Escherichia coli TOP10 (Invitrogen) and E. coli CA434 [39] (link) were cultured in Luria-Bertani (LB) medium, supplemented with chloramphenicol (25 µg/ml), where appropriate. Routine cultures of C. difficile 630 Δerm[40] (link) and C. difficile R20291 were carried out in BHIS medium (brain heart infusion medium supplemented with 5 mg/ml yeast extract and 0.1% [wt/vol] L-cysteine) [41] (link). C. difficile medium was supplemented with D-cycloserine (250 µg/ml), cefoxitin (8 µg/ml), lincomycin (20 µg/ml), and/or thiamphenicol (15 µg/ml) where appropriate. A defined minimal media [18] (link) was used as uracil-free medium when performing genetic selections. A basic nutritive mannitol broth for growth assays of C. difficile strains were prepared as follows : Proteose peptone no. 2 4% [wt/vol] (BD Diagnostics, USA), sodium phosphate dibasic 0.5%[wt/vol], potassium phosphate monobasic 0.1%[wt/vol], sodium chloride, 0.2% [wt/vol], magnesium sulfate, 0.01% [wt/vol], mannitol, 0.6% [wt/vol] with final pH at +/−7.35. For solid medium, agar was added to a final concentration of 1.0% (wt/vol). Clostridium sporogenes ATCC 15579 was cultivated in TYG media [7] (link). All Clostridium cultures were incubated in an anaerobic workstation at 37°C (Don Whitley, Yorkshire, United Kingdom). Uracil was added at 5 µg/ml, and 5-Fluoroorotic acid (5-FOA) at 2 mg/ml. All reagents, unless noted, were purchased from Sigma-Aldrich.
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