the chemical activities of each compound in S. coelicolor. Phenazine methosulfate (PMS) was found to be an effective RAC that directly activated SoxR and an effective ROS-producer that induced CatR/PerR with little thiol-perturbing activity. p-Benzoquinone was an effective RAC that directly activated SoxR, with slower ROS-producing activity, and an effective RES that induced the RsrA-SigR system. Plumbagin was an effective RAC that activated SoxR, an effective ROS-producer, and a less agile but effective RES. Diamide was an RES that effectively formed disulfides and a weak RAC that activated SoxR. Monobromobimane was a moderately effective RES and a slow producer of ROS. Interestingly, benzoquinone induced the SigR system by forming adducts on cysteine thiols in RsrA, revealing a new pathway to modulate RsrA activity. Overall, this study showed that multiple chemical activities of a reactive compound can be conveniently monitored in vivo by examining the temporal response of multiple sensitive regulators in the cell to reveal novel activities of the chemicals.

No abstract