Fig. 3.

Processing of Cwp84. (A) Detection of Cwp84 in the cwp84 mutant overexpressing either WT or the Cys116Ala protein. C. difficile 630Δerm (WT) and the cwp84 mutant harboring plasmids as indicated were grown in BHI broth to late exponential phase (OD₆₀₀ = 0.5). Cell wall extracts and culture supernatants were prepared and analyzed by SDS-PAGE, followed by Western blotting with antibodies against Cwp84 and the C-terminal His₆ tag. Plasmids: –, pMTL960; 84, pCwp84_WT; 84*, pCwp84_C116A. The upper band (84 kDa) recognized by these antibodies in the cwp84 mutant carrying pCwp84_WT is indicated (◀). The N-terminal sequence of the 77-kDa protein corresponding to the lower band was determined as SSVAY. (B) Line diagram of domain structure of Cwp84 and location of the signal peptide and propeptide domains. At the top is the domain structure of Cwp84 showing the location of the signal peptide (black rectangle), as predicted by SignalP, and the cysteine protease domain (white box), as predicted by Pfam (PF00112). The three cell wall binding motifs (PF04122) are shaded in gray. Below is the N-terminal amino acid sequence of Cwp84 showing the sites of cleavage to release the signal peptide (▾) and the mature protein (▿).