Fig. 7. GraRS-independent changes to the staphylococcal membrane also contribute to tolerance.

Phospholipids were extracted from TSB-grown and serum-adapted cultures of S. aureus JE2 WT (a), the graS::Tn mutant (b), the cls1::Tn mutant (d) and the cls2::Tn mutant (e) before being analysed by thin layer chromatography and visualised with copper sulphate and phosphoric acid. The relative phospholipid compositions were determined by quantifying spot intensities using ImageJ. Log₁₀ CFU ml⁻¹ of TSB-grown and serum-adapted cultures of S. aureus JE2 WT and the cls1::Tn and cls2::Tn mutant strains during a 6 h exposure to 80 μg ml⁻¹ daptomycin in serum (c). Log₁₀ CFU ml⁻¹ of TSB-grown and serum-adapted cultures of S. aureus JE2 WT and the cls2::Tn mutant strains during a 6 h exposure to 80 μg ml⁻¹ daptomycin in serum (f). Where appropriate, adaptation was carried out with sub-lethal concentrations of fosfomycin (dashed lines). Data in (a), (b), (d) and (e) represent the mean ± standard deviation of at least three independent experiments and were analysed by paired t-tests (* for (a), P = 0.0015 (CL), 0.0116 (PG), 0.1002 (LPG). For (b) P = 0.2642 (CL), 0.0222 (PG), 0.1069 (LPG). For (d), P = 0.0491 (CL); TSB-grown vs serum-adapted for each phospholipid). Data in (c) and (f) represent the geometric mean ± geometric standard deviation of three independent replicates and were analysed by two-way ANOVA with Dunnetts’s post-hoc test * for (c), P = 0.0008 (2 h), 0.001 (4 h) and 0.0066 (6 h) serum-adapted WT vs serum-adapted mutant. For (f), P = 0.0163 (2 h WT TSB), 0.0115 (4 h WT TSB), 0.0018 (6 h WT TSB), 0.0022 (cls2::Tn Serum 2 h), 0.0002 (cls2::Tn Serum 4 h), 0.0034 (cls2::Tn Serum 6 h), 0.0349 (cls2::Tn Serum + fosfomycin 6 h). CL, cardiolipin; PG, phosphatidylglycerol; LPG, lysyl-phosphatidylglycerol; TSB, tryptic soy broth; CFU, colony-forming units. Source data are provided as a Source Data file.