FIG. 5.

Autoradiographs showing autophosphorylation of WalK_Spn PAS domain mutant constructs, phosphoryltransfer to WalR_Spn, and WalK_Spn phosphatase of WalR_Spn∼P. Representative time courses are shown and quantitated in Fig. S5. Combined reactions of WalK_Spn autophosphorylation and WalR_Spn phosphoryltransfer were performed at 25°C in reaction mixtures containing Mg²⁺ or Ca²⁺ as described in Materials and Methods. WalK_Spn autophosphorylation reactions proceeded for 3 min before WalR_Spn was added without removal of ATP (t = 0). Reactions contained the following concentrations of proteins: 3.4 μM WalK_SpnΔN35 D133N,N136Y,L140R (N)-Sumo (A) and 2.5 μM WalK_SpnΔN35 ΔPAS[104-198] (N)-Sumo (B). Each reaction contained 9.6 μM WalR_Spn.