FIG. 3.

SpoIIAA is phosphorylated in vitro by RsbW. (A) The purified substrate AA, RsbV, or RsbS (20 μM) was incubated with [γ-³²P]ATP and the purified kinase indicated, either AB, RsbW, or RsbT (2 μM). After 30 min at 37°C, the reaction mixtures were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. (B) The AA (•), RsbV (▴), or RsbS (○) substrate was incubated with [γ-³²P]ATP and the AB kinase. Reactions were sampled after the indicated times at 37°C and analyzed by SDS-polyacrylamide gel electrophoresis. Amounts of labeled product were determined by phosphorimaging and expressed as percentages of the cognate AA substrate labeled at 30 min. (C) The AA (•), RsbV (▴), or RsbS (○) substrate was incubated with [γ-³²P]ATP and the RsbW kinase. Reactions were treated as described for panel B, with the amount of each labeled product expressed as a percentage of the cognate RsbV substrate labeled at 45 min.