Fig. 4

Rif_B not Rif_SV remarkably weakened the DNA binding affinity of RifQ. The RifQ binding affinity to the promoter regions of both rifP was tested with the addition of either Rif_B (a) or Rif_SV (b). Probes were incubated with (blue line) or without (red line) RifQ protein, and were then partially digested by DNase I. The RifQ-protected regions were indicated by dashed boxes. Rif_B but not Rif_SV appeared to weaken the RifQ binding affinity. As Rif_B and Rif_SV were dissolved in DMSO, DMSO was added in the blank group. To prevent non-specific binding between RifQ and DNA probes, sheared salmon sperm DNA was added in above reactions. c Rif_B inactivated the regulatory activity of RifQ in vivo. RT-PCR assay was employed to analyze the transcriptional levels of rifP in LYZL11 (Additional file 9: Table S1), a rifA mutant strain, which did not produce rifamycins. Either DMSO or Rif_B was added into the growth medium and samples were collected at both 12 and 24 h after rifamycin addition. As indicated in the DNase I Footprinting assay, 2.5 mM Rif_B was able to completely deactivate the RifQ binding activity, and therefore a final concentration of 2.5 mM Rif_B was added into the growth medium