Fig. 4

Reversible inhibition of the DNA binding activity of MsrR by H₂O₂ and role of Cysteine residue. a The interaction between His₆-MsrR and DNA fragments amplified from MsrR’s ORF. b The interaction between His₆-MsrR and the promoter fragment in the intergenic region between msrR and mfs (named P_msrR). c Inhibition of the DNA binding activity of MsrR by H₂O₂ and reversal of the inhibition by DTT. MsrR was prepared in three different concentrations, and aliquots were taken for EMSAs (control). Then H₂O₂ was added to the binding reaction mixture to a final concentration of 10 mM, and aliquots were taken for EMSA. In the next step DTT (a final concentration of 50 mM) was added to 10 mM H₂O₂-containing binding reaction mixture, and aliquots were taken for EMSAs. All aliquots were incubated in binding buffer, pH 8.0, with 40 ng P_msrR and then separated on an 8% native polyacrylamide gel. d The interaction between His₆-MsrR and the promoter mutating the predicted MsrR binding region (P_msrRM). e CdCl₂ was added to the binding reaction mixture to a final concentration of 0.3 mM, and the interaction between His₆-MsrR and P_msrR was performed. f The interaction between the mutated derivatives MsrR:C62S and P_msrR in the absence (left panel) or presence (right left) of 10 mM H₂O₂. Results were obtained in three independent experiments, and data show one representative experiment done in triplicate