FIG. 5.

SpdR binding to the regulatory region of cspD. (A) A DNA fragment corresponding to the regulatory region of cspD was ³²P labeled and incubated or not with increasing concentrations (0.025, 0.05, 0.1, 0.25, 0.5, and 1 μM) of purified protein as indicated. His-SpdR and the mutated version His-SpdR(D64A) were previously incubated in phosphotransfer reactions with SpdS. As a negative control, autophosphorylated SpdS was used alone. (B) Competition assays were carried out with 0.5 μM purified His-SpdR that was previously phosphorylated in vitro by SpdS. P, labeled probe; −, probe incubated with His-SpdR; S, probe incubated with His-SpdR in the presence of a 30-fold excess of unlabeled specific probe; N, probe incubated with His-SpdR in the presence of a 30-fold excess of nonspecific DNA competitor.