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Clostridium difficile  is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with  C. difficile  requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of  C. difficile  infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of  C. difficile . We validated our transposon mutagenesis approach in a model strain of  C. difficile  and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth  in vitro . We then applied this technique to the process of sporulation, an absolute requirement for  C. difficile  transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen.

High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile