Metabolite Extraction and Analysis Protocol

Cultures were harvested and suspended in an appropriate volume of 100 mM Tris-Cl (pH 8.0) and disrupted. The resultant whole-cell lysate was acidified by addition of 6 M HCl. Low-molecular-weight molecules were then extracted by adding a double volume of ethyl acetate and left overnight on a stirrer. The organic layer was then separated and evaporated to dryness, and the residual material was dissolved in a minimal volume of methanol and analyzed by information-dependent acquisition (IDA) scanning on a Sciex X500R quadrupole time of flight (QTOF) mass spectrometer fitted with an ExionLC UPHLC system using Sciex OS software with a previously reported method (45 (link), 46 (link)). The LC separation was achieved on a Gemini 5U C₁₈ column (Phenomenex; 5 μm, 50 by 4.6 mm) coupled to a Gemini guard column (Phenomenex; 4 by 3 mm, Phenomenex security cartridge). All metabolites, I to IX, were analyzed by iminodiacetic acid (IDA) scanning in both positive- and negative-ionization mode using an electrospray ionization (ESI) source with solvent systems, flow rates, and a solvent gradient described earlier (45 (link), 46 (link)). The total scan time for both the MS1 and MS2 spectra was 2.5 s, and a collision energy (volts) of 5 was used. The declustering potential and ion source voltage were set at 100 and 5,500 V respectively.