FIG 3.

SufR represses expression of the suf operon. (A) RT-qPCR analysis of the transcriptional levels of suf genes in the WT and DsufR grown on SFM for 2 or 6 days. Error bars: standard deviation of three technical replicates. ***, P < 0.001 (Student’s t test). (B) Binding of SufR-His₆ to its own promoter region by EMSA. Labeled probe (0.15 nM) and various amounts of SufR-His₆ were added to each reaction mixture. The 300-fold nonspecific competitor DNA (lane N) and unlabeled specific probe (lane S) were added to confirm the specificity of band shifts. Arrow: free probe. (C) DNase I footprinting assay of SufR on its own promoter region. Upper fluorogram, control reaction without protein. Protection fluorograms were acquired with increasing amounts of SufR-His₆ protein. (D) Nucleotide sequences of the sufR promoter region. Shaded box, the region protected by SufR; straight arrows, inverted repeat sequences; bent arrows, sufR transcriptional and translational start sites; boxes, putative −35 and −10 regions. (E) EMSAs of probe AB and the mutated probes AM and BM to identify the SufR-binding site. Mutations were introduced into the inverted repeat sequences A and B of probe AB to generate mutated probes AM and BM. Underlining, altered nucleotides.