Sample preparation for lipidomics and metabolomics experiments.

Intracellular metabolites were extracted as described previously (53). The general procedure for sample preparation was as follows. Ten milliliters of mid-log-phase culture (OD₆₀₀ of 0.5) was centrifuged at 3,220 × g for 10 min at 4°C, and the supernatant was kept for footprint analysis. After washing twice with 2 ml of 0.9% NaCl, the cell pellets were resuspended in 0.5 ml of extraction solvents. Liquid nitrogen was used to permeabilize the cells and release the intracellular metabolites. The mixtures were centrifuged at 3,220 × g for 10 min at 4°C, and 300 μl of the supernatant containing the extracted metabolites was collected in 1.5-ml centrifuge tubes and stored at −80°C. The samples were further centrifuged at 14,000 × g for 10 min at 4°C, and 200 μl of particle-free supernatant was transferred into an injection vial for liquid chromatography-mass spectrometry (LC-MS) analysis.

To evaluate the effect of different extraction solvents on the recovery of lipids from whole bacterial cells, three classic extraction methods were examined. (i) For the single-phase Bligh-Dyer method (CHCl₃/MeOH/H₂O, 1:2:0.8 [vol/vol]), the cell pellets were resuspended in 0.5 ml solvent and 300 μl of supernatant was collected using the aforementioned method. (ii) For the double-phase Bligh-Dyer method (CHCl₃/MeOH/H₂O, 1:1:0.9 [vol/vol]) (25), the cell pellets were resuspended in 380 μl of the solvent from method “i” and 100 μl CHCl₃ and 100 μl H₂O were added after vortexing and freeze-thawing to form a double-phase Bligh-dyer mixture. The lower phase (100 μl) was finally collected after centrifugation. For LC-MS analysis, 200 μl MeOH was added to the CHCl₃ extracts, which were centrifuged at 14,000 × g for 10 min at 4°C, and 200 μl of the particle-free supernatant was transferred into an LC-MS glass vial. (iii) For the method using methyl-tert-butyl ether (MTBE)/MeOH/H₂O (10:3:2.5 [vol/vol]) (26), the cell pellets were resuspended in 400 μl MTBE and 120 μl MeOH, followed by vortexing and freeze-thawing to release metabolites from the bacterial cells. Milli-Q water (100 μl) was added to the suspension to separate the phases; 300 μl of the upper phase was collected after centrifugation and dried under a nitrogen stream. For further analysis, the samples were reconstituted in 300 μl CHCl₃/MeOH (1:2 [vol/vol]) and centrifuged at 14,000 × g for 10 min at 4°C to obtain the particle-free supernatants.

The procedures for metabolic sample preparation were similar to those for lipidomic samples extracted by the single-phase Bligh-Dyer method. The only difference was the three-solvent system, CHCl₃/MeOH/H₂O (1:3:1 [vol/vol]), for extracting intracellular metabolites. For the preparation of footprint samples, an aliquot of approximately 1.5 ml of supernatant from the culture after centrifugation was rapidly filtered through a 0.22-μm filter, and the extraction solvent (250 μl) was then added. The supernatant (200 μl) was collected for LC-MS analysis after centrifugation (14,000 × g for 10 min at 4°C).