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. 2022 Mar 8;88(5):e02041-21. doi: 10.1128/aem.02041-21

FIG 2.

FIG 2

Ohr represses the transcription of its own operon. (A) Transcription levels of ohrR and ohr in the WT, ΔohrR, and CohrR. Transcription levels of evaluated genes in different strains were normalized to 16S rRNA gene (internal control). The relative transcription values of evaluated genes in the WT strain are set as 1, while those in the ΔohrR and CohrR strains are presented as fold changes. (B) EMSA verification of OhrR binding to the PohrR probe that harbors the promoter region of the ohrR-ohr operon. Labeled probe (0.2 nM) was added to each reaction mixture, and the OhrR concentration in each reaction mixture is shown above the figure. Lane S: 100-fold unlabeled specific competitive probe. Lane N: 100-fold unlabeled nonspecific competitive probe. Arrow indicates free probes, and bracket indicates the protein-DNA complexes. (C) Prodigiosin yield per cell unit (A534/OD600) for the WT and WT/ohrR-FLAG. (D) ChIP-qPCR analysis of the ohrR promoter region enrichment by C-terminal 3×FLAG-labeled OhrR in WT/ohrR-FLAG, and the WT was used as a negative control. The relative enrichment value of the ohrR promoter by OhrR is shown as %INPUT. Untreated (–tBHP): Serratia sp. ATCC 39006 cells were cross-linked without the tBHP treatment. Treated (+tBHP): Serratia sp. ATCC 39006 cells were treated with 5 mM tBHP for 30 min before cross-linking. (E) DNase I footprinting assay of the OhrR and ohrR promoter. Each reaction mixture contains 300 ng of FAM-labeled ohrR 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 (gray background) and the ohrR promoter are shown below the figure. The predicted TSS is shown by the arrow and bolding, while the start codon is also bolded. Numbers located on both sides of the nucleotide sequence indicate the distance (nt) from the predicted TSS. Experimental data are shown as means ± standard deviation (SD) (n = 3), and statistical significance was evaluated using Student’s t tests. NS, no statistical significance; ***, P < 0.001.