Fig. 4.

Characterization of a cwp13 mutant and processing of Cwp13. (A) Analysis of the effects of an insertional mutation in cwp13 by SDS-PAGE and Western blotting. C. difficile 630Δerm (WT) and the cwp13 mutant harboring plasmids as indicated were grown in BHI broth supplemented with 15 μg of thiamphenicol/ml to late exponential phase (OD₆₀₀ = 0.5). Cell wall extracts and culture supernatants were prepared and analyzed by Coomassie blue staining (top) and Western blotting with antibodies against the C-terminal His₆ tag (6% acrylamide), Cwp84 (6% acrylamide) and LMW SLP. Plasmids: –, pMTL960; 13, pCwp13_WT; 13*, pCwp13_C109A. The 77-kDa Cwp13_WT and 84-kDa Cwp13_C109A proteins detected by Coomassie blue staining and the anti-His₆ tag are indicated (◁ and ◀), and their N-terminal sequences were determined as DNSNT and APTSY, respectively. (B) Domain structure of Cwp13 and location of the signal peptide and propeptide domains. At the top is domain structure of Cwp13 showing the location of the signal peptide (black rectangle) and the cysteine protease domain (white box), as predicted by Pfam (PF00112). The cell wall anchoring domains (PF04122) are shaded in gray. Below is the N-terminal amino acid sequence of Cwp13 showing the sites of cleavage to release the signal peptide (▾) and the mature protein (▿). (C) Domain structure of SlpA. The vertical bar indicates the cleavage site of Cwp84 to produce the mature HMW SLP and LMW SLP. The black triangle (▴) shows the approximate site of cleavage, within a cell wall binding domain, by Cwp13 to generate a 47-kDa N-terminal product.