C. difficile strains used in this study.

Cell division in bacteria is mediated by the “divisome,” a multiprotein complex that synthesizes the septal peptidoglycan needed to divide one cell into two. We recently showed that the major nosocomial pathogen Clostridioides difficile assembles a divisome that is fundamentally distinct from previously studied bacteria because it lacks functional orthologs of the septal peptidoglycan-synthesizing enzymes, FtsW and FtsI. While these enzymes were previously thought to mediate cell division in all walled bacteria, C. difficile instead uses the bifunctional Class A Penicillin Binding Protein PBP1 to mediate cell division. Here, we optimized a CRISPRi-based conditional expression system to define features within PBP1 that are critical for its essential functions. Our analyses identify a novel accessory domain that is required for PBP1 function and conserved across Peptostreptococcaceae family PBP1 homologs. We further show that PBP1’s glycosyltransferase and transpeptidase activities are both strictly required for bacterial growth. While PBP1 glycosyltransferase activity is required for septum synthesis during cell division, PBP1’s transpeptidase activity is surprisingly dispensable for cell division, with TPase-deficient (PBP1TPase*) cells producing multiple aberrant septa. We demonstrate that the uncontrolled septum synthesis observed in PBP1TPase* cells depends on the non-essential Class B PBP, PBP3, but the catalytic activity of PBP3 is dispensable for this function. Since we also show that PBP3 is recruited to the divisome complex and forms a complex with PBP1, our analyses reveal a cryptic but important regulatory function for PBP3 in promoting C. difficile cell division.