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Title: The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions

Abstract

The central metabolic fluxes of Shewanella oneidensis MR-1were examined under carbon-limited (aerobic) and oxygen-limited(micro-aerobic) chemostat conditions using 13C labeled lactate as thesole carbon source. The carbon labeling patterns of key amino acids inbiomass were probed using both GC-MS and 13C-NMR. Based on the genomeannotation, a metabolic pathway model was constructed to quantify thecentral metabolic flux distributions. The model showed that thetricarboxylic acid (TCA) cycle is the major carbon metabolism route underboth conditions. The Entner-Doudoroff and pentose phosphate pathways weremainly utilized for biomass synthesis (flux below 5 percent of thelactate uptake rate). The anapleurotic reactions (pyruvate to malate andoxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt wereactive. Under carbon-limited conditions, a substantial amount of carbonwas oxidized via the highly reversible serine metabolic pathway. Fluxesthrough the TCA cycle were less whereas acetate production was more underoxygen limitation than under carbon limitation. Although fluxdistributions under aerobic, micro-aerobic, and shake-flask cultureconditions were dramatically different, the relative flux ratios of thecentral metabolic reactions did not vary significantly. Hence, S.oneidensis metabolism appears to be quite robust to environmentalchanges. Our study also demonstrates the merit of coupling GC-MS with 13CNMR for metabolic flux analysis to reduce the use of 13C labeledsubstrates and to obtain more accurate flux values.

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:
919835
Report Number(s):
LBNL-59869
R&D Project: GTL2KK; BnR: KP1102010; TRN: US200822%%585
DOE Contract Number:
DE-AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Society for Microbiology; Journal Volume: 73; Journal Issue: 3; Related Information: Journal Publication Date: 02/2007
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; AMINO ACIDS; BIOLOGICAL PATHWAYS; CARBON; CARBON SOURCES; OXYGEN; METABOLISM; micro-aerobic isotopomer model futile cycles serine metabolismflux ratio

Citation Formats

Tang, Yinjie J., Hwang, Judy S., Wemmer, David E., and Keasling, Jay D.. The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions. United States: N. p., 2006. Web.
Tang, Yinjie J., Hwang, Judy S., Wemmer, David E., & Keasling, Jay D.. The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions. United States.
Tang, Yinjie J., Hwang, Judy S., Wemmer, David E., and Keasling, Jay D.. Fri . "The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions". United States. doi:. https://www.osti.gov/servlets/purl/919835.
@article{osti_919835,
title = {The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions},
author = {Tang, Yinjie J. and Hwang, Judy S. and Wemmer, David E. and Keasling, Jay D.},
abstractNote = {The central metabolic fluxes of Shewanella oneidensis MR-1were examined under carbon-limited (aerobic) and oxygen-limited(micro-aerobic) chemostat conditions using 13C labeled lactate as thesole carbon source. The carbon labeling patterns of key amino acids inbiomass were probed using both GC-MS and 13C-NMR. Based on the genomeannotation, a metabolic pathway model was constructed to quantify thecentral metabolic flux distributions. The model showed that thetricarboxylic acid (TCA) cycle is the major carbon metabolism route underboth conditions. The Entner-Doudoroff and pentose phosphate pathways weremainly utilized for biomass synthesis (flux below 5 percent of thelactate uptake rate). The anapleurotic reactions (pyruvate to malate andoxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt wereactive. Under carbon-limited conditions, a substantial amount of carbonwas oxidized via the highly reversible serine metabolic pathway. Fluxesthrough the TCA cycle were less whereas acetate production was more underoxygen limitation than under carbon limitation. Although fluxdistributions under aerobic, micro-aerobic, and shake-flask cultureconditions were dramatically different, the relative flux ratios of thecentral metabolic reactions did not vary significantly. Hence, S.oneidensis metabolism appears to be quite robust to environmentalchanges. Our study also demonstrates the merit of coupling GC-MS with 13CNMR for metabolic flux analysis to reduce the use of 13C labeledsubstrates and to obtain more accurate flux values.},
doi = {},
journal = {American Society for Microbiology},
number = 3,
volume = 73,
place = {United States},
year = {Fri Mar 17 00:00:00 EST 2006},
month = {Fri Mar 17 00:00:00 EST 2006}
}
  • The growth and Cr(VI) reduction by Shewanella oneidensisMR-1 was examined using a mini-bioreactor system that independentlymonitors and controls pH, dissolved oxygen, and temperature for each ofits 24, 10-mL reactors. Independent monitoring and control of eachreactor in the cassette allows the exploration of a matrix ofenvironmental conditions known to influence S. oneidensis chromiumreduction. S. oneidensis MR-1 grew in minimal medium without amino acidor vitamin supplementation under aerobic conditions but required serineand glycine supplementation under anaerobic conditions. Growth wasinhibited by dissolved oxygen concentrations>80 percent. Lactatetransformation to acetate was enhanced by low concentration of dissolvedoxygen during the logarithmic growth phase. Between 11 andmore » 35oC, thegrowth rate obeyed the Arrhenius reaction rate-temperature relationship,with a maximum growth rate occurring at 35oC. S. oneidensis MR-1 was ableto grow over a wide range of pH (6-9). At neutral pH and temperaturesranging from 30-35oC, S. oneidensis MR-1 reduced 100 mu M Cr(VI) toCr(III) within 20 minutes in the exponential growth phase, and the growthrate was not affected by the addition of chromate; it reduced chromateeven faster at temperatures between 35 and 39oC. At low temperatures(<25oC), acidic (pH<6.5), or alkaline (pH>8.5) conditions, 100mu M Cr(VI) strongly inhibited growth and chromate reduction. Themini-bioreactor system enabled the rapid determination of theseparameters reproducibly and easily by performing very few experiments.Besides its use for examining parameters of interest to environmentalremediation, the device will also allow one to quickly assess parametersfor optimal production of recombinant proteins or secondarymetabolites« less
  • An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time {approx} 3 hrs), salt stressed minimal medium (doubling time {approx} 6 hrs), and minimal medium with amino acid supplementation (doubling time {approx}1.5 hrs). {sup 13}C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, ten transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposonmore » library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.« less
  • Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of wide range of electron acceptors. Here, we quantitatively assessed lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor limited growth on lactate with O2; lactate with fumarate; and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptormore » nature and availability. While being indispensible for growth, respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the TCA cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under O2 limitation but was required for anaerobic growth likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.« less
  • To study potential ecological impacts of CO{sub 2} leakage to shallow groundwater and soil/sediments from geologic CO{sub 2} sequestration (GCS) sites, this work investigated the viability and metal reduction of Shewanella oneidensis MR-1 under CO{sub 2} stress. While MR-1 could grow under high-pressure nitrogen gas (500 psi), the mix of 1% CO{sub 2} with N{sub 2 at total pressures of 15 or 150 psi significantly suppressed the growth of MR-1, compared to the N{sub 2} control. When CO{sub 2} partial pressures were over 15 psi, the growth of MR-1 stopped. The reduced bacterial viability was consistent with the pH decreasemore » and cellular membrane damage under high pressure CO{sub 2}. After exposure to 150 psi CO{sub 2} for 5 h, no viable cells survived, the cellular contents were released, and microscopy images confirmed significant cell structure deformation. However, after a relatively short exposure (25 min) to 150 psi CO{sub 2}, MR-1 could fully recover their growth within 24 h after the stress was removed, and the reduction of MnO{sub 2} by MR-1 was observed right after the stress was removed. Furthermore, MR-1 survived better if the cells were aggregated rather than suspended, or if pH buffering minerals, such as calcite, were present. To predict the cell viability under different CO{sub 2} pressures and exposure times, a two-parameter mathematical model was developed.« less
  • Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI)] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426more » regulon.« less