FIG. 4.

Mutations in yscU suppress the yscP mutant phenotype. (A) Mutations in YscU increase Yop secretion by the yscP mutant. Mutations in the cytoplasmic region of YscU were introduced in trans into the yscP yscU-double-null strain. Expression of YscU was under the control of the tac promoter, which was not induced with IPTG in these experiments. Bacteria were grown at 37°C in BHI medium lacking calcium to induce secretion. Cultures were centrifuged to separate cell pellet (P) and culture supernatant (S) fractions. Equal percentages of each fraction were separated by SDS-PAGE, and Western blots were performed by using an anti-total Yops antiserum. WT, wild type. (B) Mutations in YscU increase the cytotoxicity of the yscP mutant. Mutations in the cytoplasmic domain of YscU were introduced in trans into the yscP yscU-double-null strain. HeLa cells were infected with these strains as described in Materials and Methods, and the effect on HeLa cells was recorded. Photographs were taken at the indicated times. (C) Mutations in YscU reduce the surface localization of YscF by the yscP mutant. Mutations in the cytoplasmic region of YscU were introduced in trans into the yscP yscU-double-null strain. Bacteria were grown at 37°C in BHI medium plus calcium and were sheared to release surface organelles from the bacterial surface as described in Materials and Methods. Cultures were centrifuged to separate cell pellet and sheared fractions. Two microliters of the pellet, diluted 1:10, and 5 μl of undiluted supernatant were separated by SDS-PAGE, and Western blots were performed by using an anti-YscF antiserum.