FabT_S.pyogenes_LipidAnalysis

A FabT mutation impacts the membrane fatty acid (FA) composition. Lipids are composed of FA. The effects of the FabT mutation on membrane FA composition should also alter phospholipid metabolism. WT and mFabT strains were grown as 200 ml cultures in THY or THY-Tween until OD600 = 0.4 - 0.5. Lipid extractions and identifications were performed as described 1-4. Lipid separation was realized by normal phase HPLC (U3000 ThermoFisher Scientific) using a Inertsil Si 5μm column (150 x 2.1 mm I.D.) from GL Sciences Inc (Tokyo, Japan). Lipids were quantified using a Corona-CAD Ultra and identified by mass-spectrometry negative ionization and MS2/MS3 fragmentations (LTQ-Orbitrap Velos Pro). The adducts observed for molecular ions were: CH3COO- for MGDG, CH3COO- and H- for DGDG, H- for PG and CL. All raw data from MS/MS fragmentations can now be accessed in MZML open source format. This analysis was repeated on 3 independent experiments. 1-Kenanian, G. et al. Permissive Fatty Acid Incorporation Promotes Staphylococcal Adaptation to FASII Antibiotics in Host Environments. Cell Rep 29, 3974-3982 e3974, doi:10.1016/j.celrep.2019.11.071 (2019). 2-Abreu, S., Solgadi, A. & Chaminade, P. Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques. J Chromatogr A 1514, 54-71, doi:10.1016/j.chroma.2017.07.063 (2017). 3-Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911-917, doi:10.1139/o59-099 (1959). 4-Thedieck, K. et al. The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes. Mol Microbiol 62, 1325-1339, doi:10.1111/j.1365-2958.2006.05452.x (2006).