FIG. 8.

YscU cleavage is not required for functional Yop secretion. Four forms of YscU with different mutations (ΔNPTH, N263A, P264A, and T265A) were expressed from plasmids pML16, pML17, pML18, or pML19, respectively, in the yscU-null mutant strain YPIII(pIB75) and compared to the wild-type strain YPIII(pIB102). (A) ECL Western blot with monoclonal antibodies directed against the Flag epitope to visualize the Flag epitope containing fragments of YscU. The different forms of YscU indicated are, the full-length protein YscU (amino acids 1 to 354)_, the C-terminal cleavage product, YscU (amino acids 264 to 354). The thinner arrows point to two nonspecific bands which also appear in negative controls with Yersinia strains that do not carry any Flag-tagged constructs. ECL Western blot with polyclonal antiserum raised against the secreted Yops to analyze Yop expression and secretion was performed on whole-cell fractions (B) and supernatant fractions (C), respectively. The bacteria were cultured in BHI lacking calcium at 37°C, with or without 0.4 mM IPTG, as indicated in the figure. Molecular mass markers are shown to the left in kilodaltons.