Fig 5.

LcrF binds to the P. aeruginosa P_exoT promoter probe as a dimer. (A) EMSA using 50-bp radiolabeled probes derived from the ExsA-dependent P_exoT promoter. Probes (0.05 nM each) were incubated in the presence of 12, 36, 108, 324 nM LcrF (lanes 2 to 5) or LcrF_m (lanes 6 to 10) for 15 min at 25°C. Samples were analyzed by native polyacrylamide gel electrophoresis and phosphorimaging. (B) Diagram depicting P_exoT promoter probes with truncations that destroy binding sites 1 and/or 2. Solid and dotted lines represent native and nonnative P_exoT sequences, respectively. The adenine-rich region and conserved GnC and TGnnA sequences are indicated in bold. (C) EMSA using 60-bp radiolabeled probes derived from the ExsA-dependent P_exoT promoter. Probes (0.05 nM each) were incubated in the presence of 90 nM ExsA(A), LcrF(F), or LcrF_m for 15 min at 25°C. Samples were analyzed and imaged as described above.