FIG. 5.

Flp is secreted and processed by Y. enterocolitica. (A) Whole-cell extracts (CE) and supernatants (SN) of the Y. enterocolitica wild type, the Δflp strain, and the complemented Δflp strain and overproducing PypB from pBAD33-pypB were analyzed by Western blotting using Flp-specific antiserum. The upper part of the blot was probed with streptavidin-PO to detect the cytoplasmatic acetyl-CoA carboxylase biotin carboxyl carrier protein (AccB) to exclude contamination of supernatants with cytoplasmatic proteins. (B) Whole-cell extracts of the Y. enterocolitica wild type and E. coli DH5α carrying pWSK-pypB/flpHA and pBAD33-pypB to overproduce PypB were analyzed by Western blotting with HA-specific antibody to detect Flp-HA. Flp-HA is expressed in both strains but is processed only in Y. enterocolitica. (C) Ultracryo-electron microscopy of Y. enterocolitica overproducing PypB indicates that Flp is expressed and transported to the bacterial inner membrane. Flp was detected by using gold-labeled Flp antiserum (bar, 1 μm).