Identification of the unwinding region in theClostridioides difficilechromosomal origin of replication

Faithful DNA replication is crucial for viability of cells across all kingdoms of life. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens.Clostridioides difficileis an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination ofin silicopredictions andin vitroexperiments to demonstrate thatC. difficileemploys a bipartite origin of replication that shows DnaA-dependent melting atoriC2,located in thednaA-dnaNintergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region ofC. difficileand contributes to our understanding of the initiation of DNA replication in clostridia.