Molecular basis of coupled peptidoglycan synthesis by the FtsWIQLB divisome complex from Vibrio cholerae

Bacterial cell division requires synchronized synthesis of septal peptidoglycan by the glycosyltransferase FtsW and the transpeptidase FtsI within the divisome. How glycan polymerization and peptide crosslinking are coordinated within this assembly remains poorly understood. Here, we provide molecular insights into this coupling in the core divisome complex FtsWIQLB from Vibrio cholerae. By integrating a quantitative, native substrates-based dual-activity assay, cryo-EM, AlphaFold modeling, disulfide trapping, mass spectrometry, and chemical biology, we identify an inactive down and a putative active up conformation of FtsI with distinct interaction networks. We discover a coupling helix in FtsI that directly modulates FtsW processivity. Importantly, crosslinking requires a minimum glycan strand length, consistent with our model of coupling. Lastly, the lipid tail of lipid II is important for FtsW processivity, with short-tail analogs acting as inhibitors. Our findings suggest that substrate-driven conformational switching of FtsI spatially and temporally couples these essential enzymatic activities. Bacterial cell division requires coupled peptidoglycan synthesis by FtsW and FtsI. Here, authors uncover that conformational switching of FtsI, via a coupling helix, spatiotemporally links glycan polymerization and peptide crosslinking.