In brief, itrA was amplified from C79-13 genomic DNA and cloned into the plasmid pColdI, which was then transformed into E. coli BL21 (DE3) to express the rHis-ItrA protein. The expression protein was purified using the Ni-NTA Purification Kit (ThermoFisher, USA) according to the manufacturer’s instructions and stored at −80°C for EMSA.
EMSA was performed as follows: FAM-labeled PCR fragments encompassing the regulatory regions of ipaJ were amplified from C79-13 using the primer pairs PipaJ-EMSA-F/R (S2 Table ). Each PCR product (25 ng) was mixed with increasing concentrations of purified rHis-ItrA in a buffer containing 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 0.5 mM EDTA, 5% glycerol, 10 μg/ml BSA, and 200 ng/μl Poly(dI:dC). The reaction mixtures were incubated at 37°C for 30 min, and the protein–DNA complex was then separated by 6% native PAGE on ice. The DNA fragments were stained with ethidium bromide and displayed by Fluorescence image scans (Typhoon FLA9500, GE, USA).
EMSA was performed as follows: FAM-labeled PCR fragments encompassing the regulatory regions of ipaJ were amplified from C79-13 using the primer pairs PipaJ-EMSA-F/R (