Metabolite extraction, GC-MS, and data analysis

Metabolites were extracted from wet biomass through the methanol–water–chloroform extraction process using 100 μL of methanol/¹³C-ribitol solution to re-suspend the cells. Thereafter, the sequential addition of 100 μL of H₂O and 150 μL of chloroform was performed with vortexing for 5 min in between. After centrifugation, 150 μL of the polar phase was dried in a vacuum concentrator. For extracellular metabolites, 10 μL of cell-free culture supernatant was mixed with 10 μL of methanol/¹³C-ribitol solution and then dried in a vacuum concentrator. Derivatization and GC-MS measurement (splitless and split 1:10) of metabolites were performed as described in a previous study (Will et al., 2019). Processing of raw data was performed using the MetaboliteDetector (Neumann-Schaal et al., 2015). The peak area data were normalized to the internal standard ³C-ribitol and—for intracellular metabolites—to the determined cell number of the harvested cells. Data are available on FAIRDOMHUB (seek ID: https://fairdomhub.org/projects/348). MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/), a web-based tool, was used for analysis. The principle component analysis (PCA) was performed using the statistical analysis (one factor) module. In the heatmaps, the cluster method is Ward.D and the distance is Euclidean distance. The relative abundance of metabolites in S. Typhimurium 14028s and ΔaceB grown in TM was compared to that in MM using log₂ fold change. Four biological replicates were prepared for each treatment.