FIG 2 .

Finishing of needle elongation and substrate specificity switching are subsequent and separate events. (A) SpaS expression levels and autocleavage in the wild-type (wt) strain and indicated mutant strains with alanine substitutions in the conserved NPTH autocleavage motif were assayed by SDS-PAGE of whole-cell lysates, subsequent Western blotting, and immunodetection of the C-terminal FLAG tag of SpaS. Likewise, type III secretion into culture supernatants was profiled by immunodetection of the indicated substrate proteins. SpaS_fl, full-length SpaS. (B) Purified needle complexes of Salmonella containing wt SpaS or its autocleavage-deficient allele N258A were negatively stained on copper grids and visualized by electron microscopy. Bar, 100 nm. (C) Needle lengths of about 750 isolated needle complexes were measured for each strain, and the length distribution was illustrated in a histogram. (D) The protein content of isolated needle complexes was analyzed by SDS-PAGE, Western blotting, and immunodetection of the needle complex base proteins InvG, PrgH, and PrgK and of the inner rod protein PrgJ.