“…A sequence encoding a ArcA/CytR/CRP-His 6 fusion protein was cloned into vector pET-28a, expressed in E. coli BL21 (DE3), and purified using an Ni–NTA-Sefinose Column in accordance with the protocol provided by the manufacturer [ 37 , 39 ]. EMSA was performed by adding increasing amounts of purified phosphorylated ArcA protein (0, 1.5, 3.0, 4.5, and 6.0 µM or 0, 0.6, 1.2, 1.8 and 2.4 µM) to cytR or flrA DNA fragments (50 ng) in a binding buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM DTT, 50 mM KCl, 50 μg/mL BSA, 10% glycerol] supplemented with 20 nM acetyl phosphate [ 39 ], followed by incubation for 40 min at room temperature. Similarly, fliK DNA fragments (50 ng) were incubated with increasing amounts of 6 × His-tagged CytR or CRP protein (0, 1.2, 2.4, and 3.6 µM or 0, 2, 3, and 4 µM) in a binding buffer [20 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, 200 µM cAMP, and 10% glycerol] [ 20 ], followed by incubation for 40 min at room temperature.…”