FIG. 3.

Mutations in yscU reduce YscF export by a yscP mutant. (A) Mutations in the cytoplasmic domain of YscU were introduced in trans into the yscP yscU double null strain SAL3. Bacteria were grown at 37°C in BHI-plus-calcium medium, and YscU expression was induced with 0.2% arabinose. Cultures were sheared to remove T3SS needle structures. Sheared and cell pellet fractions were separated by centrifugation, and proteins in the sheared fraction (shearate) were precipitated with TCA. Equal percentages of sheared and cell pellet fractions were run on SDS-PAGE gels. YscF was detected by Western blotting with a polyclonal antiserum. pBAD18, vector control; pYscU-WT, plasmid expressing wild-type YscU; pYscU-Y287G, plasmid expressing YscU with the Y287G mutation; pYscU-Y317D, plasmid expressing YscU with the Y317D mutation. (B) The same fractions described above were probed with a monoclonal antibody against the cytoplasmic protein GroEL.