Monitoring Peptidoglycan Glycosyltransferase Activity

Lipid II used to monitor glycosyltransferase activity of the protein complex was dried using a nitrogen stream and dissolved in an equal volume of dimethyl sulfoxide (DMSO). The reaction and detection of glycan strands was adopted from previously published protocols^{3 (link),23 (link),42 (link),56 (link)}. Briefly, PaFtsWIQLB and EcFtsWIQLB were mixed at a final protein concentration of 1 µM in 10µL with 1 µL 10x Reaction Buffer (500 mM Tris pH 7.5, 200 mM MnCl₂), 1 µL DMSO and 1 µL Lipid II and incubated for 30 min at 25°C. Proteins were heat inactivated for 2 min at 95°C. Lipid II and glycan strands were labelled by incubating the reaction with 26 µM SaPbp4 and 20 mM BDL for 1 h at 25°C. An equal volume of Laemmli SDS-PAGE buffer was added and the mixtures were heat inactivated for 3 min at 95°C. Glycan strands were separated from Lipid II on a 4-20% Criterion TGX polyacrylamide gel (Bio Rad), run for 45 min at 200 V. After blotting onto PVDF membrane, the blot was incubated for 2 h in Superblock blocking buffer TBS (Thermo Scientific), followed by incubation with a 1:5000 dilution of IRDye800CW Streptavidin (LI_COR Bioscience) in TBS buffer at room temperature for 1 h. The blot was washed three times in PBS buffer and bands were visualised using an Odyssey CLx imaging system (Li-COR Bioscience).