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. 2021 Feb 24;10:e61525. doi: 10.7554/eLife.61525

Figure 2. The Förster resonance energy transfer (FRET) signal arises from both the glycosyltransferase and transpeptidase reactions.

(A) Peptidoglycan (PG) synthesized in reactions of PBP1BEc in the presence or absence of 1 mM ampicillin was incubated with no PG hydrolase (U), DD-endopeptidase MepM (M), or muramidase cellosyl (C), and aliquots were analysed by SDS-PAGE. Reaction conditions were the same as indicated in Figure 1B–D. (B) FRET efficiency for samples prepared as indicated in A, calculated using the (ratio)A method (see Materials and methods). Values are mean ± SD of at least three independent experiments. (C) PBP1BEc (0.5 µM) was incubated with 5 µM each of lipid II-Atto647n, lipid II-Atto550, and 14C-labelled lipid II. At indicated time points, aliquots were taken and reactions were stopped by addition of moenomycin. After measuring fluorescence (see D), the PG was digested with the muramidase cellosyl, and the resulting muropeptides were reduced with sodium borohydride and separated by HPLC. The structures of muropeptides corresponding to peaks 1–3 are shown below the chromatograms. (D) Fluorescence spectra taken with excitation at 522 nm for the samples described in C. (E) Quantification of peak 2 (GTase product, blue), peak 3 (GTase+TPase, black), or the sum of both 2 and 3 (yellow) from chromatograms in C, along with the FRET signal (red) calculated as the ratio of acceptor emission over donor emission from data in D.

Figure 2—source data 1. Numerical data to support graphs in Figure 2 and original gel images for Figure 2B.

Figure 2.

Figure 2—figure supplement 1. Effect of proportion of labelled lipid II substrates on PBP1BEc activity in detergents.

Figure 2—figure supplement 1.

Triton X-100-solubilized PBP1BEc with or without LpoB were incubated with mixtures of lipid II substrates (14C-lipid II, lipid II-Atto550, and lipid II-Atto647) with increasing molar proportions of fluorescently labelled lipid II over the total amount of substrate. Reactions were monitored by fluorescence measurements, and each version of lipid II was quantified at the end. The total concentration of lipid II was kept at 15 µM while the molar ratio of lipid II-Atto550 to lipid II-Atto647n was always 1:1. (A) Consumption of each version of lipid II, monitored as described in Materials and methods, was higher in the presence of LpoB and decreased slightly with higher amounts of labelled lipid II. In all cases, the consumption of 14C-lipid II was slightly higher than that of fluorescent lipid II substrates. (B) The proportion of fluorescent material incorporated into peptidoglycan, calculated based on the consumption data in A, did not deviate significantly from the proportion of fluorescent substrates at the start of reactions. (C) A higher proportion of fluorescent lipid II increased the final FIacceptor/FIdonor ratio, but the increase was higher in the presence of LpoB. (D) The initial slope of Förster resonance energy transfer reaction curves increased significantly with the proportion of labelled lipid II in the presence of LpoB but not in its absence. (E) Table listing the data represented in B and D. The real-time slope represents the increase in FIacceptor/FIdonor per minute at the start of reactions. All values are ± SD of three technical replicates.
Figure 2—figure supplement 1—source data 1. Numerical data to support graphs in Figure 2—figure supplement 1 and original gel images used to quantify labelled lipid II consumption.