Figure 3.

Approximately 45% of PnpR is phosphorylated (PnpR~P) in a ΔphoU2 mutant. (A) Representative Phos-tag SDS-PAGE of the encapsulated parent strain not expressing a FLAG-tagged protein (IU1781, lane 1) and of the pnpR-L-FLAG³ (IU6689, lane 2), and ΔphoU2 pnpR-L-FLAG³ (IU6687, lane 3) mutants growing exponentially in BHI broth. The gel was Western blotted with anti-FLAG antibody as described in Materials and Methods. The top two bands in all lanes are nonspecific. The upper anti-FLAG-specific band corresponds to phosphorylated PnpR~P, and the lower band corresponds to unphosphorylated PnpR. Control experiments show that the upper band is heat-sensitive, as expected for PnpR~P (Figure S4). (B) Quantification of 3 independent Phos-tag SDS-PAGE experiments. Less than 1% of PnpR was phosphorylated in the phoU2⁺ strain, whereas ≈45% of PnpR is phosphorylated (PnpR~P) in the ΔphoU2 mutant. P-value was determined by an unpaired t-test in GraphPad Prism; ^***P < 0.001.