Fig. 3.

E. coli PBP1a* variants have increased glycosyltransferase activity. (A) Purified PBP1a and its variants (5 µM) were incubated with purified E. faecalis lipid II (40 µM) for the indicated times. Reactions were stopped by incubation at 95 °C for 2 min. Products were incubated with S. aureus PBP4 (1 µM) and biotin-d-lysine (800 µM) to label the PG glycans produced. Labeled samples were then separated on an SDS/PAGE gel, transferred to a PVDF membrane, and visualized with IRDye 800CW Streptavidin. (B) The intensity of the glycan staining was quantified for each lane of each time series in A and plotted. Error bars represent the SD based on five replicates [PBP1a and PBP1a(G62S)], two replicates [PBP1a(T38P)], or three replicates [PBP1a(L146I)]. (C) Shown are the activities of each PBP1a variant relative to PBP1a(WT) at the 2-min time point. Activities were calculated by setting the activity for one of the PBP1a(WT) replicates to 1.0 and then calculating the activity of the other samples relative to it. Error bars represent the SD of the mean.