Effect of Wag31 phosphorylation on enzymatic activity of MraY and MurG. The three strains used in Fig. 1 were cultured to mid-log phase to purify a cell wall enriched envelope fraction (P60), which was then used as the sources of lipid (polyprenyl phosphate) and enzymes (MraY and MurG). 2 mg of P60 protein from each strain was incubated with 50 μM UDP-MurNAc-pentapeptide and 100 μM ATP for 5 min at 28°C, and reactions were initiated by adding 1 μCi of UDP-[14C]GlcNAc. After 1 hr, the quantity of radiolabeled lipid II (▶) was determined on TLC plates (inset) by a Phosphoimager. Data shown are from a representative experiment done in duplicate.