FIG 7.

Purified DeaD_His promotes ExsA synthesis in vitro. (A) Purified DeaD_His and DeaD_His E168A were analyzed via SDS-PAGE, followed by Coomassie blue staining, and were estimated to be >90% homogeneous. (B) The deaD mutant was transformed with expression vectors encoding untagged DeaD, DeaD_His, or DeaD_His E168A. The resulting strains were cultured under inducing conditions for T3SS gene expression and assayed for P_exsD-lacZ reporter activity and DeaD_His production by immunoblotting for the histidine tag. The reported values were normalized to the deaD mutant expressing untagged DeaD (6,230 Miller units). (C) In vitro synthesis reaction mixtures lacking template (lane 1) or containing the exsA template (lanes 2 to 7) were incubated in the absence or presence of the indicated concentrations of DeaD_His E168A (lane 3) or DeaD_His (lanes 4 to 7). Detection was based on incorporation of radiolabeled methionine and phosphorimaging. (D) In vitro synthesis reactions were performed as described above using lcrF or vfr templates. *, P < 0.05; **, P < 0.005.