Minimal synthetic medium: 5 mM KH2PO4, 10 mM Na2HPO4, 9 mM NaCl, 40 mM NH4Cl, 0.8 mM MgSO4, 0.1 mM CaCl2, 0.1 g/L thiamine, and 3 g/L glucose. Glucose and thiamine were sterilized by filtration (Minisart polyamide 0.2 μm, Sartorius, Göttingen, Germany), and other solutions were autoclaved separately.

Samples were collected using the differential method described in detail in [1]. Briefly, 120 μl of broth or filtered extracellular medium (Sartolon polyamide 0.2 μm, Sartorius) was plunged with 120 μl of fully 13C-labeled cellular extract (used as internal standard) in 5 ml ... of an ethanol/water (75:25) solution at 95 °C, incubated for 2 min, cooled on ice, and stored at −80 °C. Samples were analyzed by ion chromatography (ICS 2500 system, Dionex, Sunnyvale, CA, USA) coupled with a 4000 QTrap triple quadrupole mass spectrometer (ABSciex, Framingham, MA, USA) equipped with a Turbo V source (ABSciex) for electrospray ionization [2]. The nebulizer gas pressure was 40 psi, the desolvation gas pressure was 50 psi, the desolvation gas temperature was 650 °C, and the capillary voltage was −3.3 kV. Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), 6-phosphogluconate (6PG), combined pools of xylulose-5-phosphate, ribose-5-phosphate, and ribulose-5-phosphate (P5P), sedoheptulose-7-phosphate (S7P), phosphoenolpyruvate (PEP), and combined pools of 2- and 3-phosphoglycerate (2/3PG) were analyzed in the multiple reaction monitoring (MRM) mode, and the isotope dilution mass spectrometry (IDMS) method [3] was used to ensure accurate quantification. Fragmentation was done by collision-activated dissociation using nitrogen as the collision gas at medium pressure. The daughter ion was a phosphate group (PO3−m/z = 79 or H2PO4−m/z = 97). Three samples of broth and filtrate were collected at the mid-exponential growth phase (OD600nm ∼ 1.2) and analyzed. From these data, we were able to quantify the relative fractions of intra- and extracellular metabolites in the total pools; we calculated absolute concentrations of intracellular metabolites assuming a cell volume of 1.77 ml/gDW [4].
--------------------------------------References------------------------------
[1] H. Taymaz-Nikerel, M. de Mey, C. Ras, A. ten Pierick, R.M. Seifar, J.C. van Dam, J.J. Heijnen, W.M. van Gulik, Anal. Biochem., 386 (2009), pp. 9–19. http://doi.org/bk5b8m
[2] P. Kiefer, C. Nicolas, F. Letisse, J.-C. Portais, Anal. Biochem., 360 (2007), pp. 182–188. http://doi.org/dws46h
[3] L. Wu, M.R. Mashego, J.C. van Dam, A.M. Proell, J.L. Vinke, C. Ras, W.A. van Windenn, W.M. van Gulik, J.J. Heijnen, Anal. Biochem., 336 (2005), pp. 164–171. http://doi.org/dp4h5k
[4] C. Chassagnole, N. Noisommit-Rizzi, J.W. Schmid, K. Mauch, M. Reuss, Biotechnol. Bioeng., 79 (2002), pp. 53–73. http://doi.org/dj2nzc