Penicillin-binding proteins exhibit functional redundancy during asymmetric cell division in <i>Clostridioides difficile</i>

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the polymerization of glycan strands by shape, elongation, division, and sporulation (SEDS) family glycosyltransferases and the cross-linking of peptide cross-bridges by class B penicillin-binding proteins (bPBP). While many bacteria use distinct bPBPs to perform specialized roles during a given cellular process, some bPBPs can play redundant roles, particularly in the presence of certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs to drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, synthesizes the polar division septum and the cortex peptidoglycan layer during sporulation in the pathogen <i>Clostridioides difficile</i>. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is partially dispensable for asymmetric division. The dispensability of SpoVD's catalytic activity requires the presence of its SEDS partner, SpoVE, and another sporulation-induced bPBP, PBP3. While PBP3 localizes to the polar septum and interacts with components of the polar division machinery, the ability of PBP3 to promote division during sporulation occurs independent of its catalytic activity. Notably, this latter finding differs from previously reported modes of functional redundancy in bacteria, indicating that there are diverse mechanisms by which penicillin-binding proteins can be functionally redundant in bacteria.IMPORTANCEPeptidoglycan synthesis requires the transpeptidase activity of penicillin-binding proteins (PBPs), which have specialized functions during cell growth, division, and differentiation. However, many bacteria produce PBPs with overlapping functions, and this functional redundancy can lead to increased antibiotic resistance. While the major pathogen, <i>Clostridioides difficile</i>, requires the SpoVD PBP to form spores, we found that its transpeptidase activity is dispensable for asymmetric division, the first morphological stage of sporulation, because a sporulation-induced PBP, PBP3, partially substitutes for SpoVD's function during this stage. Since PBP3's ability to promote asymmetric division in this context does not depend on the its catalytic activity, unlike prior studies of PBP functional redundancy, our analyses highlight the diversity in mechanisms used to enable functional redundancy between PBPs.