Preparation of intracellular metabolite extracts

The sampling of intracellular metabolites was described earlier [9]. In brief, 15 OD units (1 OD units equates 1 ml at OD_600nm of 1) were sampled by using the vacuum-dependent fast-filtration approach. The filtered cells were washed twice with 5 ml cold isotonic sodium chloride solution (130 mM). After the transfer of the filter into extraction solution (60% ethanol [w/v]), the samples were shock frozen (liquid N₂) and stored at −80 °C prior extraction. To obtain the intracellular metabolite extracts, the bacterial cells were washed off the filter by shaking and vortexing. Resuspended bacteria were transferred into a 50 ml tube containing glass beads with 0.1 mm diameter (Sartorius AG). The internal standards (see Supplementary Table S1) for LC-MS and GC-MS analyses were added. Two cell disruption cycles (2 × 40 s, 6.0 m/s) were performed by using the FastPrep-24 instrument (MP Biomedicals). The transfer, washing, and centrifugation of the bacterial cell extract were described earlier [9]. The supernatant was diluted with water and stored at −80 °C for lyophilization. The dried samples were resuspended in 1.5 ml cold water and divided into two equal parts. One part of each sample was directly frozen and lyophilized for further GC-MS analysis. 150 µl ice-cold trichloromethane was added to the other part of the samples. After shaking and vortexing for ten times the samples were stored at −20 °C for 5 min and centrifuged at 4 °C and 13,000 rpm for 5 min. The upper layer of each sample was collected, frozen, and lyophilized for LC-MS analysis.