Generation of Tn Mutants in C. difficile

Plasmid pRPF215, a Himar1 mariner delivery vector^{30 (link)}, was conjugated into R20291 from E. coli SD46 and R20291 transconjugants selected on BHI agars supplemented with 250 µg/ml cycloserine (Matrix Scientific, catalog no. 072929), 8 µg/ml of cefoxitin (Chem-Impex International, catalog no. 01490), and 15 µg/ml thiamphenicol (Sigma-Aldrich, catalog no. T0261). To generate Tn mutants, R20291-pRPF215 was grown overnight in BHI broth with 15 µg/ml thiamphenicol, then subcultured into BHI to OD₆₀₀nm of 0.2. Himar1 was induced with 100 ng/ml anhydrotetracycline (ATc) (Alfa Aesar, catalog no. J66688) for mutagenesis for overnight (15 h). Tn mutants were selected by spreading culture dilutions onto pre-reduced BHI agar containing 20 µg/ml lincomycin. After incubation (24 h), colonies were collected into 96 deep well plates, to amass 7488 independent colonies. The library was screened for metronidazole-susceptible Tn mutants by growing overnight cultures in BHI containing lincomycin and spotting 2 μl inocula onto BHI agars containing 0.5 µg/ml metronidazole and 5 µg/ml of hemin using a 96-Well Bench Top Pipettor. Presumptive metronidazole-susceptible Tn mutants were confirmed by MIC testing and underwent genome and Sanger sequencing to confirm the insertion sites for ermB. Primers and strain constructs used are listed in the Supplementary Data 2 and 3.