Determination of binding efficiency of DNAs to the PhoP protein was carried out by the nitrocellulose-binding method (50). 32P-radiolabeled DNAs were incubated with a series of dilutions of the PhoP protein under the same condition used in the electrophoretic mobility shift assay at room temperature for 20 min. Final concentrations of PhoP protein were 0, 1, 2, 5, 10, and 20 μM. The samples were filtered through 0.45-μm nitrocellulose filters (HAWP, Millipore) under vacuum, and the filters were washed with 5 ml of the binding buffer, air-dried, and quantified using PhosphorImager (GE Healthcare).

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