Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications.

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.     

Gene Expression and Physiological Role of Pseudomonas aeruginosa Methionine Sulfoxide Reductases during Oxidative Stress

Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H₂O₂. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.