Fig 4.

ExsA- and LcrF-dependent activation is sensitive to nucleotide substitutions in the ExsA consensus site. (A) Sequence of the P_exoT promoter showing the conserved GnC and TGnnA sequences (highlighted in bold) and the nucleotide substitutions indicated with an arrow. (B) The PA103 exsA::Ω strain carrying the indicated P_exoT-lacZ reporters was transformed with pExsA or pLcrF. The resulting strains were cultured in the presence of EGTA and assayed for β-galactosidase activity. Activation by ExsA (open bars) and LcrF (hatched bars) is reported as the percent activity normalized to the activity of the wild-type P_exoT-lacZ reporter. (C and D) EMSAs using radiolabeled probes derived from the mutant P_exoT promoters. The nonspecific P_algD probe (Non-Sp) was included as a negative control. Probes (0.05 nM each) were incubated in the presence of 45 nM ExsA (C) or LcrF (D) for 15 min at 25°C. Samples were analyzed by native polyacrylamide gel electrophoresis and phosphorimaging. The positions of shift products 1 and 2 are indicated.