Genome-wide mapping of DsbR-binding sites
We firstly found that DsbRS TCS (encoded by PA2479-PA2480) was involved in counteracting the toxic effects of copper. The identification of two DsbR binding reigions in dsbD-dsbR intergenic region DNA led us to globally characterize all DsbR-binding loci on the chromosome of Pseudomonas aeruginosa. In order to express C-terminal flag-tagged DsbR, we first constructed the ?dsbRS::DsbR-Flag strain. We next investigated DsbR-binding to the chromosome of this strain during exponential growth by ChIP-Seq, chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing. Sequence reads obtained from three independent ChIP-Seq experiments using Flag-specific antibodies were mapped to the genome of the ?dsbRS::DsbR-Flag strain. Using MACS software, we identified 18 reproducible peaks of DsbR binding, which were enriched by >2 fold.Approximately 83% (15 peaks) are in the promoter regions of 23 genes (within 600 bp upstream of the translational start site) involved or potentially involved in various biological processes including protein folding (e.g., dsbDEG operon and dsbB), protein secretion (e.g.., hxcU, hxcP, hcpC), transport (e.g., opdT, pa2911, and pstS), and regulation of transcription (i.e., dsbRS, pa3398). Collectively, this study thus uncover a new signal transduction pathway in P. aeruginosa, DsbS/DsbR/DsbDEG and DsbB, for the regulation of Dsb system in response to copper stress. Overall design: ChIP-Seq of DsbR in the ?dsbRS::DsbR-Flag strain at the mid-log phase. Three independent experiments were performed. Input DNA (No IP) was used as a control.