skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA

Abstract

We analyzed the carbon fluxes in the central metabolism ofGeobacter 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 labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The tracer experiments showed that G. metallireducens containedcomplete biosynthesis pathways for essential metabolism, and this strainmight also have an unusual isoleucine biosynthesis route (usingacetyl-CoA and pyruvate as the precursors). The model indicated that over90 percent of the acetate was completely oxidized to CO2 via a completetricarboxylic acid (TCA) cycle while reducing iron. Pyruvate carboxylaseand phosphoenolpyruvate carboxykinase were present under theseconditions, but enzymes in the glyoxylate shunt and malic enzyme wereabsent. Gluconeogenesis and the pentose phosphate pathway were mainlyemployed for biosynthesis and accounted for less than 3 percent of totalcarbon consumption. The model also indicated surprisingly highreversibility in the reaction between oxoglutarate and succinate. Thisstep operates close to the thermodynamic equilibrium possibly becausesuccinate is synthesized via a transferase reaction, and the conversionof oxoglutarate to succinatemore » is a rate limiting step for carbonmetabolism. These findings enable a better understanding of therelationship between genome annotation and extant metabolic pathways inG. metallireducens.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Ernest Orlando Lawrence Berkeley NationalLaboratory, Berkeley, CA (US)
Sponsoring Org.:
USDOE Director. Office of Science. Biological andEnvironmental Research
OSTI Identifier:
918684
Report Number(s):
LBNL-63533
Journal ID: ISSN 0099-2240; AEMIDF; R&D Project: VGTLTH; BnR: KP1102010; TRN: US200819%%405
DOE Contract Number:
DE-AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 73; Journal Issue: 12; Related Information: Journal Publication Date: June 2007
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; AMINO ACIDS; BINDING ENERGY; BIOLOGICAL PATHWAYS; BIOSYNTHESIS; CARBON; CARBON SOURCES; IN VIVO; METABOLISM; REACTION KINETICS; labeled acetate minimal medium isoleucine TCA cycle acetyl-CoAtransferase

Citation Formats

Tang, Yinjie J., Chakraborty, Romy, Garcia-Martin, Hector, Chu,Jeannie, Hazen, Terry C., and Keasling, Jay D. Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA. United States: N. p., 2007. Web. doi:10.1128/AEM.02986-06.
Tang, Yinjie J., Chakraborty, Romy, Garcia-Martin, Hector, Chu,Jeannie, Hazen, Terry C., & Keasling, Jay D. Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA. United States. doi:10.1128/AEM.02986-06.
Tang, Yinjie J., Chakraborty, Romy, Garcia-Martin, Hector, Chu,Jeannie, Hazen, Terry C., and Keasling, Jay D. Mon . "Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA". United States. doi:10.1128/AEM.02986-06. https://www.osti.gov/servlets/purl/918684.
@article{osti_918684,
title = {Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA},
author = {Tang, Yinjie J. and Chakraborty, Romy and Garcia-Martin, Hector and Chu,Jeannie and Hazen, Terry C. and Keasling, Jay D.},
abstractNote = {We analyzed the carbon fluxes in the central metabolism ofGeobacter 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 labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The tracer experiments showed that G. metallireducens containedcomplete biosynthesis pathways for essential metabolism, and this strainmight also have an unusual isoleucine biosynthesis route (usingacetyl-CoA and pyruvate as the precursors). The model indicated that over90 percent of the acetate was completely oxidized to CO2 via a completetricarboxylic acid (TCA) cycle while reducing iron. Pyruvate carboxylaseand phosphoenolpyruvate carboxykinase were present under theseconditions, but enzymes in the glyoxylate shunt and malic enzyme wereabsent. Gluconeogenesis and the pentose phosphate pathway were mainlyemployed for biosynthesis and accounted for less than 3 percent of totalcarbon consumption. The model also indicated surprisingly highreversibility in the reaction between oxoglutarate and succinate. Thisstep operates close to the thermodynamic equilibrium possibly becausesuccinate is synthesized via a transferase reaction, and the conversionof oxoglutarate to succinate is a rate limiting step for carbonmetabolism. These findings enable a better understanding of therelationship between genome annotation and extant metabolic pathways inG. metallireducens.},
doi = {10.1128/AEM.02986-06},
journal = {Applied and Environmental Microbiology},
number = 12,
volume = 73,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • We analyzed the carbon fluxes in the central metabolism ofGeobacter 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 labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The model indicated that over 90 percent of the acetate wascompletely oxidized to CO2 via a complete tricarboxylic acid (TCA) cyclewhile reducing iron. Pyruvatemore » carboxylase and phosphoenolpyruvatecarboxykinase were present under these conditions, but enzymes in theglyoxylate shunt and malic enzyme were absent. Gluconeogenesis and thepentose phosphate pathway were mainly employed for biosynthesis andaccounted for less than 3 percent of total carbon consumption. The modelalso indicated surprisingly high reversibility in the reaction betweenoxoglutarate and succinate. This step operates close to the thermodynamicequilibrium possibly because succinate is synthesized via a transferasereaction, and its product, acetyl-CoA, inhibits the conversion ofoxoglutarate to succinate. These findings enable a better understandingof the relationship between genome annotation and extant metabolicpathways in G. metallireducens.« less
  • Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and fresh-water environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal med-ium with [3-13C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23-0.29 h31) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo-steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance ofmore » metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636-640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate.« less
  • Plutonium (Pu), a key contaminant at sites associated with the manufacture of nuclear weapons and with nuclear-energy wastes, can be precipitated to 'immobilized' plutonium phases in systems that promote bioreduction. Ferric iron (Fe 3+) is often present in contaminated sites, and its bioreduction to ferrous iron (Fe 2+) may be involved in the reduction of Pu to forms that precipitate. Alternately, Pu can be reduced directly by the bacteria. Besides Fe, contaminated sites often contain strong complexing ligands, such as nitrilotriacetic acid (NTA). We used biogeochemical modeling to interpret the experimental fate of Pu in the absence and presence ofmore » ferric iron (Fe 3+) and NTA under anaerobic conditions. In all cases, Shewanella alga BrY (S. alga) reduced Pu(V)(PuO 2 +) to Pu(III), and experimental evidence indicates that Pu(III) precipitated as PuPO 4(am). In the absence of Fe 3+ and NTA, reduction of PuO 2 + was directly biotic, but modeling simulations support that PuO 2 + reduction in the presence of Fe 3+ and NTA was due to an abiotic stepwise reduction of PuO 2 + to Pu4+, followed by reduction of Pu 4+ to Pu 3+, both through biogenically produced Fe 2+. This means that PuO 2 + reduction was slowed by first having Fe 3+ reduced to Fe 2+. Modeling results also show that the degree of PuPO 4(am) precipitation depends on the NTA concentration. While precipitation out-competes complexation when NTA is present at the same or lower concentration than Pu, excess NTA can prevent precipitation of PuPO 4(am).« less
  • Uranium bioremediation has emerged as a potential strategy of cleanup of radionuclear contamination worldwide. An integrated geochemical & microbial community model is a promising approach to predict and provide insights into the bioremediation of a complicated natural subsurface. In this study, an integrated column-scale model of uranium bioremediation was developed, taking into account long-term interactions between biotic and abiotic processes. It is also combined with a comprehensive thermodynamic analysis to track the fate and cycling of biogenic species. As compared with other bioremediation models, the model increases the resolution of the connection of microbial community to geochemistry and establishes directmore » quantitative correlation between overall community evolution and geochemical variation, thereby accurately predicting the community dynamics under different sedimentary conditions. The thermodynamic analysis examined a recently identified homogeneous reduction of U(VI) by Fe(II) under dynamic sedimentary conditions across time and space. It shows that the biogenic Fe(II) from Geobacter metabolism can be removed rapidly by the biogenic sulphide from sulfate reducer metabolism, hence constituting one of the reasons that make the abiotic U(VI) reduction thermodynamically infeasible in the subsurface. Further analysis indicates that much higher influent concentrations of both Fe(II) and U(VI) than normal are required to for abiotic U(VI) reduction to be thermodynamically feasible, suggesting that the abiotic reduction cannot be an alternative to the biotic reduction in the remediation of uranium contaminated groundwater.« less