Lawrence Berkeley National Laboratory

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the 1st or 2nd position was the sole carbon source, and Fe-NTA was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/gdw/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl-CoA and pyruvate as the precursors). The model indicated that over 90 percent of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid (TCA) cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3 percent of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.