FIG. 6.

The proteolytic cleavage of YscU is independent of a functional TTSS. ECL Western blot with monoclonal antibodies directed against the Flag epitope was employed to visualize the cleavage of YscU in whole-cell fractions in different bacteria: E. coli K-12 strain S17-1λpir, the plasmid-cured strain YPIII, and the yscU-null mutant YPIII(pIB75), all containing pML13 expressing full-length YscU tailed with a Flag tag. The different forms of YscU indicated in the figure are the full-length protein YscU (amino acids 1 to 354) and the C-terminal cleavage product YscU (amino acids 264 to 354). The thinner arrow points to a protein band recognized by the Flag antibodies that appears also in negative controls, i.e., Yersinia strains where no Flag-tagged construct is present. The Yersinia strains were cultured at 37°C in calcium-depleted medium, and the E. coli strain was grown in LB at 37°C; all cultures were supplemented with 0.4 mM IPTG. Molecular mass markers are shown to the left in kilodaltons.