Fig. 4

Binding of CepR to the cefD-cmcI promoter regions. a Expression and purification of His-tagged CepR analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lane 1, control sample. Lane 2, His-tagged CepR induced by the addition of IPTG. Lane 3, purified fusion protein. Lane M, protein marker. b Electrophoretic mobility shift assay analysis of the binding of His-tagged CepR to the promoter regions of cefD-cmcI. The P1, P2, and P3 cefD-cmcI intergenic regions were 208 bp, 315 bp, and 318 bp, respectively. The probes were incubated either with no protein ( − ) or with 3.0 μg of CepR ( + ).1.0 µg polydI/dC was used as a competitor. c A fixed amount of probe P3 was incubated with no CepR (Lane 1), 0.5–3.0 µg of CepR (Lanes 2–5), or 3.0 μg of CepR and 100-fold excess of unlabeled specific probe (Lane 6). 1.0 µg polydI/dC was used as a competitor. d Positions of P1–P3 cefD-cmcI intergenic regions in the cephamycin C biosynthetic gene cluster