In vitro glycosyltransferase activity assay

Glycosyltransferase activity assays were performed using a protocol adapted from (35). Briefly, a 10× MTG buffer [125 mM HEPES (pH7.5), 20 mM MnCl₂, 2.5 mM Tween 80] was prepared. Master mixes of the reactions were prepared such that the final conditions were as follows: 0.1 µM purified His-PBP2a variant, 1× MTG buffer, 2% DMSO, 10 µM lipid II purified from E. faecalis as described in (33), and 200 µM cephalexin to block transpeptidase activity. Cephalexin was not included in the experiments presented in Fig. S4.

Reactions were initiated by the addition of lipid II and immediately placed at 25°C. Aliquots (10 µL) of the reaction mixture were withdrawn at the indicated times and stopped by heat denaturation for 5 min at 95°C. Following inactivation, recombinant S. aureus PBP4 (5 µM final concentration) (34) and biotinylated D-lysine (200 µM final concentration; Sigma) were added, and the samples were incubated for 1 hour at 37°C. Reactions were quenched by the addition of 15 µL of 2× SDS sample buffer with 10% β-mercaptoethanol, and 5 µL of the reaction was resolved by SDS-PAGE on a 4%–20% acrylamide gel for 80 min at 35 mA. The samples were transferred to a PVDF membrane and washed with TBS (Tris-buffered saline). The membrane was incubated in 0.4% paraformaldehyde for 1 hour, washed with 1× PBS, and then blocked for 1 hour in SuperBlock (Thermo). The membrane was then incubated in SuperBlock with IRDye 800CW streptavidin (diluted 1:5,000) for 1 hour at room temperature. Membranes were washed three times in TBS/Tween 20 and once in TBS and imaged using an Odyssey CLx System (LI-COR Biosciences).