Defined [3-13C] sodium L-lactate (98%, Cambridge Isotope, USA) minimal medium (30 mM, pH = 7, buffered with 20 mM PIPES). For salt stress experiments, 330 mM NaCl was added to the medium. To enhance growth, a standard amino acid mix (containing 17 unlabeled amino acids, without tryptophan, glutamine, and asparagine) was added to the medium for a final concentration of 25 μM for each amino acid.

The GC-MS protocol for isotopomer measurement has been described previously [1-3]. In brief, the protein in biomass from the early exponential growth phase (OD600 = 0.3–0.4) was hydrolyzed to free amino acids in 6 M HCl at 100ºC for 24 h. GC-MS samples were prepared in 100 µ ... L tetrahydrofuran (THF) and 100 µL N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (Sigma-Aldrich, USA) and baked at 65–80 ºC for one hour. GC-MS analysis was carried out using a gas chromatograph (DB5 column, HP6890 series, Agilent Inc, USA) equipped with a mass spectrometer (5973 Network, Agilent Inc, USA). Two types of positively charged species were used in flux calculation: unfragmented amino acids, [M-57], and fragmented amino acids without α carboxyl group, [M-159] [4].
The metabolic network used to calculate the metabolic flux profile of S. oneidensisis MR-1 included the tricarboxylic acid (TCA) cycle (including the glyoxylate shunt), C1 metabolism, the Entner-Doudoroff (ED) pathway, gluconeogenesis, and the pentose phosphate (PP) pathway. The algorithm for metabolic flux calculation has been described in detail in our previous papers [5,6].

------------------References------------------
[1] Tang YJ, Pingitore F, Mukhopadhyay A, Phan R, Hazen TC, Keasling, JD. Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using GC-MS and FT-ICR mass spectrometry. J Bacteriol. 2007;189:940–949. http://doi.org/c2s4qx
[2] Tang YJ, Meadows AL, Keasling JD. A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion. Biotechnol Bioeng. 2007;96:125–133. http://doi.org/br93c2
[3] Tang YJ, Ashcroft M, Chen D, Min G, Kim C, Murkhejee B, Larabell C, Keasling JD, Chen FF. Charge-associated effects of fullerene derivatives on microbial structural integrity and central metabolism. Nano Lett. 2007;7:754–760. http://doi.org/bd8662
[4] Wahl SA, Dauner M, Wiechert, W. New tools for mass isotopomer data evaluation in 13C flux analysis: mass isotope correction, data consistency checking, and precursor relationships. Biotechnol Bioeng. 2004;85:259–268. http://doi.org/c7pdsj
[5] Tang YJ, Martin HG, Dehal PS, Deutschbauer A, Llora X, Meadows A, Arkin A, Keasling JD. Metabolic flux analysis of Shewanella spp. reveals evolutionary robustness in central carbon metabolism. Biotechnol Bioeng. 2009;102:1161–1169. http://doi.org/bd6rqx
[6] Tang YJ, Hwang JS, Wemmer D, Keasling JD. Shewanella oneidensis MR-1 fluxome under various oxygen conditions. Appl Environ Microbiol. 2007;73:718–729. http://doi.org/b2v3mx