Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

Abstract

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13 C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fenitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/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 coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO 2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) 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% 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.Geobacter species are known to be one of the dominant groups of microorganisms mediating iron reduction in the environment (21). They have been found to be ubiquitous in a myriad of subsurface environments. Detailed studies of their metabolism have revealed them to be capable of bioremediation of several heavy metals, including uranium, plutonium, technetium, and vanadium, as well as biodegradation of several organic contaminants, including monoaromatic hydrocarbons (16,17,26). More recently, Geobacter species have been used to generate electricity from waste organic matter (2, 15, 19). These unique metabolisms make Geobacter species important players in the contaminated subsurface environment (18). Geobacter metallireducens was the first iron-reducing organism isolated that coupled complete oxidation of organic acids with reduction of iron oxides (20,21). It completely oxidizes organic carbons such as fatty acids, alcohols, and monoaromatic compounds via the tricarboxylic acid (TCA) cycle (3, 20) coupled with the reduction of iron. The genomes of several Geobacter species have been sequenced, and proteome data are also available (5, 24). While the genome sequence and proteome are important for understanding Geobacter, they are not necessarily accurate represe...

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

“…Similar results were observed in E. coli respiring with acetate, with 30% of flux from isocitrate going to the glyoxylate shunt and 70% through the lower TCA cycle (35). A 13 C-labeling study of Geobacter metallireducens showed high TCA cycle flux from acetate while respiring with Fe 3ϩ with zero flux through the glyoxylate shunt (36). However, this depiction was misleading as there is nothing in the genome sequence to suggest that the glyoxylate shunt exists in G. metallireducens and rather pyruvate synthase is likely critical to assimilate acetate.…”

In Silico Geobacter sulfurreducens Metabolism and Its Representation in Reactive Transport Models

“…2). The fluxes are consistent with those of 13 C tracer experiments observed in the isotopic data of Tang et al (43), which showed that the TCA cycle encompassed ϳ90% of the acetate uptake flux, with an additional ϳ8% of acetate flowing through the TCA cycle being used for amino acid and lipid production, and the remainder of the acetate uptake flux passed through the pentose-phosphate pathway and gluconeogenesis. Along with the FB model, the kinetic model predicted that for lower acetate uptake rates, acetate is channeled preferentially into the TCA cycle, leading to low growth efficiency.…”

Evaluation of a Genome-Scale In Silico Metabolic Model for Geobacter metallireducens by Using Proteomic Data from a Field Biostimulation Experiment

“…Within the data sets, there was good correspondence in general trends between in silico relative fluxes and protein abundances. Previous studies have indicated that up to 90% of carbon flux through Geobacter strains is directed through respiratory pathways rather than toward biosynthesis (57). Consistent with this observation, the highest in silico fluxes and protein abundances were observed for proteins associated with the activation of acetate (via an acetyl coenzyme A [acetylCoA] hydrolase/transferase enzyme) and the incorporation of acetyl-CoA into the tricarboxylic acid (TCA) cycle (via citrate synthase).…”