Fig 4.

(A) Map of the P_exoT promoter showing the ExsA consensus binding sequence, and the conserved GnC and TGnnA sequences (bold with larger typeface) located within binding site 1. The nucleotide substitutions for each of the mutant P_exoT reporters used in the genetic loss-of-contact experiments are indicated with arrows. (B) The PA103 exsA::Ω strain carrying the mutant P_exoT-lacZ reporters and expressing wt ExsA from an arabinose-inducible expression vector was grown in the presence of EGTA and 0.1% arabinose and assayed for β-galactosidase activity. *, P < 0.05. (C) The PA103 exsA::Ω strain carrying either a P_exsC-lacZ (open bars) or a P_exoT-lacZ (hatched bars) transcriptional reporter was transformed with vectors expressing wt ExsA or the indicated ExsA alanine substitution mutants. The resulting strains were cultured under inducing (+EGTA) conditions for T3SS gene expression with 0.1% arabinose and assayed for β-galactosidase activity. The reported values are the percent activity for each alanine substitution mutant relative to wt ExsA. Immunoblots demonstrating the steady-state expression levels of ExsA and the alanine scanning mutants are shown as insets. The asterisk indicates a cross-reactive band that served as a loading control.