Large‐scale
            <sup>13</sup>
            C‐flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in
            <i>Escherichia coli</i>

Rijsewijk, Bart Rudolf Boudewijn Haverkorn van; Nanchen, Annik; Nallet, Sophie; Kleijn, Roelco J.; Sauer, Uwe

doi:10.1038/msb.2011.9

Cited by 156 publications

(180 citation statements)

References 76 publications

(152 reference statements)

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“…Successful applications of 13 C-labeling and gas chromatographytime-of-flight-mass spectrometry (GC-TOF-MS) have been reported for a range of species and tissues, including Escherichia coli (Yuan et al, 2006;Haverkorn van Rijsewijk et al, 2011), Saccharomyces cerevisiae (Birkemeyer et al, 2005), photoautotrophic cyanobacteria, Synechocystis sp (Huege et al, 2011;Young et al, 2011), Arabidopsis thaliana (Huege et al, 2007;Williams et al, 2010), soybean (Glycine max) embryos (Sriram et al, 2004), Brassica napus (Schwender et al, 2004b(Schwender et al, , 2006, and potato (Solanum tuberosum) tubers (Roessner-Tunali et al, 2004). However, to date, there are relatively few reports that use 13 CO 2 to study metabolism in photosynthesizing plant tissue.…”

Section: Introductionmentioning

confidence: 99%

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

et al. 2013

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Photosynthesis is the basis for life, and its optimization is a key biotechnological aim given the problems of population explosion and environmental deterioration. We describe a method to resolve intracellular fluxes in intact Arabidopsis thaliana rosettes based on time-dependent labeling patterns in the metabolome. Plants photosynthesizing under limiting irradiance and ambient CO 2 in a custom-built chamber were transferred into a 13 CO 2 -enriched environment. The isotope labeling patterns of 40 metabolites were obtained using liquid or gas chromatography coupled to mass spectrometry. Labeling kinetics revealed striking differences between metabolites. At a qualitative level, they matched expectations in terms of pathway topology and stoichiometry, but some unexpected features point to the complexity of subcellular and cellular compartmentation. To achieve quantitative insights, the data set was used for estimating fluxes in the framework of kinetic flux profiling. We benchmarked flux estimates to four classically determined flux signatures of photosynthesis and assessed the robustness of the estimates with respect to different features of the underlying metabolic model and the time-resolved data set.

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Section: Introductionmentioning

confidence: 99%

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

et al. 2013

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“…It has been well described that E. coli cells first utilize glucose and excrete acetate, and then they take up and utilize acetate subsequent to the depletion of glucose (26,27). Under glucose-depleted conditions, a series of sequential peaks of luciferase activity appeared from the lag phase to the end of cell growth.…”

mentioning

confidence: 99%

“…The activation of Acs for the utilization of acetate is well defined as the requirement of transcriptional activation of acs by CRP as well as the deacetylation of Acs by CobB (26). Such regulation can change the rate and direction of the metabolic carbon flux, depending on the intracellular and extracellular environments (27)(28)(29).…”

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confidence: 99%

“…Metabolome analyses, using time of flight mass spectrometry or gas chromatography-mass spectrometry systems, have been used to quantify intermediate metabolites (30,31). Moreover, carbon metabolic flux has been analyzed using 13 C-labeled metabolites (27). In addition to these powerful methods to analyze intermediates from cell extracts, some other methods have enabled the observation of intracellular intermediate metabolites in living cells (32,33).…”

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confidence: 99%

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Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Shimada

Tanaka

2016

Appl Environ Microbiol

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Regulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employing Vibrio fischeri luciferase to probe the intracellular metabolic condition in Escherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells. IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used a Vibrio fischeri luciferase monitoring system to probe the intracellular metabolic condition in Escherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells. M olecular mechanisms of cellular metabolic regulation have been the subject of extensive studies, as understanding these mechanisms is important not only for basic biology but also for practical applications, such as improving the yield o...

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“…In line with this, metabolic flux studies using various bacteria, including aerobic C. glutamicum (52) and B. subtilis (67), the facultative anaerobes E. coli (68) and Basfia succiniciproducens (69), and the anaerobes Lactobacillus plantarum (70) and Lactococcus lactis (71), identified the EMP pathway as the major catabolic pathway during growth on glucose. It has been proposed that the ED pathway may not play a major role in glucose metabolism but instead primarily functions in the breakdown of sugar acids that cannot be metabolized through the EMP pathway (14,72).…”

Section: Discussionmentioning

confidence: 89%

Large-Scale ¹³ C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

Klingner

Bartsch

Dogs

et al. 2015

Appl Environ Microbiol

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e Marine bacteria form one of the largest living surfaces on Earth, and their metabolic activity is of fundamental importance for global nutrient cycling. Here, we explored the largely unknown intracellular pathways in 25 microbes representing different classes of marine bacteria that use glucose: Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia of the Bacteriodetes phylum. We used 13 C isotope experiments to infer metabolic fluxes through their carbon core pathways. Notably, 90% of all strains studied use the Entner-Doudoroff (ED) pathway for glucose catabolism, whereas only 10% rely on the Embden-Meyerhof-Parnas (EMP) pathway. This result differed dramatically from the terrestrial model strains studied, which preferentially used the EMP pathway yielding high levels of ATP. Strains using the ED pathway exhibited a more robust resistance against the oxidative stress typically found in this environment. An important feature contributing to the preferential use of the ED pathway in the oceans could therefore be enhanced supply of NADPH through this pathway. The marine bacteria studied did not specifically rely on a distinct anaplerotic route, but the carboxylation of phosphoenolpyruvate (PEP) or pyruvate for fueling of the tricarboxylic acid (TCA) cycle was evenly distributed. The marine isolates studied belong to clades that dominate the uptake of glucose, a major carbon source for bacteria in seawater. Therefore, the ED pathway may play a significant role in the cycling of mono-and polysaccharides by bacterial communities in marine ecosystems. Marine bacteria influence global environmental dynamics in fundamental ways by controlling the biogeochemistry and productivity of the oceans (1). Due to their importance, marine microorganisms have been studied intensively (2). In particular, their mechanisms for metabolizing carbon and other nutrients have attracted attention, because they directly or indirectly affect the biogeochemical status of seawater (3). A prominent nutrient in seawater is glucose, the most abundant free neutral aldose (4). Current estimates of glucose concentrations in seawater indicate an almost ubiquitous distribution in the oceans in a nanomolar range (5). Particularly, large amounts of glucose are available in coastal habitats, e.g., during bloom situations (6). In fact, a large fraction (Ͼ30%) of bacterial growth can be supported by this monosaccharide in some oceans (7,8). Furthermore, glucose is the dominant component of dissolved polysaccharides, which constitute up to 15% of marine dissolved organic matter (9). The turnover of the (monomeric and polymeric) glucose pool in different oceanic regions ranges from days to months, and glucose assimilation in marine surface waters may represent up to 40% of bacterial carbon production (5). Taken together, bacteria that use glucose are common in the sea (10), and glucose is a representative model nutrient to monitor carbon uptake by heterotrophic marine bacteria (11). At this point, questions that arise from current knowledge concern...

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Large‐scale ¹³ C‐flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli

Abstract: The authors analyze the role transcription plays in regulating bacterial metabolic flux. Of 91 transcriptional regulators studied, 2/3 affect absolute fluxes, but only a small number of regulators control the partitioning of flux between different metabolic pathways.

Cited by 156 publications

References 76 publications

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Large-Scale ¹³ C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

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Large‐scale 13 C‐flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli

Abstract: The authors analyze the role transcription plays in regulating bacterial metabolic flux. Of 91 transcriptional regulators studied, 2/3 affect absolute fluxes, but only a small number of regulators control the partitioning of flux between different metabolic pathways.

Cited by 156 publications

References 76 publications

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Large-Scale 13 C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

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Large‐scale ¹³ C‐flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli

Large-Scale ¹³ C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose