Fig. 2: Cells expressing MraY(T23P) accumulate lipid II.
a, Schematic representation of the method used to isolate and analyse lipid II from bacterial cells. b–e, Representative extracted ion chromatograms of lipid II (EIC) and quantification of EICs for P. aeruginosa (b,c) or E. coli (d,e) strains expressing the indicated MraY variant. Three independent replicates of the extractions were performed and lipid II levels quantified using the area of the peak from the extracted ion chromatogram using the Agilent software. Dots represent the values obtained for the biological replicates; bars and error bars indicate mean ± s.d. For PAO1: MraYT23P vs MraYWT in PAO1 *P = 0.0219, PAO1 ΔponB ΔlpoA **P = 0.007; for MG1655: MraYT23P vs MraYWT in MG1655 *P = 0.0456, MG1655 ΔponA ΔlpoB ponB[E313D], NS (not significant), (unpaired two-tailed t-test) but the trends for the individual samples were consistent with the other experiments with PaMraYT23P production promoting the highest levels of lipid II and empty vector the least. f, Schematic representation of the MraY enzyme assay. g, Representative time course showing the production of uridine in assays containing purified MraY or MraYT23P as indicated. The assay was repeated at least twice with two independent preparations of protein. NaBH4, sodium borohydride; H3PO4, phosphoric acid.
