FIG. 4.

Optimization of the MraY-MurG coupled assay. (A to D) Membranes of E. coli AMA1004 ponB::Spc^r were incubated with UDP-MurNAc(pp) and UDP-[3H]GlcNAc at 37°C. (A) Time dependence; 4 μg of membrane protein was used, and the reaction was terminated at different times. (B) Protein dependence; various quantities of membrane protein were used, and the reaction was terminated after 5 min. (C) Effects of tunicamycin (IC₅₀, ∼0.3 μg/ml) and nisin (IC₅₀, ∼16 μg/ml). (D) Effect of vancomycin (IC₅₀, ∼9 μM). In panels C and D, the assay was performed under the standard conditions described in Materials and Methods. (E) MraY-MurG assay in wild-type membranes in the presence of 0.3 μM moenomycin. Shown are the effects of tunicamycin (T; 4 μg/ml), nisin (N; 312 μg/ml), vancomycin (V; 300 μM), penicillin G (P; 200 μM), and bacitracin (B; 4 U/ml). The error bars indicate SEM.