The PqrR Transcriptional Repressor of Pseudomonas aeruginosa Transduces Redox Signals via an Iron-Containing Prosthetic Group▿ †

Inducible defenses against oxidative stress are coordinated by redox-sensitive transcription factors that transduce oxidative damage into differential gene expression. The opportunistic human pathogen Pseudomonas aeruginosa has evolved under physiological and host-derived sources of oxidative stress. Previous work showed that the pqrABC and pqrR genes of P. aeruginosa, all lacking known functions, were induced by treatment of three different isolates of P. aeruginosa with paraquat (PQ), a superoxide-producing agent. Insertional mutation of the pqrABCR genes resulted in hypersensitive phenotypes to a variety of oxidants, although the hypersensitivity to PQ was marginal. Mutation of pqrR and complementation assays showed that PqrR regulated the pqrABC genes in response to PQ. PqrR, a member of the MarR family of transcriptional regulators, contains a C-terminal region with four conserved cysteines, which suggested redox-regulated transcriptional activity. Purified PqrR bound to two discrete sites at the pqrA and pqrR regulatory regions. The in vitro DNA binding activity of PqrR was decreased by exposure to air and reconstituted by treatment with dl-dithiothreitol. Elemental analysis and preliminary electron paramagnetic resonance experiments showed that PqrR contains iron. Interestingly, site-directed mutagenesis of C-terminal cysteines demonstrated that the four conserved cysteine residues are essential for in vivo redox sensing by PqrR

Defining a rob Regulon in Escherichia coli by Using Transposon Mutagenesis

The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor

The solution structure of the oxidative stress-related protein YggX from Escherichia coli

YggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N-terminal antiparallel β-sheets and three α-helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding

Rapid Changes in Gene Expression Dynamics in Response to Superoxide Reveal SoxRS-Dependent and Independent Transcriptional Networks

Background. SoxR and SoxS constitute an intracellular signal response system that rapidly detects changes in superoxide levels and modulates gene expression in E. coli. A time series microarray design was used to identify co-regulated SoxRSdependent and independent genes modulated by superoxide minutes after exposure to stress. Methodology/Principal Findings. soxS mRNA levels surged to near maximal levels within the first few minutes of exposure to paraquat, a superoxideproducing compound, followed by a rise in mRNA levels of known SoxS-regulated genes. Based on a new method for determining the biological significance of clustering results, a total of 138 genic regions, including several transcription factors and putative sRNAs were identified as being regulated through the SoxRS signaling pathway within 10 minutes of paraquat treatment. A statistically significant two-block SoxS motif was identified through analysis of the SoxS-regulated genes. The SoxRS-independent response included members of the OxyR, CysB, IscR, BirA and Fur regulons. Finally, the relative sensitivity to superoxide was measured in 94 strains carrying deletions in individual, superoxide-regulated genes. Conclusions/ Significance. By integrating our microarray time series results with other microarray data, E. coli databases and the primary literature, we propose a model of the primary transcriptional response containing 226 protein-coding and sRNA sequences. From the SoxS dependent network the first statistically significant SoxS-related motif was identified