Identifying decomposition products in extracts of cellular metabolites

Kimball, Elizabeth; Rabinowitz, Joshua D.

doi:10.1016/j.ab.2006.07.038

Cited by 75 publications

(53 citation statements)

References 43 publications

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“…For metabolome sampling, filter-grown cells were quenched by dropping them into Ϫ75°C methanol. Extraction of metabolites was conducted by using a series of methanol/water steps (19,20). LC-MS/MS analysis included selected reaction-monitoring scans for Ϸ170 metabolites; of these, 68 gave quantitative data in at least one organism (the others were below the limit of quantitation or too unstable to yield reliable information).…”

Section: Resultsmentioning

confidence: 99%

Conservation of the metabolomic response to starvation across two divergent microbes

Brauer

Yuan

Bennett

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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We followed 68 cellular metabolites after carbon or nitrogen starvation of Escherichia coli and Saccharomyces cerevisiae, using a filter-culture methodology that allows exponential growth, nondisruptive nutrient removal, and fast quenching of metabolism. Dynamic concentration changes were measured by liquid chromatography-tandem mass spectrometry and viewed in clustered heat-map format. The major metabolic responses anticipated from metabolite-specific experiments in the literature were observed as well as a number of novel responses. When the data were analyzed by singular value decomposition, two dominant characteristic vectors were found, one corresponding to a generic starvation response and another to a nutrient-specific starvation response that is similar in both organisms. Together these captured a remarkable 72% of the metabolite concentration changes in the full data set. The responses described by the generic starvation response vector (42%) included, for example, depletion of most biosynthetic intermediates. The nutrient-specific vector (30%) included key responses such as increased phosphoenolpyruvate signaling glucose deprivation and increased ␣-ketoglutarate signaling ammonia deprivation. Metabolic similarity across organisms extends from the covalent reaction network of metabolism to include many elements of metabolome response to nutrient deprivation as well.Escherichia coli ͉ metabolomics ͉ nitrogen/carbon metabolism ͉ Saccharomyces cerevisiae ͉ starvation response T he pathways of cellular metabolism are close to identical across widely divergent organisms (1). The prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae share essentially the same metabolic network (2) despite radically different compartmentation. The concentrations and fluxes of metabolites depend on the interactions among this conserved network structure, the cellular environment, and species-specific factors, such as the location, activities, and regulation of metabolic enzymes. Except for a few classic examples, e.g., central carbon metabolism in E. coli and yeast (3-5) and nitrogen assimilation in bacteria (6-8) the effect of environmental nutrient perturbations on the cellular metabolome have not been directly measured.We explored exponentially growing E. coli and S. cerevisiae cultures suddenly deprived of their carbon or nitrogen sources. Metabolic composition can change in as little as a few seconds (9, 10). To get accurate snapshots of the metabolome, we devised a method of growing cells directly on filters to avoid time-consuming procedures (e.g., centrifugation or filtration) before quenching of biochemical activity (11). Transfer of a filter from a plate containing growth medium into cold organic solvent quickly quenches metabolism; transfer to a plate with a different medium composition allows a quick change of the nutrient environment (Fig. 1).For measurement of a number of metabolites simultaneously, we used a liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (LC-MS/...

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Section: Resultsmentioning

confidence: 99%

Conservation of the metabolomic response to starvation across two divergent microbes

Brauer

Yuan

Bennett

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

244

209

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“…To characterize the intracellular metabolome, cells from 3 ml of exponentially growing cultures were collected on nylon filters (0.45-m pore size), and metabolites were extracted from the cells by immediately submerging filters in 2 ml of Ϫ20°C acetonitrile-methanol-water (40:40:20) (34,46). Extracts were centrifuged and the supernatant solutions were analyzed by reverse-phase high-performance liquid chromatography (HPLC) mass spectrometry (MS).…”

Section: Methodsmentioning

confidence: 99%

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Aristilde

Lewis

Park

et al. 2015

Appl Environ Microbiol

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Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.

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“…HNWP04700). Filters containing the cells are then immediately submerged into 1.5 ml of Ϫ20°C acetonitrile-methanol-water (40:40:20), which quenches metabolism and extracts metabolites (27,36). The filtering and quenching steps were performed inside the anaerobic chamber described above.…”

Section: Methodsmentioning

confidence: 99%

Metabolome Remodeling during the Acidogenic-Solventogenic Transition in Clostridium acetobutylicum

Amador‐Noguez

Brasg

Xiao

et al. 2011

Appl Environ Microbiol

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113

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The fermentation carried out by the biofuel producer Clostridium acetobutylicum is characterized by two distinct phases. Acidogenesis occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). Solventogenesis initiates as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol). Using metabolomics, isotope tracers, and quantitative flux modeling, we have mapped the metabolic changes associated with the acidogenic-solventogenic transition. We observed a remarkably ordered series of metabolite concentration changes, involving almost all of the 114 measured metabolites, as the fermentation progresses from acidogenesis to solventogenesis. The intracellular levels of highly abundant amino acids and upper glycolytic intermediates decrease sharply during this transition. NAD(P)H and nucleotide triphosphates levels also decrease during solventogenesis, while low-energy nucleotides accumulate. These changes in metabolite concentrations are accompanied by large changes in intracellular metabolic fluxes. During solventogenesis, carbon flux into amino acids, as well as flux from pyruvate (the last metabolite in glycolysis) into oxaloacetate, decreases by more than 10-fold. This redirects carbon into acetyl coenzyme A, which cascades into solventogenesis. In addition, the electron-consuming reductive tricarboxylic acid (TCA) cycle is shutdown, while the electron-producing oxidative (clockwise) right side of the TCA cycle remains active. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources (carbon and reducing power) from biomass production into solvent production.The anaerobic bacterium Clostridium acetobutylicum is a promising biofuel producer due to its capacity to ferment a variety of carbohydrates into acetone, butanol, and ethanol. The metabolism of this organism is characterized by two sequential phases. The first, the acidogenic phase, occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). The second, the solventogenic phase, occurs as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol) and the partial re-assimilation of previously produced acids (12,34). A comprehensive understanding of the mechanisms that control the transition into the solventogenic state would be an important step toward the commercial production of solvents using this anaerobic bacterium. Despite numerous research efforts, however, they remain incompletely understood (1,13,19,33).Initial studies of the acidogenic-solventogenic transition focused on investigating the biochemistry and transcriptional regulation of the pathways directly involved in acid and solvent production (12,18,21,25). The acidogenic/solventogenic pathways, however, constitute only a fraction of the metabolic network, and their functionality relies on the supply of substrates (e.g., acetyl coenzyme A [acetyl-CoA], ATP, NADH, and NADPH) from core metabolic pathways. Ther...

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Identifying decomposition products in extracts of cellular metabolites

Cited by 75 publications

References 43 publications

Conservation of the metabolomic response to starvation across two divergent microbes

Conservation of the metabolomic response to starvation across two divergent microbes

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Metabolome Remodeling during the Acidogenic-Solventogenic Transition in Clostridium acetobutylicum

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