Methoximation pyridine hydrochloride

Both the sample bacterial preparation and GC-MS analysis were performed as described previously (23 (link)). Briefly, 10 high- and 10 low-virulent strains were cultured in the LB medium with 200 rpm at 37℃ until reaching an optical density at 600 nm (OD600) of 1.0. The aliquot of 10-mL cells was quenched with precooled methanol (Sigma Aldrich) and by ultrasonication. Ribitol (0.1 mg/mL, Sigma Aldrich) was added as an internal standard. The aliquot of the 500-µL supernatant was separated with 12,000 g at 4°C for 10 min and dried by a vacuum centrifugation dryer (Labconco). Methoximation–pyridine hydrochloride (Sigma Aldrich) was added to the dried fraction above and continuously shaken with 200 rpm at 30°C for 90 min. Eighty microliters of N-methyl-N-trimethylsilyltrifluoroacetamide (Sigma Aldrich) was added and incubated at 37°C for 30 min. The data were analyzed using an Agilent 7890A GC and an Agilent 5975C VL MSD detector (Agilent Technologies). The compounds were identified by Agilent Chrom Station software (Agilent Technologies) and the National Institute of Standards and Technology (NIST) library. Every sample was analyzed in duplicate.

Metabolic profiles were detected by GC-MS-based metabolomics as described previously [9 (link)]. Samples from different treatments with Ala or/and Gent were prepared as follows: Bacteria were collected, washed with saline, and adjusted to an OD₆₀₀ nm value of 1.0 to remove M9 medium. Then, 10-mL resuspension was collected by centrifugation at 2,300 × g at 4°C and transferred into 1.5 mL QSP microtube, immediately quenched with −80°C pre-cooled methanol (Sigma). Bacterial metabolites were extracted after repeated freeze-thaw cycles of liquid nitrogen. The 0.1 mg/mL ribose was added as the internal standard and the resuspension was sonicated for 10 min at the 200 W working power. The supernatant was separated by centrifugation at 4°C and 14,200 × g for 10 min and placed in a 37°C vacuum centrifuge dryer (Labconco, USA) to evaporate the methanol. Dry extracts were performed on GC-MS analysis. The dried samples were added to 80 μL of 20 mg/mL methoximation-pyridine hydrochloride (Sigma-Aldrich). After sonication, the reaction was performed at 37°C for 3 h. Subsequently, 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA, Sigma) was added and the reaction was performed at 37°C for 30 min. GC-MS data were detected by Thermo Scientific Trace DSQ II.