A reducing system of the superoxide sensor SoxR in Escherichia coli

Abstract

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.

Functional Analysis of SoxR Residues Affecting Transduction of Oxidative Stress Signals into Gene Expression

“…The resulting mutant strain and the parent EH46 strain were transformed with pSE380-based vectors containing the WT soxR gene or the mutant alleles encoding the proteins mentioned above, and the transcriptionactivating abilities of the various proteins in the two strains was determined by assaying ␤-galactosidase activity from the soxS promoter-lacZ reporter. As previously observed (40), WT SoxR showed significantly higher activity in an rsxC background, which indicates loss of regulation (Fig. 4).…”

“…In this work we also explored the effect of a recently identified reducing system on SoxR (40). The products of the rsx operon and the rseC gene are partially responsible for maintaining SoxR in a reduced state in the absence of oxidative stress (40), although no direct interactions have been identified between any of the gene products and SoxR.…”

“…The products of the rsx operon and the rseC gene are partially responsible for maintaining SoxR in a reduced state in the absence of oxidative stress (40), although no direct interactions have been identified between any of the gene products and SoxR. We considered the possibility that the in vivo transcription defect displayed by certain SoxR mutant proteins might be caused by excessive reduction of these proteins by the rsx/rseC system.…”

“…A complex of genes involved in partially maintaining the SoxR [2Fe-2S] centers in the reduced state was recently identified and comprises six members of the rsx operon and the rseC gene product (40). Deletion of any one of these genes increased the basal activity of SoxR by 20% (40).…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…Roe and colleagues showed that the rsxABCDGE and rseC genes mediate SoxR reduction (40). They encode membrane-bound proteins that resemble members of the Rhodobacer capsulatus Rnf complex, which drives electrons onto nitrogenase.…”