Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in .

Peptidoglycan (PG) is a giant macromolecule that completely surrounds bacterial cells and prevents lysis in hypo-osmotic environments. This net-like macromolecule is made of glycan strands linked to each other by two types of transpeptidases that form either 4→3 (PBPs) or 3→3 (LDTs) cross-links. Previously, we devised a heavy isotope-based PG full labeling method coupled to mass spectrometry to determine the mode of insertion of new subunits into the expanding PG network (Atze et al., 2022). We showed that PG polymerization operates according to different modes for the formation of the septum and of the lateral cell walls, as well as for bacterial growth in the presence or absence of β-lactams in engineered strains that can exclusively rely on LDTs for PG cross-linking when drugs are present. Here, we apply our method to the resolution of the kinetics of the reactions leading to the covalent tethering of the Braun lipoprotein (Lpp) to PG and the subsequent hydrolysis of that same covalent link. We find that Lpp and disaccharide-peptide subunits are independently incorporated into the expanding lateral cell walls. Newly synthesized septum PG appears to contain small amounts of tethered Lpp. LDTs did mediate intense shuffling of Lpp between PG stems leading to a dynamic equilibrium between the PG-tethered and free forms of Lpp.