M9 medium contained (per liter of deionized water) 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4 · 2H2O, and 3.0 g KH2PO4. The following components were sterilized separately and then added (per liter of final volume): 2 ml of 1 M MgSO4, 1 ml of 0.1 M CaCl2, 0.3 ml of 1 mM filter-sterilized thiamine HCl, and 10 ml of a trace element solution containing (per liter) 1 g of FeCl3 · 6H2O, 0.18 g ZnSO4 · 7H2O, 0.12 g CuCl2 · 2H2O, 0.12 g MnSO4 · H2O, and 0.18 g CoCl2 · 6H2O. Sterilized glucose was added to a final concentration of 1 g per liter.

Metabolic-flux ratio analysis by GC-MS - Samples for gas chromatographymass spectrometry (GC-MS) analysis were prepared as described previously [1]. Briefly, the 13C-labeled chemostat cultures were harvested after the OD600 was stable for at least two volume changes (the min ... imum continuous operation was seven volume changes). Cell pellets were hydrolyzed in 6MHCl at 105°C for 24 h in sealed microtubes. The hydrolysates were dried under a stream of air at around 60°C and then derivatized at 85°C in 30 µl dimethylformamide (Fluka, Switzerland) and 30 µl N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide with 1% (vol/vol) tert-butyldimethylchlorosilane (Fluka, Switzerland) for 60 min [2]. Derivatized amino acids were analyzed on a series 8000 GC combined with an MD 800 mass spectrometer (Fisons Instruments, Beverly, MA). The GC-MSderived mass isotope distributions of proteinogenic amino acids were then corrected for naturally occurring isotopes [1]. The corrected mass distributions were related to the in vivo metabolic activities obtained with previously described algebraic equations and statistical-data treatment of metabolic-flux ratio analysis [1] using the software Fiat Flux [3].

------------References---------------
[1] Fischer, E., and U. Sauer. 2003. Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur. J. Biochem. 270: 880–891. http://doi.org/dg3q64
[2] Fischer, E., N. Zamboni, and U. Sauer. 2004. High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. Anal. Biochem. 325:308–316. http://doi.org/c6kx35
[3] Zamboni, N., E. Fischer, and U. Sauer. 2005. FiatFlux—a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6:209. http://doi.org/bj2zk2