FIG. 3.

EHEC O157 produces an O-antigen G4C. Different EHEC strains were grown overnight in LB at 30°C and processed for electron microscopy as previously described (32). Representative ferritin-stained thin sections are shown in panel A. The strains tested include wild-type (wt) EPEC; wzy, etk, and etp mutants; and complemented mutants, as indicated above each frame. In panel B, total polysaccharides of EHEC were extracted and the G4C-PS was separated from the LPS as described in Materials and Methods. LPS O-antigen levels were analyzed by SDS-PAGE and Western blotting with anti-O157 antibody (B, upper panel, O-PS). Aliquots of the LPS-free G4C-PS preparation (10 μl) were spotted onto a membrane and dried, and the amount of G4C-PS was estimated by dot blotting with anti-O157antiserum (B, lower panel). Care was taken to apply equivalent amounts of material derived from the same numbers of bacteria to all of the lanes and spots. The genotypes of the strains analyzed are indicated above the lanes.