FIG. 7.

Subcellular localization studies with HrcN and HrcL. HrcN, HrcL, and HpaB colocalize to the bacterial membranes upon activation of the T3S system. Strains 85* (wild type [wt]) 85*ΔhrcL (ΔhrcL), 85*ΔhrcN (ΔhrcN), and 85E*ΔhrcU (ΔhrcU) were grown in minimal medium A supplemented with sucrose and Casamino Acids under secretion-permissive (pH 5.3) and nonpermissive (pH 7.0) conditions. Membrane (M) and soluble (S) fractions were separated by ultracentrifugation. For the analysis of HrcL, HpaB, and HpaC, proteins were synthesized as C-terminally c-Myc epitope-tagged derivatives. Total protein extracts (TE), M, and S were analyzed by immunoblotting using antibodies directed against HrcN, HrcC, and the c-Myc epitope.