Expression of TcdE isoforms in C. difficile. (A) TcdE sequence. All possible translation start sites are indicated as Met1, Met25, and Met27; the potential Shine-Dalgarno (SD) sequences are underlined, and mutated nucleotides in the specified constructs are in boldface type. (B) Cytoplasmic membrane protein analysis of C. difficile strains expressing various TcdE isoforms. Western blots were probed with anti-TcdE, anti-6×His, or anti-ATPase beta-subunit antibodies.