Compound discoverer 2.1 sp1

B. thailandensis Pbur and B. thailandensis Pbur burG::Kan were independently grown in 300 ml baffled Erlenmeyer flasks filled with 100 ml of MM9 medium that had been supplemented with either tetracycline (45 μg ml⁻¹) or tetracycline (45 μg ml⁻¹) plus kanamycin (150 μg ml⁻¹) at 30 °C with orbital shaking for 24 h. Subsequently, the cells were harvested from a 50 ml aliquot by centrifugation (8,000g, for 15 min). The resulting cell pellet was resuspended in 25 ml methanol, sonicated and subsequently incubated for 60 min at room temperature. The centrifugation and extraction steps were repeated a second time and both obtained fractions were combined and concentrated in vacuo to yield crude extracts. These extracts were dissolved in 4 ml of methanol, and 100 µl of the resulting solution was then diluted with 200 µl methanol and filtered through a PTFE syringe filter. Subsequently the extracts were subjected to LC-HRMS analysis for a metabolomics analysis using the software Compound Discoverer 2.1 SP1 (Thermo Fisher Scientific). Both genotypes were analysed using a metabolomics workflow with and without a pattern scoring node to filter for sulfur-containing metabolites. The obtained metabolic profiles were compared using differential analysis.

Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, ¹³C, and ¹⁵N isotopes were performed using MAVEN (Princeton, NJ, USA),⁷⁸ against an in house library of deuterated lipid standards (SPLASH® LIPIDOMIX® Mass Spec Standard, Avanti Lipids) and in house libraries of 3,000 unlabeled (MSMLS, IROATech, Bolton, MA, USA; IroaTech ; product A2574 by ApexBio; standard compounds for central carbon and nitrogen pathways from SIGMA Aldrich, St Louis, MO, USA) and labeled standards (see below for the latter). Discovery mode analysis was performed with standard workflows using Compound Discoverer 2.1 SP1 (Thermo Fisher Scientific, San Jose, CA). From these analyses, metabolite IDs or unique chemical formulae were determined from high-resolution accurate intact mass, isotopic patterns, identification of eventual adducts (e.g., Na + or K+, etc.) and MS^{2 (link)} fragmentation spectra against the KEGG pathway, HMDB, ChEBI, and ChEMBL databases. Additional untargeted lipidomics analyses were performed with the software LipidSearch (Thermo Fisher, Bremen, Germany).