FIG 3.

The DEAD motif is critical for T3SS gene expression. (A) The deaD E168Q and deaD E168A alleles were cloned into pJN105 and assayed for their ability to complement a deaD mutant for P_exsD-lacZ reporter activity. The reported values were normalized to the deaD mutant carrying pDeaD (4,671 Miller units). (B) PA14 transposon mutants with insertion in annotated RNA-helicases were cultured under inducing conditions for T3SS gene expression and whole-cell lysates were analyzed for ExsA production by immunoblotting. A cross-reactive band (indicated with an asterisk) served as a loading control. (C) An expression plasmid for the PA103 equivalent of the PA14 12760 RNA helicase, which shares 44% identity with DeaD, was introduced into the deaD mutant. The resulting strain was assayed P_exsD-lacZ reporter activity. The reported values were normalized to wt PA103 carrying pJN105 (4,133 Miller units). *, P < 0.05; **, P < 0.005.