Cell wall hydrolysis promotes a second wave of transpeptidation to achieve cell separation following septation in Bacillus subtilis.

Septation and cell separation occur as distinct events during cell division in many Gram-positive bacteria. The process first involves synthesis of a complete, multilayered peptidoglycan (PG) septum dividing the cell, which is subsequently hydrolyzed to facilitate physical separation. Using fluorescent D-amino acids and high-resolution microscopy, we identify a previously unrecognized, post-septational wave of transpeptidation that crosslinks septal PG during cell separation in Bacillus subtilis. Notably, this activity does not involve new PG synthesis, but instead remodels pre-existing septal PG. The transpeptidase PBPH plays a key role in this process, and its activity and localization at the separating septum depend on PG hydrolysis by the endopeptidase LytF. Disruption of this interplay impairs cell separation. Our findings reveal a mechanism whereby the coordinated activities of PG hydrolysis and transpeptidation ensure successful cytokinesis. This work expands the current model of cell division by identifying post-septational transpeptidation as a key step in septal resolution and pole formation.