Synthetic medium (48 mM Na2HPO4, 22 mM KH2PO4, 10 mM NaCl, 45 mM (NH4)2SO4, 4 g/L glucose) supplemented with 1 mM MgSO4, 1 mg/mL thiamine, 0.056 mg/L CaCl2, 0.08 mg/L FeCl3, 0.01 mg/L MnCl2·4H2O, 0.017 mg/L ZnCl2, 0.0043 mg/L CuCl2·2H2O, 0.006 mg/L CoCl2·2H2O, and 0.06 mg/L Na2MoO4·2H2O.

Extracellular metabolites: glucose and acetate, were measured by enzymatic assay kits (F‐kit, Roche Diagnostics, Germany). ... Intracellular metabolites: sample preparation was carried out using the modified method described by Ohashi et al. [1] An aliquot of culture broth containing 0.015 g of cells was passed through a 0.45‐μm pore size filter (Millipore). The cells on the filter were washed with 20 mL of Milli‐Q water at 37°C and metabolism was stopped by submerging in 4 mL of methanol at 4°C containing 2 μM 2‐(N‐morpholino)ethanesulfonic acid, 2 μM trimesate, 2 μM methionine sulfone and 2 μM 3‐aminopyrrolidine as internal standard. Quantities of 4 mL of chloroform and 1.6 mL of Milli‐Q water were added to the solution, which was then fully mixed. The solution was centrifuged at 2,300g for 5 min at 4°C, and the separated methanol layer was filtered by centrifugation through a Millipore 5‐kDa cutoff filter to remove high‐molecular‐weight compounds. The filtrate was lyophilized and then dissolved in 50 μL of Milli‐Q water before CE‐TOFMS analysis. CE‐TOFMS experiments were performed on an Agilent CE capillary electrophoresis system (Agilent Technologies, Germany) and an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA). The CE‐TOFMS conditions used for anionic metabolite analysis have been described elsewhere [2,3]. All measurements were performed three times.
------------------------------------------References--------------------------------------- [1] Sohashi Y, Hirayama A, Ishikawa T, Nakamura S, Shimizu K, Ueno Y, Tomita M, Soga T. Depiction of metabolome changes in histidine‐starved Escherichia coli by CE‐TOFMS. Mol Biosyst. 2008; 4: 135–147. http://doi.org/fsn7dx [2] Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, Kakazu Y, Ishikawa T, Robert M, Nishioka T, Tomita M. Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption. J Biol Chem. 2006; 281: 16768–16776. http://doi.org/fch7nt [3] Soga T, Ohashi Y, Ueno Y, Naraoka H, Tomita M, Nishioka T. Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. J Proteome Res. 2003; 2: 488–494. http://doi.org/fgcc2v