FIG. 1.

Multiple regions of ExoU are required for cytotoxicity in yeast. A. Transposon-based linker insertion mutagenesis was employed to randomly insert five amino acids throughout ExoU. The location of each insertion within the 687-amino-acid ExoU protein is depicted by a white bar. Insertions that abolished cytotoxicity in yeast are depicted as black bars extending above the protein. Five regions, represented as R1, R2, R3, R4, and R5, were chosen for further characterization. B. The effects of representative single-amino-acid substitutions in ExoU on yeast viability are shown. As was the case for wild-type ExoU, a K115A substitution did not affect the growth of yeast under conditions that repress expression of ExoU (SC-Glc agar) but prevented growth under conditions that induced expression of ExoU (SC-Gal agar). In contrast, a S142A substitution prevented ExoU-mediated killing during growth on inducing SC-Gal agar. C. Amino acids within regions 1 to 5 that resulted in a loss of cytotoxicity when changed are shown in boldface type and are underlined. All other listed amino acids did not abolish cytotoxicity when changed.