FIG. 5.

YscP acts upstream of YopN. (A) The yopN yscP double mutant is impaired in Yop secretion. The yopN yscP-null strain YPIII(pIB8769) and the yopN-null strain YPIII(pIB82) were grown at 37°C in BHI medium with or without calcium to induce secretion. Bacteria were centrifuged to separate cell pellet and supernatant fractions. Equal amounts of supernatant fractions were separated by SDS-PAGE and stained with Coomassie brilliant blue. WT, wild type. (B) Surface localization of YscF in the yopN yscP mutant. 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 on SDS-PAGE gels, and Western blots were performed by using an anti-YscF antiserum.