Young-Sang Koh

A reducing system of the superoxide sensor SoxR in Escherichia coli

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.

Regulation of the ribA gene encoding GTP cyclohydrolase II by the soxRS locus in Escherichia coli.

We isolated a promoter that is inducible by paraquat, a superoxide-generating agent, fromEscherichia coli using the promoter-probe plasmid pRS415. Sequence analysis revealed that the promoter derives from theribA gene encoding GTP cyclohydrolase II, which is the first enzyme in the biosynthetic pathway of riboflavin. We fused thelacZ gene with theribA promoter to monitor the expression of the gene in the single-copy state. LacZ expression from theribA promoter was induced about eight-fold by 200 µM paraquat. Other known superoxide generators, menadione and plumbagin, also induced the expression ofβ-galactosidase in the fusion strain. On the other hand, no significant induction was observed following treatment with hydrogen peroxide, ethanol or heat shock. Induction ofβ-galactosidase was significantly reduced by the introduction of a Δsox-8::cat orsoxS3::Tn10 mutation into the fusion strain, indicating that theribA gene is a member of thesoxRS regulon. The transcriptional start site was determined by primer extension analysis and putative binding sites for SoxS in both orientations were identified. GTP cyclohydrolase II activity in soluble extracts ofE. coli increased more than three-fold on treatment with paraquat. This increase was dependent on thesoxRS locus, and reflects the increase in transcript levels. However, flavin pools did not change significantly. A possible role forribA induction during superoxide stress is discussed.

Dual regulation of the paraquat‐inducible gene pqi‐5 by SoxS and RpoS in Escherichia coli

The pqi‐5 gene, producing a probable membrane protein of unknown function, has been reported to be a member of the soxRS regulon. The SoxRS‐dependent induction of pqi‐5 by paraquat occurs only during the exponential phase. The expression of pqi‐5 increased in the absence of paraquat during the stationary phase or under conditions of carbon or phosphate starvation. This increase was regulated at the transcriptional level by RpoS (σs), which recognized the second promoter (P2) approx. 5 nucleotides upstream from the promoter (P1) used at the exponential phase. Studies with a series of 5’deletions revealed that the paraquat‐responsive element resides between ‐52 and ‐42 nucleotides upstream from the P1 start site, whose nucleotide sequence matches closely to other SoxS‐binding sequences. The stationary‐phase induction required sequences up to position ‐42, which correspond to the 5’border of the putative ‐35 hexamer for the P2 promoter. The binding of the purified SoxS protein to the pqi‐5 promoter upstream sequences was demonstrated by gel mobility‐shift and DNase I protection assays. The transcription from P1 promoter by EσD was activated by purified SoxS in vitro, as was observed in vivo. The dual regulation of pqi‐5 by SoxS at the exponential phase and RpoS at the stationary phase is the first to be reported among the members of the soxRS regulon, suggesting that this gene might indeed play some role under stressful conditions.

The reversed SoxS-binding site upstream of the ribA promoter in Escherichia coli

Abstract The ribA gene, encoding GTP cyclohydrolase II in Escherichia coli, is a member of the soxRS regulon, which is induced by superoxide-generating agents. By evaluating lacZ expression driven by the ribA promoter carrying different lengths of upstream region in a monolysogen, we found that the superoxide-responsive element resides between 56 and 94 nucleotides upstream of the transcriptional start site. Purified SoxS protein bound to this region and protected nucleotides between positions −80 and −58 from degradation by DNase I. This region contains a putative SoxS-binding sequence (soxbox) in reverse orientation. The SoxS protein interacted specifically with four guanine residues within the soxbox sequence, as demonstrated by methylation interference analysis. These results clearly indicate that SoxS binds to the reversed soxbox sequence in the ribA gene, while in other known genes of the soxRS regulon it binds to the normally oriented soxbox. Possible modes of interaction between SoxS and RNA polymerase are discussed.