Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute (Hinxton, UK). Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer (10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC (Roche)). After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer (0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB) was added, and samples were heated to 96uC for 5 mins. Total cell lysates (amounts corrected for OD 600 ) were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in PBST buffer (phosphate buffered saline with 0.1% v/v Tween-20) containing 5% membrane blocking reagent (Amersham Biosciences). To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection (Amersham Bioscience), or a goatanti-mouse-biotin-conjugated secondary antibody (Dako) followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody (Jackson). Detection was done using on a Typhoon instrument (GE Healthcare). Background corrected peak volumes were quantified using ImageQuant TL (Amersham Biosciences).