SoxRS-Regulated Expression and Genetic Analysis of theyggXGene ofEscherichia coli

Abstract

Genomic studies with bacteria have identified redox-responsive genes without known roles in counteracting oxidative damage. Previous transcriptional profiling showed that expression of one such gene, yggX, was activated by superoxide stress in Escherichia coli. Here we show that this activation could be mimicked by artificial expression of the regulatory protein SoxS. Northern analysis confirmed the transcriptional activation of yggX by oxidative stress or SoxS expression but not in response to the related MarA or Rob proteins. Northern analysis showed that mltC, which codes for a peptidoglycan hydrolase and is positioned immediately downstream of yggX, was also regulated by oxidative stress or ectopic expression of SoxS. Purified SoxS protein bound to the predicted yggX promoter region, between positions 223 and 163 upstream from the yggX translational start site. Within this region, a 20-bp sequence was found to be necessary for oxidative stress-mediated activation of yggX transcription. A yggX deletion strain was hypersensitive to the redox-cycling agent paraquat, and a plasmid expressing YggX complemented the sensitivity of the deletion strain. Under exposure to paraquat, the yggX deletion strain showed a deficiency in aconitase activity compared to the isogenic wild-type strain, while expression of YggX from a multicopy plasmid increased the aconitase levels above those of the wild-type strain. These results demonstrate the direct regulation of the yggX gene by the redox-sensing SoxRS system and provide further evidence for the involvement of yggX in protection of iron-sulfur proteins against oxidative damage.

The importance of the Rcs phosphorelay in the survival and pathogenesis of the enteropathogenic yersiniae

“…The microarray data indicate that the YPIII pIB1 Rcs phosphorelay is a major regulator of stressresponse genes, presumably due to its ability to respond to perturbations of the membrane as an early indication of stress. The Rcs regulon includes genes encoding DNA repair enzymes, mutY, mutH, mutT and deoB, along with YPO0917 and YPO0953, which are paralogues of YggE and YggX that have a function in restoring physiological defects of E. coli after oxidative stress (Pomposiello et al, 2003;Skovran et al, 2004;Kim et al, 2005).…”

Expression Analysis of Up-Regulated Genes Responding to Plumbagin inEscherichia coli

“…A stretch of sequence containing a putative marbox was observed in the regions upstream of fldA, mdaB, nfnB, nfo, sodA, and yggX. These sequences have been proposed to be the controlling elements, but so far only that of yggX has been characterized by binding of SoxS to a promoter-containing fragment and those of mdaB and nfnB have been mapped by fragment deletion in a promoter assay (28,32). It is worth noting that those characterizations were not based upon the effects of plumbagin.…”

“…Similar to nfo, ygfZ and sodA lost their reactions toward plumbagin when bacterial soxS was deleted (data not shown). Although not tested, fldA and yggX are presumably in the same category since they have been known to be regulated by SoxS (32,37).…”

“…To confirm the effects of plumbagin treatment on gene expression, a mutation was introduced into the mapped marbox sequence upstream of mdaB (28,32). In the pMH-mdaB construct, the putative core element GCAC of the marbox ( pression level of MdaB from pMH-mdaBm was fivefold decreased relative to that from pMH-mdaB (compare lanes 1 and 3 in Fig.…”

“…Therefore, a single base mutation of the marbox sequence drastically decreased the basal level of MdaB expression, as well as the plumbagin induction effect. In a similar approach, the proposed marbox of nfo (28,32) was mutated (Fig. 4A) and the core-containing element CGCAT in pMH-nfo was mutated to TCTAG in pMH-nfom.…”