FIG 4.
OhrR directly represses the transcription of pigA-O genes. (A) Prodigiosin yield per cell unit (A534/OD600). (B) Expression analysis of C-terminal 3×FLAG-labeled PigA in WT/pigA-FLAG and ΔohrR/pigA-FLAG strains. Untreated (–tBHP): cells were grown in LB medium. Treated (+tBHP): cells were grown in LB medium supplemented with 0.05 mM tBHP. (C) ChIP-qPCR analysis of the pig operon promoter region enrichment by FLAG-labeled OhrR in WT/ohrR-FLAG. The WT was used as negative control. The relative enrichment of the pig operon promoter by OhrR is shown as %INPUT. Untreated (–tBHP): cells without the tBHP treatment were cross-linked. Treated (+tBHP): cells were treated with 5 mM tBHP for 30 min before cross-linking. (D) EMSA verification of OhrR binding to Ppig probe that harbors the promoter region of the pig operon. Labeled probe (0.2 nM) was added to each reaction mixture. OhrR concentrations in each reaction mixture are shown above the figure. Lane S, 100-fold unlabeled specific competitive probe. Lane N, 100-fold unlabeled nonspecific competitive probe. The arrow indicates free probes, and the bracket indicates protein-DNA complexes. (E) DNase I footprinting assay of OhrR and the pig operon promoter. Each reaction mixture contained 300 ng of FAM-labeled pig promoter probe, and the OhrR concentration is shown to the left of the figure (control: without addition of OhrR protein). The nucleotide sequences of the protected region (bolded residues) and the pig promoter are shown below the figure. Arrows indicate inverted repeats. The TSS of pigA is shown by an arrow and bolding. Numbers located on both sides of the nucleotide sequence indicate the distance (nt) from the TSS. The −35 and −10 regions are shown within a box. Shaded aera: ROP1 box. (F) Identification of the OhrR binding site in the pig promoter. EMSA of the association between OhrR and Ppig probe, in addition to the mutated Ppig probe (Ppig-mut). OhrR concentrations in each reaction mixture and nucleotide sequences of the predicted binding site and the mutated binding site are shown above the figure. The arrow indicates free probes, and the bracket indicates protein-DNA complexes. (G) Prodigiosin yield and pigA transcription levels in WT/PaacC1-pig and ΔohrR/PaacC1-pig strains. Transcription levels of genes in different strains were determined and normalized to 16S rRNA gene (internal control). The transcription value of pigA in WT/PaacC1-pig is set as 1 and the value of ΔohrR/PaacC1-pig is shown as fold change. Experimental data are shown as means ± SD (n = 3), and significant differences between data sets were evaluated with Student’s t tests. NS, no statistical significance; ***, P < 0.001.