Analyses of cell wall synthesis in Clostridioides difficile reveal a diversification in cell division mechanisms in endospore-forming bacteria.

Current models of bacterial cell division assume that the core synthases of the multiprotein divisome complex, FtsW-FtsI, are the primary drivers of septal peptidoglycan (PG) synthesis. These enzymes are typically encoded in the highly conserved division and cell wall ( dcw ) cluster and are considered to be universally essential for cell division. Here, we combine bioinformatics analyses with functional characterization in the pathogen Clostridioides difficile to show that dcw -encoded PG synthases have undergone a surprising specialization in the sole endospore-forming phylum, Firmicutes, to fulfill sporulation-specific roles. We describe a novel role for these enzymes in synthesizing septal PG during the sporulation-specific mode of cell division in C. difficile . Although these enzymes are directly regulated by canonical divisome components during this process, dcw -encoded PG synthases and their divisome regulators are unexpectedly dispensable for cell division during normal growth. Instead, C. difficile uses its sole bifunctional class A penicillin-binding protein (aPBP) to drive cell division, revealing a previously unreported role for this class of PG synthases as the core divisome enzyme. Collectively, our findings reveal how the emergence of endosporulation in the Firmicutes phylum was a key driver for the functional repurposing of an otherwise universally conserved cellular process such as cell division. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a divisome that differs markedly from previously studied bacteria, thus representing an attractive, unique target for therapeutic purposes.