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. Author manuscript; available in PMC: 2018 Apr 23.
Published in final edited form as: Nat Chem. 2018 Jan 8;10(3):363–371. doi: 10.1038/nchem.2919

Figure 1. Schematic representation of peptidoglycan biosynthesis and assays used to measure the translocation of lipid II.

Figure 1

a, Synthesis of the peptidoglycan begins with the formation of soluble precursors, produced from a series of cytoplasmic reactions, catalyzed by MurA-MurF. MraY-MurG complete the synthesis of the precursor lipid II. Lipid II is then translocated to the periplasm by different families of flippases (such as FtsW or RodA, MurJ, and Amj), where it is incorporated into the pre-existing cell wall by penicillin binding proteins and additional factors (1–3). b, Assay used to measure the in vitro translocation of lipid II in liposomes (12). In this case, liposomes are loaded with donor-labeled lipid II (lipid II with green bulb) and when lipid II is flipped across the membrane (left panel with FtsW loaded), then an acceptor-labeled vancomycin derivative (U shaped box in brown) binds to it and a fluorescence signal is created. In this assay, fluorescence signal was created for lipisomes loaded with FtsW (left panel) but not when loaded with MurJ (right panel). c, Assay used to measure the in vivo translocation of lipid II in living E. coli (13). In this case, purified ColM toxin was added to actively growing E. coli cells. ColM cleaved lipid II product (disaccharide-pentapeptide) was then quantified in the periplasm. In this assay, flipping was observed only for MurJ (right panel) but not for FtsW (left panel).