FIGURE 6.

Pilus assembly by PilE mCherry in a retraction-deficient background. The ability of PilE mCherry to support pilus assembly in a retraction-deficient strain was tested in the ΔfimU pilE ΔMPP pilT mutant that lacks surface piliation in the absence of fimU and pilE. A, pilus assembly was probed by shearing proteins from the surface of the cells and analyzing the surface fractions by SDS-PAGE. The flagellin band is used as a loading control. B, incorporation of PilE mCherry into pili was examined by Western blot analysis of the surface fractions above and probing for PilE using an α-PilE peptide antibody (1:1000). Arrows indicate the bands of interest. The samples were probed with an antibody to inner membrane protein PilO (1:5000) as a control for cell lysis; the last lane is PAO1 lysate as a positive control for PilO. C, intracellular levels of PilE and PilO were probed by Western blot analysis with α-PilE peptide antibody (1:1000 dilution) and α-PilO antibody (1:5000), respectively.