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Sample records for mr-1 metabolism reveals

  1. Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals previously uncharacterized machinery for lactate utilization

    SciTech Connect

    Pinchuk, Grigoriy E.; Rodionov, Dmitry A.; Yang, Chen; Li, Xiaoqing; Osterman, Andrei L.; Dervyn, Etienne; Geydebrekht, Oleg V.; Reed, Samantha B.; Romine, Margaret F.; Collart, Frank R.; Scott, J.; Fredrickson, Jim K.; Beliaev, Alex S.

    2009-02-24

    The ability to utilize lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D- or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. Using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D- and L-lactate dehydrogenase enzymes. The predicted D-LDH gene (dldD, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by three genes with previously unknown functions (lldEGF, SO1520-19-18). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dldD and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is the first described example of a multi-subunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.

  2. Invariability of Central Metabolic Flux Distribution in Shewanella oneidensis MR-1 Under Environmental or Genetic Perturbations

    SciTech Connect

    Tang, Yinjie; Martin, Hector Garcia; Deutschbauer, Adam; Feng, Xueyang; Huang, Rick; Llora, Xavier; Arkin, Adam; Keasling, Jay D.

    2009-04-21

    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 transposon 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.

  3. Hydrogen Metabolism in Shewanella oneidensis MR-1

    PubMed Central

    Meshulam-Simon, Galit; Behrens, Sebastian; Choo, Alexander D.; Spormann, Alfred M.

    2007-01-01

    Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions. PMID:17189435

  4. Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: a Tool for Data Analysis and Hypothesis Generation

    SciTech Connect

    Pinchuk, Grigoriy E.; Hill, Eric A.; Geydebrekht, Oleg V.; De Ingeniis, Jessica; Zhang, Xiaolin; Osterman, Andrei; Scott, James H.; Reed, Samantha B.; Romine, Margaret F.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.; Reed, Jennifer L.

    2010-06-24

    Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and flexibility of the electron transfer networks as well as central and peripheral carbon metabolism pathways. To understand the factors contributing to the ecophysiological success of Shewanellae, the metabolic network of S. oneidensis MR-1 was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify futile cycles, (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism

  5. Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation

    PubMed Central

    Hill, Eric A.; Geydebrekht, Oleg V.; De Ingeniis, Jessica; Zhang, Xiaolin; Osterman, Andrei; Scott, James H.; Reed, Samantha B.; Romine, Margaret F.; Konopka, Allan E.; Beliaev, Alexander S.; Fredrickson, Jim K.

    2010-01-01

    Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems

  6. Investigations of Structure and Metabolism within Shewanella oneidensis MR-1 Biofilms

    SciTech Connect

    Mclean, Jeffrey S.; Majors, Paul D.; Reardon, Catherine L.; Bilskis, Christina L.; Reed, Samantha B.; Romine, Margaret F.; Fredrickson, Jim K.

    2008-07-01

    Biofilms are known to possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments within that may ultimately drive species diversity, determine biofilm structure and also the spatial arrangement of the community members. Using noninvasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated anaerobic reduction kinetics, structural variation, and the stratification of metabolism within live biofilms of the facultative anaerobic dissimilatory metal-reducing Shewanella oneidensis strain MR-1. Biofilms were pregrown using a defined minimal media in a homebuilt constant depth film fermenter and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. The sample was subjected to various, rapidly switched substrate/ anaerobic electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate electron acceptors). Localized NMR spectroscopy was used to non-invasively monitored the spectra of hydrogen-containing metabolites at high temporal resolution (4.5 min) under oxygen-limited conditions. Anaerobic reduction was immediately observed upon switching feed solutions indicate that no gene induction (transcriptional response) was needed for MR-1 to switch between fumarate, dimethyl sulfoxide (DMSO) and nitrate electron acceptors. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate structural changes in response to the availability of electron acceptor and also the outcome of metabolic competition under oxygen-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how this possibly translates to the environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described here.

  7. Involvement of Shewanella oneidensis MR-1 LuxS in Biofilm Development and Sulfur Metabolism

    SciTech Connect

    Learman, Deric R.; Yi, Haakrho; Brown, Steven D.; Martin, Stanton L.; Geesey, Gill G.; Stevens, Ann M.; Hochella, Michael F.

    2009-01-05

    The role of LuxS in Shewanella oneidensis MR-1 has been examined by transcriptomic profiling, biochemical, and physiological experiments. The results indicate that a mutation in luxS alters biofilm development, not by altering quorum-sensing abilities but by disrupting the activated methyl cycle (AMC). The S. oneidensis wild type can produce a luminescence response in the AI-2 reporter strain Vibrio harveyi MM32. This luminescence response is abolished upon the deletion of luxS. The deletion of luxS also alters biofilm formations in static and flowthrough conditions. Genetic complementation restores the mutant biofilm defect, but the addition of synthetic AI-2 has no effect. These results suggest that AI-2 is not used as a quorum-sensing signal to regulate biofilm development in S. oneidensis. Growth on various sulfur sources was examined because of the involvement of LuxS in the AMC. A mutation in luxS produced a reduced ability to grow with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and to recycle homocysteine. These data suggest that LuxS is important to metabolizing methionine and the AMC in S. oneidensis.

  8. Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in Shewanella oneidensis MR-1

    SciTech Connect

    Cruz-Garza, Claribel; Murray, Alison E.; Rodrigues, Jorge L.M.; Gralnick, Jeffrey A.; McCue, Lee Ann; Romine, Margaret F.; Loffler, F. E.; Tiedje, James M.

    2011-03-30

    EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is not well understood. The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down regulated at least 2-fold and the EtrA7-1 mutant grew poorly with fumarate and dimethyl sulfoxide (DMSO), suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down regulation of genes implicated in aerobic metabolism. In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and confers physiological advantages to strain MR-1 under certain growth conditions. In conjunction with other regulators, EtrA fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1.

  9. Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1

    PubMed Central

    Alves, Mónica N.; Neto, Sónia E.; Alves, Alexandra S.; Fonseca, Bruno M.; Carrêlo, Afonso; Pacheco, Isabel; Paquete, Catarina M.; Soares, Cláudio M.; Louro, Ricardo O.

    2015-01-01

    The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB–OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC. PMID:26175726

  10. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-01

    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 acceptor 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.

  11. Involvement of cytochrome c CymA in the anaerobic metabolism of RDX by Shewanella oneidensis MR-1.

    PubMed

    Perreault, Nancy N; Crocker, Fiona H; Indest, Karl J; Hawari, Jalal

    2012-02-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitramine explosive commonly used for military applications that is responsible for severe soil and groundwater contamination. In this study, Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically (3.5 µmol·h(-1)·(g protein)(-1)) via two initial routes: (1) sequential N-NO(2) reductions to the corresponding nitroso (N-NO) derivatives (94% of initial RDX degradation) and (2) denitration followed by ring cleavage. To identify genes involved in the anaerobic metabolism of RDX, a library of ~2500 mutants of MR-1 was constructed by random transposon mutagenesis and screened for mutants with a reduced ability to degrade RDX compared with the wild type. An RDX-defective mutant (C9) was isolated that had the transposon inserted in the c-type cytochrome gene cymA. C9 transformed RDX at ~10% of the wild-type rate, with degradation occurring mostly via early ring cleavage caused by initial denitration leading to the formation of methylenedinitramine, 4-nitro-2,4-diazabutanal, formaldehyde, nitrous oxide, and ammonia. Genetic complementation of mutant C9 restored the wild-type phenotype, providing evidence that electron transport components have a role in the anaerobic reduction of RDX by MR-1. PMID:22260206

  12. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-30

    Shewanella oneidensis MR-1 is a facultative anaerobe growing by coupling organic matter oxidation to reduction of wide range of electron acceptors. Here we quantitatively assessed lactate and pyruvate metabolism of these bacteria under three distinct conditions: electron acceptor limited growth on lactate with O2 and fumarate, and pyruvate fermentation, which does not sustain growth but allows cells to survive for prolonged period. 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 all ATP needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute much 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, and TCA cycle did not contribute significantly to substrate oxidation. Pyruvate dehydrogenase reaction was not involved in lactate metabolism under O2 limitation, however was important for anaerobic growth probably supplying reducing equivalents for biosynthesis. Unexpectedly, obtained results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination between substrate-level phosphorylation and a respiratory process, 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). Based on involved enzymes localization we hypothesize that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  13. Redox and pH Microenvironments within Shewanella oneidensis MR-1 Biofilms Reveal an Electron Transfer Mechanism

    PubMed Central

    Babauta, Jerome T.; Nguyen, Hung Duc; Beyenal, Haluk

    2011-01-01

    The goal of this research was to quantify the variations in redox potential and pH in Shewanella oneidensis MR-1 biofilms respiring on electrodes. We grew S. oneidensis MR-1 on a graphite electrode, which was used to accept electrons for microbial respiration. We modified well-known redox and pH microelectrodes with a built-in reference electrode so that they could operate near polarized surfaces and quantified the redox potential and pH profiles in these biofilms. In addition, we used a ferri-/ferrocyanide redox system in which electrons were only transferred by mediated electron transfer to explain the observed redox potential profiles in biofilms. We found that regardless of the polarization potential of the biofilm electrode, the redox potential decreased toward the bottom of the biofilm. In a fully redox-mediated control system (ferri-/ferrocyanide redox system), the redox potential increased toward the bottom when the electrode was the electron acceptor. The opposite behavior of redox profiles in biofilms and the redox-controlled system is explained by S. oneidensis MR-1 biofilms not being redox-controlled when they respire on electrodes. The lack of a significant variation in pH implies that there is no proton transfer limitation in S. oneidensis MR-1 biofilms and that redox potential profiles are not caused by pH. PMID:21648431

  14. Integrated Analysis of Protein Complexes and Regulatory Networks Involved in Anaerobic Energy Metabolism of Shewanella Oneidensis MR-1

    SciTech Connect

    Tiedje, James M.

    2005-06-01

    Anaerobic Nitrate Reduction. Nitrate is an extensive co-contaminant at some DOE sites making metal and radionuclide reduction problematic. Hence, we sought to better understand the nitrate reduction pathway and its control in S. oneidensis MR-1. It is not known whether the nitrate reduction is by denitrification or dissimilatory nitrate reduction into ammonium (DNRA). By both physiological and genetic evidence, we proved that DNRA is the nitrate reduction pathway in this organism. Using the complete genome sequence of S. oneidensis MR-1, we identified a gene encoding a periplasmic nitrate reductase based on its 72% sequence identity with the napA gene in E. coli. Anaerobic growth of MR-1 on nitrate was abolished in a site directed napA mutant, indicating that NapA is the only nitrate reductase present. The anaerobic expression of napA and nrfA, a homolog of the cytochrome b552 nitrite reductase in E. coli, increased with increasing nitrate concentration until a plateau was reached at 3 mM KNO3. This indicates that these genes are not repressed by increasing concentrations of nitrate. The reduction of nitrate can generate intermediates that can be toxic to the microorganism. To determine the genetic response of MR-1 to high concentrations of nitrate, DNA microarrays were used to obtain a complete gene expression profile of MR-1 at low (1 mM) versus high (40 mM) nitrate concentrations. Genes encoding transporters and efflux pumps were up-regulated, perhaps as a mechanism to export toxic compounds. In addition, the gene expression profile of MR-1, grown anaerobically with nitrate as the only electron acceptor, suggested that this dissimilatory pathway contributes to N assimilation. Hence the nitrate reduction pathway could serve a dual purpose. The role of EtrA, a homolog of Fnr (global anaerobic regulator in E. coli) was examined using an etrA deletion mutant we constructed, S. oneidensis EtrA7-1.

  15. Transcriptome analysis reveals response regulator SO2426-mediated gene expression in Shewanella oneidensis MR-1 under chromate challenge

    SciTech Connect

    Chourey, Karuna; Wei, Wei; Wan, Xiu-Feng; Thompson, Dorothea K.

    2008-01-01

    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 SO2426 regulon.

  16. Genes That Enhance the Ecological Fitness of Shewanella oneidensis MR-1 in Sediments Reveal the Value of Antibiotic Resistance▿ †

    PubMed Central

    Groh, Jennifer L.; Luo, Qingwei; Ballard, Jimmy D.; Krumholz, Lee R.

    2007-01-01

    Environmental bacteria persist in various habitats, yet little is known about the genes that contribute to growth and survival in their respective ecological niches. Signature-tagged mutagenesis (STM) of Shewanella oneidensis MR-1 coupled with a screen involving incubations of mutant strains in anoxic aquifer sediments allowed us to identify 47 genes that enhance fitness in sediments. Gene functions inferred from annotations provide us with insight into physiological and ecological processes that environmental bacteria use while growing in sediment ecosystems. Identification of the mexF gene and other potential membrane efflux components by STM demonstrated that homologues of multidrug resistance genes present in pathogens are required for sediment fitness of nonpathogenic bacteria. Further studies with a mexF deletion mutant demonstrated that the multidrug resistance pump encoded by mexF is required for resistance to antibiotics, including chloramphenicol and tetracycline. Chloramphenicol-adapted cultures exhibited mutations in the gene encoding a TetR family regulatory protein, indicating a role for this protein in regulating expression of the mexEF operon. The relative importance of mexF for sediment fitness suggests that antibiotic efflux may be a required process for bacteria living in sediment systems. PMID:17114320

  17. Transcriptome analysis reveals response regulator SO2426-mediated gene expression in Shewanella oneidensis MR-1 under chromate challenge

    PubMed Central

    Chourey, Karuna; Wei, Wei; Wan, Xiu-Feng; Thompson, Dorothea K

    2008-01-01

    Background 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 SO2426 regulon. Results Inactivation of so2426 by an in-frame deletion resulted in enhanced chromate sensitivity and a reduced capacity to remove extracellular Cr(VI) relative to the parental strain. Time-resolved microarray analysis was used to compare transcriptomic profiles of wild-type and SO2426-deficient mutant S. oneidensis under conditions of chromate exposure. In total, 841 genes (18% of the arrayed genome) were up- or downregulated at least twofold in the Δso2426 mutant for at least one of six time-point conditions. Hierarchical cluster analysis of temporal transcriptional profiles identified a distinct cluster (n = 46) comprised of co-ordinately regulated genes exhibiting significant downregulated expression (p < 0.05) over time. Thirteen of these genes encoded proteins associated with transport and binding functions, particularly those involved in Fe transport and homeostasis (e.g., siderophore biosynthetic enzymes, TonB-dependent receptors, and the iron-storage protein ferritin). A conserved hypothetical operon (so1188-so1189-so1190), previously identified as a potential target of Fur-mediated repression, as well as a putative bicyclomycin resistance gene (so2280) and cation efflux family protein gene (so2045) also were repressed in the

  18. Oxygen-dependent autoaggregation in Shewanella oneidensis MR-1

    SciTech Connect

    Mclean, Jeffrey S.; Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Bilskis, Christina L.; Zakrajsek, Brian A.; Hill, Eric A.; Saffarini, Daad; Romine, Margaret F.; Gorby, Yuri A.; Fredrickson, Jim K.; Beliaev, Alex S.

    2008-07-01

    In aerobic chemostat cultures maintained at 50% dissolved O2 tension (123.5 µM dissolved O2), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl2 and retained this multi-cellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein, and glycoconjugates. Upon onset of O2-limited growth (dissolved O2 below detection) however, the Ca2+-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O2-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O2 concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologs of metal reduction genes, including mtrD (SO1782), mtrE (SO1781), and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H2O2. Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

  19. Revealed aspect of metabolic osteoarthritis.

    PubMed

    Chadha, Rishmeen

    2016-12-01

    In current scenario there is an emerging trend to consider osteoarthritis (OA) phenotypes based on their risk factors and therefore offering potential for targeted therapies. OA prevalence with other coexistence diseases, such as systemic arterial hypertension, cardiovascular disease, diabetes, and dyslipidemia are increasing. Majority of OA associated with MetS, despite overweight of patients, the occurrence of OA in joints do not bear load suggest, the chronic inflammation status existing in patients with MetS can alter the metabolism of cartilage, regardless of excessive weight. This paper indicates that factors responsible for metabolic syndrome has clear role in cartilage degeneration and further degradation. PMID:27453640

  20. Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H2

    SciTech Connect

    Kreuzer, Helen W.; Hill, Eric A.; Moran, James J.; Bartholomew, Rachel A.; Hui, Yang; Hegg, Eric L.

    2014-03-01

    Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data is consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organism, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

  1. Metabolomics reveals metabolic biomarkers of Crohn's disease

    SciTech Connect

    Jansson, J.K.; Willing, B.; Lucio, M.; Fekete, A.; Dicksved, J.; Halfvarson, J.; Tysk, C.; Schmitt-Kopplin, P.

    2009-06-01

    The causes and etiology of Crohn's disease (CD) are currently unknown although both host genetics and environmental factors play a role. Here we used non-targeted metabolic profiling to determine the contribution of metabolites produced by the gut microbiota towards disease status of the host. Ion Cyclotron Resonance Fourier Transform Mass Spectrometry (ICR-FT/MS) was used to discern the masses of thousands of metabolites in fecal samples collected from 17 identical twin pairs, including healthy individuals and those with CD. Pathways with differentiating metabolites included those involved in the metabolism and or synthesis of amino acids, fatty acids, bile acids and arachidonic acid. Several metabolites were positively or negatively correlated to the disease phenotype and to specific microbes previously characterized in the same samples. Our data reveal novel differentiating metabolites for CD that may provide diagnostic biomarkers and/or monitoring tools as well as insight into potential targets for disease therapy and prevention.

  2. Transcriptome Analysis of Early Surface-Associated Growth of Shewanella oneidensis MR-1

    PubMed Central

    Gödeke, Julia; Binnenkade, Lucas; Thormann, Kai M.

    2012-01-01

    Bacterial biofilm formation starts with single cells attaching to a surface, however, little is known about the initial attachment steps and the adaptation to the surface-associated life style. Here, we describe a hydrodynamic system that allows easy harvest of cells at very early biofilm stages. Using the metal ion-reducing gammaproteobacterium Shewanella oneidensis MR-1 as a model organism, we analyzed the transcriptional changes occurring during surface-associated growth between 15 and 60 minutes after attachment. 230 genes were significantly upregulated and 333 were downregulated by a factor of ≥2. Main functional categories of the corresponding gene products comprise metabolism, uptake and transport, regulation, and hypothetical proteins. Among the genes highly upregulated those implicated in iron uptake are highly overrepresented, strongly indicating that S. oneidensis MR-1 has a high demand for iron during surface attachment and initial biofilm stages. Subsequent microscopic analysis of biofilm formation under hydrodynamic conditions revealed that addition of Fe(II) significantly stimulated biofilm formation of S. oneidensis MR-1 while planktonic growth was not affected. Our approach to harvest cells for transcriptional analysis of early biofilm stages is expected to be easily adapted to other bacterial species. PMID:22860070

  3. Metabolic profiling reveals key metabolic features of renal cell carcinoma

    PubMed Central

    Catchpole, Gareth; Platzer, Alexander; Weikert, Cornelia; Kempkensteffen, Carsten; Johannsen, Manfred; Krause, Hans; Jung, Klaus; Miller, Kurt; Willmitzer, Lothar; Selbig, Joachim; Weikert, Steffen

    2011-01-01

    Abstract Recent evidence suggests that metabolic changes play a pivotal role in the biology of cancer and in particular renal cell carcinoma (RCC). Here, a global metabolite profiling approach was applied to characterize the metabolite pool of RCC and normal renal tissue. Advanced decision tree models were applied to characterize the metabolic signature of RCC and to explore features of metastasized tumours. The findings were validated in a second independent dataset. Vitamin E derivates and metabolites of glucose, fatty acid, and inositol phosphate metabolism determined the metabolic profile of RCC. α-tocopherol, hippuric acid, myoinositol, fructose-1-phosphate and glucose-1-phosphate contributed most to the tumour/normal discrimination and all showed pronounced concentration changes in RCC. The identified metabolic profile was characterized by a low recognition error of only 5% for tumour versus normal samples. Data on metastasized tumours suggested a key role for metabolic pathways involving arachidonic acid, free fatty acids, proline, uracil and the tricarboxylic acid cycle. These results illustrate the potential of mass spectroscopy based metabolomics in conjunction with sophisticated data analysis methods to uncover the metabolic phenotype of cancer. Differentially regulated metabolites, such as vitamin E compounds, hippuric acid and myoinositol, provide leads for the characterization of novel pathways in RCC. PMID:19845817

  4. The Electrogenic Bacterium Shewanella Oneidensis MR-1 and its Mutants with Increased Reducing Capacity

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Mordkovich, N. N.; Shakulov, R. S.; Debabov, V. G.

    2013-02-01

    Mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited an increased reducing activity and a higher rate of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S.oneidensis MR-1 will be used in microbial fuel cells (MFC) to enhance energy production from organic compounds. The strain S. oneidensis MR-1 and its mutants with an increased electron production will be used as a good source of bioelectricity in MFC in the experiments on the International Space Station.

  5. Aggrandizing power output from Shewanella oneidensis MR-1 microbial fuel cells using calcium chloride.

    PubMed

    Fitzgerald, Lisa A; Petersen, Emily R; Gross, Benjamin J; Soto, Carissa M; Ringeisen, Bradley R; El-Naggar, Mohamed Y; Biffinger, Justin C

    2012-01-15

    There are several interconnected metabolic pathways in bacteria essential for the conversion of carbon electron sources directly into electrical currents using microbial fuel cells (MFCs). This study establishes a direct exogenous method to increase power output from a Shewanella oneidensis MR-1 containing MFC by adding calcium chloride to the culture medium. The current output from each CaCl(2) concentration tested revealed that the addition of CaCl(2) to 1400 μM increased the current density by >80% (0.95-1.76 μA/cm(2)) using sodium lactate as the sole carbon source. Furthermore, polarization curves showed that the maximum power output could be increased from 157 to 330 μW with the addition of 2080 μM CaCl(2). Since the conductivity of the culture medium did not change after the addition of CaCl(2) (confirmed by EIS and bulk conductivity measurements), this increase in power was primarily biological and not based on ionic effects. Thus, controlling the concentration of CaCl(2) is a pathway to increase the efficiency and performance of S. oneidensis MR-1 MFCs. PMID:22154401

  6. Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor

    PubMed Central

    Reantragoon, Rangsima; Kjer-Nielsen, Lars; Patel, Onisha; Chen, Zhenjun; Illing, Patricia T.; Bhati, Mugdha; Kostenko, Lyudmila; Bharadwaj, Mandvi; Meehan, Bronwyn; Hansen, Ted H.; Godfrey, Dale I.

    2012-01-01

    Mucosal-associated invariant T (MAIT) cells express a semiinvariant αβ T cell receptor (TCR) that binds MHC class I–like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vβ2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR–MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR β chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR–MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR–CD1d-antigen interaction, in which both the α and β chain of the NKT TCR is required for ligation above the F′-pocket of CD1d. PMID:22412157

  7. High-syn conformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures of Shewanella oneidensis MR-1 uridine phosphorylase in the free form and in complex with uridine.

    PubMed

    Safonova, Tatyana N; Mikhailov, Sergey N; Veiko, Vladimir P; Mordkovich, Nadezhda N; Manuvera, Valentin A; Alekseev, Cyril S; Kovalchuk, Mikhail V; Popov, Vladimir O; Polyakov, Konstantin M

    2014-12-01

    Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP from Shewanella oneidensis MR-1 (SoUP) was performed in Escherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-syn conformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine. PMID:25478848

  8. The mxd operon in Shewanella oneidensis MR-1 is induced in response to starvation and regulated by ArcS/ArcA and BarA/UvrY

    PubMed Central

    2013-01-01

    Background S. oneidensis MR-1 is a dissimilatory metal-reducing bacterium. Under anoxic conditions S. oneidensis MR-1 attaches to and uses insoluble minerals such as Fe(III) and Mn(IV) oxides as electron acceptors. In the laboratory, S. oneidensis MR-1 forms biofilms under hydrodynamic flow conditions on a borosilicate glass surface; formation of biofilms was previously found to be dependent on the mxd gene cluster (mxdABCD). Results This study revealed environmental and genetic factors regulating expression of the mxd genes in S. oneidensis MR-1. Physiological experiments conducted with a S. oneidensis MR-1 strain carrying a transcriptional lacZ fusion to the mxd promoter identified electron donor starvation as a key factor inducing mxd gene expression. Tn5 mutagenesis identified the ArcS/ArcA two-component signaling system as a repressor of mxd expression in S. oneidensis MR-1 under planktonic conditions. Biofilms of ∆arcS and ∆arcA strains carrying a transcriptional gfp -reporter fused to the mxd promoter revealed a reduced mxd expression, suggesting that ArcS/ArcA are necessary for activation of mxd expression under biofilm conditions. Biofilms of ∆arcS and ∆arcA mutants were unable to form a compact three-dimensional structure consistent with a low level of mxd expression. In addition, BarA/UvrY was identified as a major regulator of mxd expression under planktonic conditions. Interestingly, biofilms of ∆barA and ∆uvrY mutants were able to form three-dimensional structures that were, however, less compact compared to wild type biofilms. Conclusions We have shown here that the mxd genes in S. oneidensis MR-1 are controlled transcriptionally in response to carbon starvation and by the ArcS/ArcA and the BarA/UvrY signaling system. BarA might function as a sensor to assess the metabolic state of the cell, including carbon starvation, leading to expression of the mxd operon and therefore control biofilm formation. PMID:23705927

  9. Purification and Characterization of [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

    SciTech Connect

    Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.; Heald, Steve M.; Dohnalkova, Alice; Sybirna, Kateryna; Bottin, Herve; Squier, Thomas C.; Zachara, John M.; Fredrickson, Jim K.

    2011-08-02

    The γ-proteobacterium Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that was implicated in both H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned into a protein expression vector. The resulting plasmid was transformed into a MR-1 mutant deficient in H2 formation. Expression of MR-1 [NiFe]-H2ase in trans restored the mutant’s ability to produce H2 at 37% of that for wild type. Following expression, MR-1 [NiFe]-H2ase was purified to near homogeneity. The purified MR-1 [NiFe]-H2ase could couple H2 oxidation to reduction of Tc(VII) and methyl viologen directly. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated Tc(VII) but not methyl viologen reductions. Under the conditions tested, Tc(VII) reduction was complete in Tris buffer but not in HEPES buffer. The reduced Tc(IV) was soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc(IV) precipitates formed in HEPES buffer were packed with crystallites. Although X-ray absorption near-edge spectroscopy measurements confirmed that the reduction products found in both buffers were Tc(IV), extended X-ray adsorption fine-structure measurements revealed that these products were very different. While the product in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2•nH2O. These results shows for the first time that MR-1 [NiFe]-H2ase is a bidirectional enzyme that catalyzes both H2 formation and oxidation as well as Tc(VII) reduction directly by coupling H2 oxidation.

  10. Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses

    SciTech Connect

    Gao, Haichun; Wang, Xiaohu; Yang, Zamin Koo; Palzkill, Timothy; Zhou, Jizhong

    2008-01-01

    The Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in Escherichia coli. Shewanella oneidensis MR-1 contains a gene encoding a putative ArcA homolog with {approx} 81% amino acid sequence identity to the E. coli ArcA protein but not a full-length arcB gene. To understand the role of ArcA in S. oneidensis, an arcA deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O{sub 2}. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (p < 0.05) by the mutation. In contrast to E. coli ArcA, the protein appears to be dispensable in regulation of the TCA cycle in S. oneidensis. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli. These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

  11. Environmental and genetic perturbations reveal different networks of metabolic regulation

    PubMed Central

    Greenberg, Anthony J; Hackett, Sean R; Harshman, Lawrence G; Clark, Andrew G

    2011-01-01

    Progress in systems biology depends on accurate descriptions of biological networks. Connections in a regulatory network are identified as correlations of gene expression across a set of environmental or genetic perturbations. To use this information to predict system behavior, we must test how the nature of perturbations affects topologies of networks they reveal. To probe this question, we focused on metabolism of Drosophila melanogaster. Our source of perturbations is a set of crosses among 92 wild-derived lines from five populations, replicated in a manner permitting separate assessment of the effects of genetic variation and environmental fluctuation. We directly assayed activities of enzymes and levels of metabolites. Using a multivariate Bayesian model, we estimated covariance among metabolic parameters and built fine-grained probabilistic models of network topology. The environmental and genetic co-regulation networks are substantially the same among five populations. However, genetic and environmental perturbations reveal qualitative differences in metabolic regulation, suggesting that environmental shifts, such as diet modifications, produce different systemic effects than genetic changes, even if the primary targets are the same. PMID:22186737

  12. NeuCode Proteomics Reveals Bap1 Regulation of Metabolism.

    PubMed

    Baughman, Joshua M; Rose, Christopher M; Kolumam, Ganesh; Webster, Joshua D; Wilkerson, Emily M; Merrill, Anna E; Rhoads, Timothy W; Noubade, Rajkumar; Katavolos, Paula; Lesch, Justin; Stapleton, Donald S; Rabaglia, Mary E; Schueler, Kathy L; Asuncion, Raymond; Domeyer, Melanie; Zavala-Solorio, Jose; Reich, Michael; DeVoss, Jason; Keller, Mark P; Attie, Alan D; Hebert, Alexander S; Westphall, Michael S; Coon, Joshua J; Kirkpatrick, Donald S; Dey, Anwesha

    2016-07-12

    We introduce neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology. PMID:27373151

  13. Global Molecular and Morphological Effects of 24-Hour Chromium(VI) Exposure on Shewanella oneidensis MR-1

    PubMed Central

    Chourey, Karuna; Thompson, Melissa R.; Morrell-Falvey, Jennifer; VerBerkmoes, Nathan C.; Brown, Steven D.; Shah, Manesh; Zhou, Jizhong; Doktycz, Mitchel; Hettich, Robert L.; Thompson, Dorothea K.

    2006-01-01

    The biological impact of 24-h (“chronic”) chromium(VI) [Cr(VI) or chromate] exposure on Shewanella oneidensis MR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic characterization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (peptidoglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment. PMID:16957260

  14. Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1

    SciTech Connect

    Chourey, Karuna; Thompson, Melissa R; Morrell-Falvey, Jennifer L; Verberkmoes, Nathan C; Brown, Steven D; Shah, Manesh B; Zhou, Jizhong; Doktycz, Mitchel John; Hettich, Robert {Bob} L; Thompson, Dorothea K

    2006-01-01

    The biological impact of 24-h ("chronic") chromium(VI) [Cr(VI) or chromate] exposure on ShewanellaoneidensisMR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic charac terization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (pepti doglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment.

  15. Environmental metabolomics reveal geographic variation in aerobic metabolism and metabolic substrates in Mongolian gerbils (Meriones unguiculatus).

    PubMed

    Shi, Yao-Long; Chi, Qing-Sheng; Liu, Wei; Fu, He-Ping; Wang, De-Hua

    2015-06-01

    Mongolian gerbils (Meriones unguiculatus) have a large-scale distribution in northern China. Geographic physiological variations which related to energy and water metabolism are critical to animals' local adaptation and distribution. However, the underlying biochemical mechanism of such variation and its role in adaptation remains largely unknown. We used GC-MS metabolomics approach to investigate the biochemical adaptation of Mongolian gerbils from xeric (desert), transition (desert steppe) and mesic (typical steppe) environments. Gerbils in desert population had lower resting metabolic rate (RMR) and total evaporative water loss (TEWL) than mesic population. Serum metabolomics revealed that concentrations of five tricarboxylic acid cycle intermediates (citrate, cis-aconitate, α-ketoglutarate, fumarate and malate) were lower in desert population than mesic population. Gastrocnemius metabolomics and citrate synthase activity analysis showed a lower concentration of citrate and lower citrate synthase activity in desert population. These findings suggest that desert dwelling gerbils decrease RMR and TEWL via down-regulation of aerobic respiration. Gastrocnemius metabolomics also revealed that there were higher concentrations of glucose and glycolytic intermediates, but lower concentrations of lipids, amino acids and urea in desert population than mesic population. This geographic variation in metabolic substrates may enhance metabolic water production per oxygen molecule for desert population while constraining aerobic respiration to reduce RMR and TEWL. PMID:25817427

  16. Metabolomics reveals insect metabolic responses associated with fungal infection.

    PubMed

    Xu, Yong-Jiang; Luo, Feifei; Gao, Qiang; Shang, Yanfang; Wang, Chengshu

    2015-06-01

    The interactions between insects and pathogenic fungi are complex. We employed metabolomic techniques to profile insect metabolic dynamics upon infection by the pathogenic fungus Beauveria bassiana. Silkworm larvae were infected with fungal spores and microscopic observations demonstrated that the exhaustion of insect hemocytes was coupled with fungal propagation in the insect body cavity. Metabolomic analyses revealed that fungal infection could significantly alter insect energy and nutrient metabolisms as well as the immune defense responses, including the upregulation of carbohydrates, amino acids, fatty acids, and lipids, but the downregulation of eicosanoids and amines. The insect antifeedant effect of the fungal infection was evident with the reduced level of maclurin (a component of mulberry leaves) in infected insects but elevated accumulations in control insects. Insecticidal and cytotoxic mycotoxins like oosporein and beauveriolides were also detected in insects at the later stages of infection. Taken together, the metabolomics data suggest that insect immune responses are energy-cost reactions and the strategies of nutrient deprivation, inhibition of host immune responses, and toxin production would be jointly employed by the fungus to kill insects. The data obtained in this study will facilitate future functional studies of genes and pathways associated with insect-fungus interactions. PMID:25895944

  17. Shewanella oneidensis MR-1-Induced Fe(III) Reduction Facilitates Roxarsone Transformation.

    PubMed

    Chen, Guowei; Ke, Zhengchen; Liang, Tengfang; Liu, Li; Wang, Gang

    2016-01-01

    Although microbial activity and associated iron (oxy)hydroxides are known in general to affect the environmental dynamics of 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), the mechanistic understanding of the underlying biophysico-chemical processes remains unclear due to limited experimental information. We studied how Shewanella oneidensis MR-1 -a widely distributed metal-reducing bacterium, in the presence of dissolved Fe(III), affects roxarsone transformations and biogeochemical cycling in a model aqueous system. The results showed that the MR-1 strain was able to anaerobically use roxarsone as a terminal electron acceptor and to convert it to a single product, 3-amino-4-hydroxybenzene arsonic acid (AHBAA). The presence of Fe(III) stimulated roxarsone transformation via MR-1-induced Fe(III) reduction, whereby the resulting Fe(II) acted as an efficient reductant for roxarsone transformation. In addition, the subsequent secondary Fe(III)/Fe(II) mineralization created conditions for adsorption of organoarsenic compounds to the yielded precipitates and thereby led to arsenic immobilization. The study provided direct evidence of Shewanella oneidensis MR-1-induced direct and Fe(II)-associated roxarsone transformation. Quantitative estimations revealed a candidate mechanism for the early-stage environmental dynamics of roxarsone in nature, which is essential for understanding the environmental dynamics of roxarsone and successful risk assessment. PMID:27100323

  18. Shewanella oneidensis MR-1-Induced Fe(III) Reduction Facilitates Roxarsone Transformation

    PubMed Central

    Chen, Guowei; Ke, Zhengchen; Liang, Tengfang; Liu, Li; Wang, Gang

    2016-01-01

    Although microbial activity and associated iron (oxy)hydroxides are known in general to affect the environmental dynamics of 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), the mechanistic understanding of the underlying biophysico-chemical processes remains unclear due to limited experimental information. We studied how Shewanella oneidensis MR-1 –a widely distributed metal-reducing bacterium, in the presence of dissolved Fe(III), affects roxarsone transformations and biogeochemical cycling in a model aqueous system. The results showed that the MR-1 strain was able to anaerobically use roxarsone as a terminal electron acceptor and to convert it to a single product, 3-amino-4-hydroxybenzene arsonic acid (AHBAA). The presence of Fe(III) stimulated roxarsone transformation via MR-1-induced Fe(III) reduction, whereby the resulting Fe(II) acted as an efficient reductant for roxarsone transformation. In addition, the subsequent secondary Fe(III)/Fe(II) mineralization created conditions for adsorption of organoarsenic compounds to the yielded precipitates and thereby led to arsenic immobilization. The study provided direct evidence of Shewanella oneidensis MR-1-induced direct and Fe(II)-associated roxarsone transformation. Quantitative estimations revealed a candidate mechanism for the early-stage environmental dynamics of roxarsone in nature, which is essential for understanding the environmental dynamics of roxarsone and successful risk assessment. PMID:27100323

  19. Metabolic phenotyping reveals a lipid mediator response to ionizing radiation.

    PubMed

    Laiakis, Evagelia C; Strassburg, Katrin; Bogumil, Ralf; Lai, Steven; Vreeken, Rob J; Hankemeier, Thomas; Langridge, James; Plumb, Robert S; Fornace, Albert J; Astarita, Giuseppe

    2014-09-01

    Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine. PMID:25126707

  20. Identification of Mobile Elements and Pseudogenes in the Shewanella oneidensis MR-1 Genome

    SciTech Connect

    Romine, Margaret F.; Carlson, Timothy; Norbeck, Angela D.; McCue, Lee Ann; Lipton, Mary S.

    2008-05-01

    Shewanella oneidensis MR-1 is the first of 22 different Shewanella spp. whose genomes have been or are being sequenced and thus serves as the model organism for studying the functional repertoire of the Shewanella genus. The original MR-1 genome annotation revealed a large number of transposase genes and pseudogenes, indicating that many of the genome’s functions may be decaying. Comparative analyses of the sequenced Shewanella strains suggest that 209 genes in MR-1 have in-frame stop codons, frameshifts, or interruptions and/or are truncated and that 65 of the original pseudogene predictions were erroneous. Among the decaying functions are that of one of three chemotaxis clusters, type I pilus production, starch utilization, and nitrite respiration. Many of the mutations could be attributed to members of 41 different types of insertion sequence (IS) elements and three types of miniature inverted-repeat transposable elements identified here for the first time. The high copy numbers of individual mobile elements (up to 71) are expected to promote large-scale genome recombination events, as evidenced by the displacement of the algA promoter. The ability of MR-1 to acquire foreign genes via reactions catalyzed by both the integron integrase and the ISSod25-encoded integrases is suggested by the presence of attC sites and genes whose sequences are characteristic of other species downstream of each site. This large number of mobile elements and multiple potential sites for integrasemediated acquisition of foreign DNA indicate that the MR-1 genome is exceptionally dynamic, with many functions and regulatory control points in the process of decay or reinvention.

  1. Survival of Shewanella oneidensis MR-1 after UV radiation exposure.

    PubMed

    Qiu, Xiaoyun; Sundin, George W; Chai, Benli; Tiedje, James M

    2004-11-01

    We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB, UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among Shewanella strains examined, with D37 and D10 values of 5.6 and 16.5% of Escherichia coli K-12 values. Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1 was observed following doses of 3.3 J of UVC m(-2), 568 J of UVB m(-2), 25 kJ of UVA m(-2), and 558 J of solar UVB m(-2), respectively. Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively. A significant UV mutability to rifampin resistance was detected in both UVC- and UVB-treated samples, with the mutation frequency in the range of 10(-5) to 10(-6). Unlike in E. coli, the expression levels of the nucleotide excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage inducible in MR-1. Complementation of Pseudomonas aeruginosa UA11079 (uvrA deficient) with uvrA of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude. Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents. PMID:15528503

  2. Adoptive T Cell Therapy Targeting CD1 and MR1

    PubMed Central

    Guo, Tingxi; Chamoto, Kenji; Hirano, Naoto

    2015-01-01

    Adoptive T cell immunotherapy has demonstrated clinically relevant efficacy in treating malignant and infectious diseases. However, much of these therapies have been focused on enhancing, or generating de novo, effector functions of conventional T cells recognizing HLA molecules. Given the heterogeneity of HLA alleles, mismatched patients are ineligible for current HLA-restricted adoptive T cell therapies. CD1 and MR1 are class I-like monomorphic molecules and their restricted T cells possess unique T cell receptor specificity against entirely different classes of antigens. CD1 and MR1 molecules present lipid and vitamin B metabolite antigens, respectively, and offer a new front of targets for T cell therapies. This review will cover the recent progress in the basic research of CD1, MR1, and their restricted T cells that possess translational potential. PMID:26052329

  3. Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency

    PubMed Central

    Weng, Rui; Shen, Sensen; Tian, Yonglu; Burton, Casey; Xu, Xinyuan; Liu, Yi; Chang, Cuilan; Bai, Yu; Liu, Huwei

    2015-01-01

    Serotonin is an important neurotransmitter that broadly participates in various biological processes. While serotonin deficiency has been associated with multiple pathological conditions such as depression, schizophrenia, Alzheimer’s disease and Parkinson’s disease, the serotonin-dependent mechanisms remain poorly understood. This study therefore aimed to identify novel biomarkers and metabolic pathways perturbed by serotonin deficiency using metabolomics approach in order to gain new metabolic insights into the serotonin deficiency-related molecular mechanisms. Serotonin deficiency was achieved through pharmacological inhibition of tryptophan hydroxylase (Tph) using p-chlorophenylalanine (pCPA) or genetic knockout of the neuronal specific Tph2 isoform. This dual approach improved specificity for the serotonin deficiency-associated biomarkers while minimizing nonspecific effects of pCPA treatment or Tph2 knockout (Tph2-/-). Non-targeted metabolic profiling and a targeted pCPA dose-response study identified 21 biomarkers in the pCPA-treated mice while 17 metabolites in the Tph2-/- mice were found to be significantly altered compared with the control mice. These newly identified biomarkers were associated with amino acid, energy, purine, lipid and gut microflora metabolisms. Oxidative stress was also found to be significantly increased in the serotonin deficient mice. These new biomarkers and the overall metabolic pathways may provide new understanding for the serotonin deficiency-associated mechanisms under multiple pathological states. PMID:26154191

  4. Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms

    PubMed Central

    2012-01-01

    Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed “extended” overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and 13C-metabolic flux analysis. PMID:22963408

  5. Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency

    NASA Astrophysics Data System (ADS)

    Weng, Rui; Shen, Sensen; Tian, Yonglu; Burton, Casey; Xu, Xinyuan; Liu, Yi; Chang, Cuilan; Bai, Yu; Liu, Huwei

    2015-07-01

    Serotonin is an important neurotransmitter that broadly participates in various biological processes. While serotonin deficiency has been associated with multiple pathological conditions such as depression, schizophrenia, Alzheimer’s disease and Parkinson’s disease, the serotonin-dependent mechanisms remain poorly understood. This study therefore aimed to identify novel biomarkers and metabolic pathways perturbed by serotonin deficiency using metabolomics approach in order to gain new metabolic insights into the serotonin deficiency-related molecular mechanisms. Serotonin deficiency was achieved through pharmacological inhibition of tryptophan hydroxylase (Tph) using p-chlorophenylalanine (pCPA) or genetic knockout of the neuronal specific Tph2 isoform. This dual approach improved specificity for the serotonin deficiency-associated biomarkers while minimizing nonspecific effects of pCPA treatment or Tph2 knockout (Tph2-/-). Non-targeted metabolic profiling and a targeted pCPA dose-response study identified 21 biomarkers in the pCPA-treated mice while 17 metabolites in the Tph2-/- mice were found to be significantly altered compared with the control mice. These newly identified biomarkers were associated with amino acid, energy, purine, lipid and gut microflora metabolisms. Oxidative stress was also found to be significantly increased in the serotonin deficient mice. These new biomarkers and the overall metabolic pathways may provide new understanding for the serotonin deficiency-associated mechanisms under multiple pathological states.

  6. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  7. Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism

    PubMed Central

    Nardozza, Simona; MacRae, Elspeth A.; Sulpice, Ronan; Clearwater, Michael J.

    2013-01-01

    Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography–mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species. PMID:24058160

  8. [Somatic polyploidy associated metabolic changes revealed by modular biology].

    PubMed

    Anatskaia, O V; Vinogradov, A E

    2010-01-01

    Excessive somatic polyploidy usually accompanies physiologic and pathologic overload and it is generally accepted as a symptom of pathology. At the same time, polyploidy cells exist in most fungal, plant, mollusk, fish, bird and mammalian tissues confirming their great evolutionary success. The secret of this success remains enigmatic. Since transcriptome rearrangements usually start with metabolic flux redistribution, we decided to investigate firstly the effects of polyploidy on cell metabolism. Using multitest approach of modular biology and databases Entrez Gene, RefSeq, GNF SymAtlas, Gene Ontology, KEGG, BioCarta; MsigDb, Reactome, GenMAPP, and HumanCyc, we performed detailed comparison of metabolic genes expression in human and mouse organs with reciprocal pattern of polyploidy (i. e. in the heart and in the liver). Pairwise criss-cross comparison of diploid vs. polyploid organs allowed removing species- and tissue-specific effects. From our results, polyploidy is associated with rearrangements of main metabolic pathways. We found deep depression of mitochondrial processes, features of autophagia, and increased carbohydrate degradation and lipid biosynthesis. Taken together, these changes pointed to the energy and oxygen deprivation. We also found clear indications of enhanced oxidative stress protection. The major of them are triggering of pentose-phosphate pathway, depression of mitochondria-cytoplasm electron shuttles, and impartment of electron flows across 1 (NADH dehydrogenase) and IV (cytochrome c-oxydase) breath complexes. We suggest that all these changes are necessary for the increase in metabolic plasticity and for the protection of replicating DNA from oxidative damage. PMID:20302017

  9. Metabolic profiling reveals ethylene mediated metabolic changes and a coordinated adaptive mechanism of 'Jonagold' apple to low oxygen stress.

    PubMed

    Bekele, Elias A; Beshir, Wasiye F; Hertog, Maarten L A T M; Nicolai, Bart M; Geeraerd, Annemie H

    2015-11-01

    Apples are predominantly stored in controlled atmosphere (CA) storage to delay ripening and prolong their storage life. Profiling the dynamics of metabolic changes during ripening and CA storage is vital for understanding the governing molecular mechanism. In this study, the dynamics of the primary metabolism of 'Jonagold' apples during ripening in regular air (RA) storage and initiation of CA storage was profiled. 1-Methylcyclopropene (1-MCP) was exploited to block ethylene receptors and to get insight into ethylene mediated metabolic changes during ripening of the fruit and in response to hypoxic stress. Metabolic changes were quantified in glycolysis, the tricarboxylic acid (TCA) cycle, the Yang cycle and synthesis of the main amino acids branching from these metabolic pathways. Partial least square discriminant analysis of the metabolic profiles of 1-MCP treated and control apples revealed a metabolic divergence in ethylene, organic acid, sugar and amino acid metabolism. During RA storage at 18°C, most amino acids were higher in 1-MCP treated apples, whereas 1-aminocyclopropane-1-carboxylic acid (ACC) was higher in the control apples. The initial response of the fruit to CA initiation was accompanied by an increase of alanine, succinate and glutamate, but a decline in aspartate. Furthermore, alanine and succinate accumulated to higher levels in control apples than 1-MCP treated apples. The observed metabolic changes in these interlinked metabolites may indicate a coordinated adaptive strategy to maximize energy production. PMID:26031836

  10. Cellular hallmarks reveal restricted aerobic metabolism at thermal limits

    PubMed Central

    Neves, Aitana; Busso, Coralie; Gönczy, Pierre

    2015-01-01

    All organisms live within a given thermal range, but little is known about the mechanisms setting the limits of this range. We uncovered cellular features exhibiting signature changes at thermal limits in Caenorhabditis elegans embryos. These included changes in embryo size and shape, which were also observed in Caenorhabditis briggsae, indicating evolutionary conservation. We hypothesized that such changes could reflect restricted aerobic capacity at thermal limits. Accordingly, we uncovered that relative respiration in C. elegans embryos decreases at the thermal limits as compared to within the thermal range. Furthermore, by compromising components of the respiratory chain, we demonstrated that the reliance on aerobic metabolism is reduced at thermal limits. Moreover, embryos thus compromised exhibited signature changes in size and shape already within the thermal range. We conclude that restricted aerobic metabolism at the thermal limits contributes to setting the thermal range in a metazoan organism. DOI: http://dx.doi.org/10.7554/eLife.04810.001 PMID:25929283

  11. Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

    PubMed Central

    Gödeke, Julia; Paul, Kristina; Lassak, Jürgen; Thormann, Kai M

    2011-01-01

    Shewanella oneidensis MR-1 is capable of forming highly structured surface-attached communities. By DNase I treatment, we demonstrated that extracellular DNA (eDNA) serves as a structural component in all stages of biofilm formation under static and hydrodynamic conditions. We determined whether eDNA is released through cell lysis mediated by the three prophages LambdaSo, MuSo1 and MuSo2 that are harbored in the genome of S. oneidensis MR-1. Mutant analyses and infection studies revealed that all three prophages may individually lead to cell lysis. However, only LambdaSo and MuSo2 form infectious phage particles. Phage release and cell lysis already occur during early stages of static incubation. A mutant devoid of the prophages was significantly less prone to lysis in pure culture. In addition, the phage-less mutant was severely impaired in biofilm formation through all stages of development, and three-dimensional growth occurred independently of eDNA as a structural component. Thus, we suggest that in S. oneidensis MR-1 prophage-mediated lysis results in the release of crucial biofilm-promoting factors, in particular eDNA. PMID:20962878

  12. Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

    SciTech Connect

    Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.; Blow, Matthew; Korlach, Jonas; Deutschbauer, Adam; Malmstrom, Rex

    2013-08-30

    We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns. However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.

  13. Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

    PubMed Central

    Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.; Blow, Matthew; Korlach, Jonas; Deutschbauer, Adam

    2013-01-01

    We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication. PMID:23995632

  14. The Shewanella oneidensis MR-1 Fluxome under Various OxygenConditions

    SciTech Connect

    Tang, Yinjie J.; Hwang, Judy S.; Wemmer, David E.; Keasling, Jay D.

    2006-03-17

    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.

  15. Metabolic Differences in Microbial Cell Populations Revealed by Nanophotonic Ionization

    SciTech Connect

    Walker, Bennett; Antonakos, Cory; Retterer, Scott T; Vertes, Akos

    2013-01-01

    ellular differences are linked to cell differentiation, the proliferation of cancer and to the development of drug resistance in microbial infections. Due to sensitivity limitations, however, large- scale metabolic analysis at the single cell level is only available for cells significantly larger in volume than Saccharomyces cerevisiae (~30 fL). Here we demonstrate that by a nanophotonic ionization platform and mass spectrometry, over one hundred up to 108 metabolites, or up to 18% of the known S. cerevisiae metabolome, can be identified in very small cell populations (n < 100). Under ideal conditions, r Relative quantitation of up to 4% of the metabolites is achieved at the single cell level.

  16. Multidimensional Profiling Platforms Reveal Metabolic Dysregulation caused by Organophosphorus Pesticides

    PubMed Central

    Medina-Cleghorn, Daniel; Heslin, Ann; Morris, Patrick J.; Mulvihill, Melinda M.; Nomura, Daniel K.

    2014-01-01

    We are environmentally exposed to countless synthetic chemicals on a daily basis with an increasing number of these chemical exposures linked to adverse health effects. However, our understanding of the (patho)physiological effects of these chemicals remains poorly understood, due in-part to a general lack of effort to systematically and comprehensively identify the direct interactions of environmental chemicals with biological macromolecules in mammalian systems in vivo. Here, we have used functional chemoproteomic and metabolomic platforms to broadly identify direct enzyme targets that are inhibited by widely used organophosphorus (OP) pesticides in vivo in mice and to determine metabolic alterations that are caused by these chemicals. We find that these pesticides directly inhibit over 20 serine hydrolases in vivo leading to widespread disruptions in lipid metabolism. Through identifying direct biological targets of OP pesticides, we show heretofore unrecognized modes of toxicity that may be associated with these agents and underscore the utility of utilizing multidimensional profiling approaches to obtain a more complete understanding of toxicities associated with environmental chemicals. PMID:24205821

  17. Metaproteomic analysis reveals microbial metabolic activities in the deep ocean

    NASA Astrophysics Data System (ADS)

    Wang, Da-Zhi; Xie, Zhang-Xian; Zhang, Shu-Feng; Wang, Ming-Hua; Zhang, Hao; Kong, Ling-Fen; Lin, Lin

    2016-04-01

    The deep sea is the largest habitat on earth and holds many and varied microbial life forms. However, little is known about their metabolic activities in the deep ocean. Here, we characterized protein profiles of particulate (>0.22 μm) and dissolved (between 10 kDa and 0.22 μm) fractions collected from the deep South China Sea using a shotgun proteomic approach. SAR324, Alteromonadales and SAR11 were the most abundant groups, while Prasinophyte contributed most to eukaryotes and cyanophage to viruses. The dominant heterotrophic activity was evidenced by the abundant transporters (33%). Proteins participating in nitrification, methanogenesis, methyltrophy and CO2 fixation were detected. Notably, the predominance of unique cellular proteins in dissolved fraction suggested the presence of membrane structures. Moreover, the detection of translation proteins related to phytoplankton indicated that other process rather than sinking particles might be the downward export of living cells. Our study implied that novel extracellular activities and the interaction of deep water with its overlying water could be crucial to the microbial world of deep sea.

  18. Biotransformation and biomethylation of arsenic by Shewanella oneidensis MR-1.

    PubMed

    Wang, Juan; Wu, Mingyin; Lu, Gan; Si, Youbin

    2016-02-01

    The resistance of Shewanella oneidensis MR-1 to toxic arsenic was investigated by measuring the growth of the bacteria in the presence of As(III) and As(V) in different growth media. The bacteria were shown to biotransform arsenic through the partial methylation of inorganic arsenic into methylated metabolites. This biotransformation of inorganic arsenic by S. oneidensis MR-1 was affected by the methyl donor, the composition of the medium, and the presence of Fe(III). The relative content of methylated arsenic in the medium containing S-adenosyl methionine as the methyl donor was greater than that in the medium containing methylcobalamin. The biotransformation process driven by Fe-reducing bacteria, and occurred in combination with microbially mediated As-Fe reduction in the presence of Fe(III). The results demonstrate that S. oneidensis MR-1 methylates inorganic arsenic into less toxic organoarsenic compounds. This process has potential applications in the bioremediation of environmental arsenic, and the results provide new insights into the control of in situ arsenic pollution. PMID:26692509

  19. Functional Specificity of Extracellular Nucleases of Shewanella oneidensis MR-1

    PubMed Central

    Heun, Magnus; Binnenkade, Lucas; Kreienbaum, Maximilian

    2012-01-01

    Bacterial species such as Shewanella oneidensis MR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases of S. oneidensis MR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease of Shewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg2+ or Mn2+) were required for function. endA is cotranscribed with phoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion of endA abolished both extracellular degradation of DNA by S. oneidensis MR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM, endA deletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases of S. oneidensis exert specific functions required under different conditions. PMID:22492434

  20. Phenotype-based cell-specific metabolic modeling reveals metabolic liabilities of cancer.

    PubMed

    Yizhak, Keren; Gaude, Edoardo; Le Dévédec, Sylvia; Waldman, Yedael Y; Stein, Gideon Y; van de Water, Bob; Frezza, Christian; Ruppin, Eytan

    2014-01-01

    Utilizing molecular data to derive functional physiological models tailored for specific cancer cells can facilitate the use of individually tailored therapies. To this end we present an approach termed PRIME for generating cell-specific genome-scale metabolic models (GSMMs) based on molecular and phenotypic data. We build >280 models of normal and cancer cell-lines that successfully predict metabolic phenotypes in an individual manner. We utilize this set of cell-specific models to predict drug targets that selectively inhibit cancerous but not normal cell proliferation. The top predicted target, MLYCD, is experimentally validated and the metabolic effects of MLYCD depletion investigated. Furthermore, we tested cell-specific predicted responses to the inhibition of metabolic enzymes, and successfully inferred the prognosis of cancer patients based on their PRIME-derived individual GSMMs. These results lay a computational basis and a counterpart experimental proof of concept for future personalized metabolic modeling applications, enhancing the search for novel selective anticancer therapies. PMID:25415239

  1. Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus

    DOE PAGESBeta

    Payyavula, Raja S.; Tschaplinski, Timothy J.; Jawdy, Sara; Sykes, Robert; Tuskan, Gerald A.; Kalluri, Udaya C.

    2014-10-07

    Background: UDP-glucose pyrophopharylase (UGPase) is a sugar metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate glucose. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in woody plants such as Populus is poorly understood. Results: We characterized the functional role of UGPase in Populus deltoides by overexpressing a native gene. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of UGPase results in perturbations inmore » primary as well as secondary metabolism resulting in reduced sugar and starch levels and increased phenolics such as caffeoyl- and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. Conclusions: These results demonstrate that UGPase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism outside of cell wall biosynthesis of Populus.« less

  2. Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus

    SciTech Connect

    Payyavula, Raja S.; Tschaplinski, Timothy J.; Jawdy, Sara; Sykes, Robert; Tuskan, Gerald A.; Kalluri, Udaya C.

    2014-10-07

    Background: UDP-glucose pyrophopharylase (UGPase) is a sugar metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate glucose. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in woody plants such as Populus is poorly understood. Results: We characterized the functional role of UGPase in Populus deltoides by overexpressing a native gene. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of UGPase results in perturbations in primary as well as secondary metabolism resulting in reduced sugar and starch levels and increased phenolics such as caffeoyl- and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. Conclusions: These results demonstrate that UGPase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism outside of cell wall biosynthesis of Populus.

  3. Phenotype-based cell-specific metabolic modeling reveals metabolic liabilities of cancer

    PubMed Central

    Le Dévédec, Sylvia; Waldman, Yedael Y; Stein, Gideon Y; van de Water, Bob

    2014-01-01

    Utilizing molecular data to derive functional physiological models tailored for specific cancer cells can facilitate the use of individually tailored therapies. To this end we present an approach termed PRIME for generating cell-specific genome-scale metabolic models (GSMMs) based on molecular and phenotypic data. We build >280 models of normal and cancer cell-lines that successfully predict metabolic phenotypes in an individual manner. We utilize this set of cell-specific models to predict drug targets that selectively inhibit cancerous but not normal cell proliferation. The top predicted target, MLYCD, is experimentally validated and the metabolic effects of MLYCD depletion investigated. Furthermore, we tested cell-specific predicted responses to the inhibition of metabolic enzymes, and successfully inferred the prognosis of cancer patients based on their PRIME-derived individual GSMMs. These results lay a computational basis and a counterpart experimental proof of concept for future personalized metabolic modeling applications, enhancing the search for novel selective anticancer therapies. DOI: http://dx.doi.org/10.7554/eLife.03641.001 PMID:25415239

  4. Metabolic Flux Analysis of Shewanella spp. Reveals Evolutionary Robustness in Central Carbon Metabolism

    SciTech Connect

    Tang, Yinjie J.; Martin, Hector Garcia; Dehal, Paramvir S.; Deutschbauer, Adam; Llora, Xavier; Meadows, Adam; Arkin, Adam; Keasling, Jay D.

    2009-08-19

    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 of 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.

  5. Candida milleri species reveals intraspecific genetic and metabolic polymorphisms.

    PubMed

    Vigentini, Ileana; Antoniani, Davide; Roscini, Luca; Comasio, Andrea; Galafassi, Silvia; Picozzi, Claudia; Corte, Laura; Compagno, Concetta; Dal Bello, Fabio; Cardinali, Gianluigi; Foschino, Roberto

    2014-09-01

    Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897(T) and CBS5658(T). Most isolates were ascribed to C. milleri, even if a strong relatedness was confirmed with C. humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2 Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C. milleri species is provided thus representing an important feature for the development of technological applications in bakery industries. PMID:24929720

  6. Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.

    PubMed

    Jha, Abhishek K; Huang, Stanley Ching-Cheng; Sergushichev, Alexey; Lampropoulou, Vicky; Ivanova, Yulia; Loginicheva, Ekaterina; Chmielewski, Karina; Stewart, Kelly M; Ashall, Juliet; Everts, Bart; Pearce, Edward J; Driggers, Edward M; Artyomov, Maxim N

    2015-03-17

    Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes. PMID:25786174

  7. Serum Metabolic Profiling Reveals Altered Metabolic Pathways in Patients with Post-traumatic Cognitive Impairments

    PubMed Central

    Yi, Lunzhao; Shi, Shuting; Wang, Yang; Huang, Wei; Xia, Zi-an; Xing, Zhihua; Peng, Weijun; Wang, Zhe

    2016-01-01

    Cognitive impairment, the leading cause of traumatic brain injury (TBI)-related disability, adversely affects the quality of life of TBI patients, and exacts a personal and economic cost that is difficult to quantify. The underlying pathophysiological mechanism is currently unknown, and an effective treatment of the disease has not yet been identified. This study aimed to advance our understanding of the mechanism of disease pathogenesis; thus, metabolomics based on gas chromatography/mass spectrometry (GC-MS), coupled with multivariate and univariate statistical methods were used to identify potential biomarkers and the associated metabolic pathways of post-TBI cognitive impairment. A biomarker panel consisting of nine serum metabolites (serine, pyroglutamic acid, phenylalanine, galactose, palmitic acid, arachidonic acid, linoleic acid, citric acid, and 2,3,4-trihydroxybutyrate) was identified to be able to discriminate between TBI patients with cognitive impairment, TBI patients without cognitive impairment and healthy controls. Furthermore, associations between these metabolite markers and the metabolism of amino acids, lipids and carbohydrates were identified. In conclusion, our study is the first to identify several serum metabolite markers and investigate the altered metabolic pathway that is associated with post-TBI cognitive impairment. These markers appear to be suitable for further investigation of the disease mechanisms of post-TBI cognitive impairment. PMID:26883691

  8. Integrated analysis of transcript-level regulation of metabolism reveals disease-relevant nodes of the human metabolic network

    PubMed Central

    Galhardo, Mafalda; Sinkkonen, Lasse; Berninger, Philipp; Lin, Jake; Sauter, Thomas; Heinäniemi, Merja

    2014-01-01

    Metabolic diseases and comorbidities represent an ever-growing epidemic where multiple cell types impact tissue homeostasis. Here, the link between the metabolic and gene regulatory networks was studied through experimental and computational analysis. Integrating gene regulation data with a human metabolic network prompted the establishment of an open-sourced web portal, IDARE (Integrated Data Nodes of Regulation), for visualizing various gene-related data in context of metabolic pathways. Motivated by increasing availability of deep sequencing studies, we obtained ChIP-seq data from widely studied human umbilical vein endothelial cells. Interestingly, we found that association of metabolic genes with multiple transcription factors (TFs) enriched disease-associated genes. To demonstrate further extensions enabled by examining these networks together, constraint-based modeling was applied to data from human preadipocyte differentiation. In parallel, data on gene expression, genome-wide ChIP-seq profiles for peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding protein (CEBP) α, liver X receptor (LXR) and H3K4me3 and microRNA target identification for miR-27a, miR-29a and miR-222 were collected. Disease-relevant key nodes, including mitochondrial glycerol-3-phosphate acyltransferase (GPAM), were exposed from metabolic pathways predicted to change activity by focusing on association with multiple regulators. In both cell types, our analysis reveals the convergence of microRNAs and TFs within the branched chain amino acid (BCAA) metabolic pathway, possibly providing an explanation for its downregulation in obese and diabetic conditions. PMID:24198249

  9. Integrated analysis of transcript-level regulation of metabolism reveals disease-relevant nodes of the human metabolic network.

    PubMed

    Galhardo, Mafalda; Sinkkonen, Lasse; Berninger, Philipp; Lin, Jake; Sauter, Thomas; Heinäniemi, Merja

    2014-02-01

    Metabolic diseases and comorbidities represent an ever-growing epidemic where multiple cell types impact tissue homeostasis. Here, the link between the metabolic and gene regulatory networks was studied through experimental and computational analysis. Integrating gene regulation data with a human metabolic network prompted the establishment of an open-sourced web portal, IDARE (Integrated Data Nodes of Regulation), for visualizing various gene-related data in context of metabolic pathways. Motivated by increasing availability of deep sequencing studies, we obtained ChIP-seq data from widely studied human umbilical vein endothelial cells. Interestingly, we found that association of metabolic genes with multiple transcription factors (TFs) enriched disease-associated genes. To demonstrate further extensions enabled by examining these networks together, constraint-based modeling was applied to data from human preadipocyte differentiation. In parallel, data on gene expression, genome-wide ChIP-seq profiles for peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding protein (CEBP) α, liver X receptor (LXR) and H3K4me3 and microRNA target identification for miR-27a, miR-29a and miR-222 were collected. Disease-relevant key nodes, including mitochondrial glycerol-3-phosphate acyltransferase (GPAM), were exposed from metabolic pathways predicted to change activity by focusing on association with multiple regulators. In both cell types, our analysis reveals the convergence of microRNAs and TFs within the branched chain amino acid (BCAA) metabolic pathway, possibly providing an explanation for its downregulation in obese and diabetic conditions. PMID:24198249

  10. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

    PubMed

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R; Fox, Paul L; Gerson, Stanton L; Hoppel, Charles L; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. PMID:26595448

  11. Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells

    PubMed Central

    Armitage, Emily G.; Kotze, Helen L.; Allwood, J. William; Dunn, Warwick B.; Goodacre, Royston; Williams, Kaye J.

    2015-01-01

    Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments. PMID:26508589

  12. Metabolic profiling of somatic tissues from Monochamus alternatus (Coleoptera: Cerambycidae) reveals effects of irradiation on metabolism.

    PubMed

    Qu, Liangjian; Wang, Lijuan; Wang, Qinghua; Wang, Yuzhu; Zhang, Yongan

    2014-01-01

    A high-level of sexual sterility is of importance for the sterile insect technique (SIT). However, the use of high-dose-intensity gamma radiation to induce sterility has negative impacts not only on reproductive cells but also on somatic cells. In this study, we investigated the metabolite differences in somatic tissues between non-irradiated, 20-Gy-irradiated, and 40-Gy-irradiated male Monochamus alternatus, an important vector of the pathogenic nematode, Bursaphelenchus xylophilus, which kills Asian pines. The results showed that metabolite levels changed moderately in the 20-Gy samples but were markedly altered in the 40-Gy samples compared with the non-irradiated samples. Twenty-six and 53 metabolites were disturbed by 20-Gy and 40-Gy radiation, respectively. Thirty-six metabolites were found to be markedly altered in the 40-Gy samples but were not changed significantly in the 20-Gy samples. The comprehensive metabolomic disorders induced by 40-Gy radiation dysregulated six metabolic pathways involved in the life process. The findings presented in this manuscript will contribute to our knowledge of the characteristic metabolic changes associated with gamma-radiation-induced damage to somatic cells and will allow for better exploration of the SIT for the control of this target pest. PMID:24937685

  13. Metabolic Profiling of Somatic Tissues from Monochamus alternatus (Coleoptera: Cerambycidae) Reveals Effects of Irradiation on Metabolism

    PubMed Central

    Qu, Liangjian; Wang, Lijuan; Wang, Qinghua; Wang, Yuzhu; Zhang, Yongan

    2014-01-01

    A high-level of sexual sterility is of importance for the sterile insect technique (SIT). However, the use of high-dose-intensity gamma radiation to induce sterility has negative impacts not only on reproductive cells but also on somatic cells. In this study, we investigated the metabolite differences in somatic tissues between non-irradiated, 20-Gy-irradiated, and 40-Gy-irradiated male Monochamus alternatus, an important vector of the pathogenic nematode, Bursaphelenchus xylophilus, which kills Asian pines. The results showed that metabolite levels changed moderately in the 20-Gy samples but were markedly altered in the 40-Gy samples compared with the non-irradiated samples. Twenty-six and 53 metabolites were disturbed by 20-Gy and 40-Gy radiation, respectively. Thirty-six metabolites were found to be markedly altered in the 40-Gy samples but were not changed significantly in the 20-Gy samples. The comprehensive metabolomic disorders induced by 40-Gy radiation dysregulated six metabolic pathways involved in the life process. The findings presented in this manuscript will contribute to our knowledge of the characteristic metabolic changes associated with gamma-radiation-induced damage to somatic cells and will allow for better exploration of the SIT for the control of this target pest. PMID:24937685

  14. Shewregdb: Database and visualization environment for experimental and predicted regulatory information in Shewanella oneidensis mr-1

    SciTech Connect

    Syed, Mustafa; Karpinets, Tatiana V.; Leuze, Mike; Kora, Guruprasad; Romine, Margaret F.; Uberbacher, Edward

    2009-10-15

    Shewanella oneidensis MR-1 is an important model organism for environmental research as it has an exceptional metabolic and respiratory versatility regulated by a complex regulatory network. We have developed a database to collect experimental and computational data relating to regulation of gene and protein expression and a visualization environment that enables integration of these data types. The regulatory information in the database was collected from the published literature and different Internet resources. It includes predictions of DNA regulator binding sites, sigma factor binding sites, transcription units, operons, promoters, and RNA regulators including non-coding RNAs, riboswitches, and different types of terminators. A visualization environment based on GBrowser was developed for accessing the collected information and for its overlaying with experimental data (experimental results from studies employing microarrays, proteomics, and/or gene mutagenesis) and other genome annotations.

  15. Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT(-/-) mice.

    PubMed

    Stockebrand, Malte; Nejad, Ali Sasani; Neu, Axel; Kharbanda, Kusum K; Sauter, Kathrin; Schillemeit, Stefan; Isbrandt, Dirk; Choe, Chi-Un

    2016-08-01

    Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking L-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT(-/-) (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes. PMID:26940723

  16. Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1

    PubMed Central

    Clark, Iain C.; Melnyk, Ryan A.; Youngblut, Matthew D.; Carlson, Hans K.; Iavarone, Anthony T.

    2015-01-01

    ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. PMID:25991681

  17. Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation

    PubMed Central

    Brown, Ashley R.; Correa, Elon; Xu, Yun; AlMasoud, Najla; Pimblott, Simon M.; Goodacre, Royston; Lloyd, Jonathan R.

    2015-01-01

    Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear. PMID

  18. Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation.

    PubMed

    Brown, Ashley R; Correa, Elon; Xu, Yun; AlMasoud, Najla; Pimblott, Simon M; Goodacre, Royston; Lloyd, Jonathan R

    2015-01-01

    Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear. PMID

  19. Comparative metagenomic analyses reveal viral-induced shifts of host metabolism towards nucleotide biosynthesis

    PubMed Central

    2014-01-01

    Background Viral genomes often contain metabolic genes that were acquired from host genomes (auxiliary genes). It is assumed that these genes are fixed in viral genomes as a result of a selective force, favoring viruses that acquire specific metabolic functions. While many individual auxiliary genes were observed in viral genomes and metagenomes, there is great importance in investigating the abundance of auxiliary genes and metabolic functions in the marine environment towards a better understanding of their role in promoting viral reproduction. Results In this study, we searched for enriched viral auxiliary genes and mapped them to metabolic pathways. To initially identify enriched auxiliary genes, we analyzed metagenomic microbial reads from the Global Ocean Survey (GOS) dataset that were characterized as viral, as well as marine virome and microbiome datasets from the Line Islands. Viral-enriched genes were mapped to a “global metabolism network” that comprises all KEGG metabolic pathways. Our analysis of the viral-enriched pathways revealed that purine and pyrimidine metabolism pathways are among the most enriched pathways. Moreover, many other viral-enriched metabolic pathways were found to be closely associated with the purine and pyrimidine metabolism pathways. Furthermore, we observed that sequential reactions are promoted in pathways having a high proportion of enriched genes. In addition, these enriched genes were found to be of modular nature, participating in several pathways. Conclusions Our naïve metagenomic analyses strongly support the well-established notion that viral auxiliary genes promote viral replication via both degradation of host DNA and RNA as well as a shift of the host metabolism towards nucleotide biosynthesis, clearly indicating that comparative metagenomics can be used to understand different environments and systems without prior knowledge of pathways involved. PMID:24666644

  20. Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

    PubMed Central

    Lee, Su Hyeon; Kim, Eung Ju; Lee, Dong-Hyoung; Lee, Won-Yong; Chung, Bong Chul

    2016-01-01

    Background Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. Methods Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. Results The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. Conclusions Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism. PMID:27139607

  1. Ontogeny of Hepatic Energy Metabolism Genes in Mice as Revealed by RNA-Sequencing

    PubMed Central

    Renaud, Helen J.; Cui, Yue Julia; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D.

    2014-01-01

    The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age). The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5–Day 5 (perinatal-enriched), Day 10–Day 20 (pre-weaning-enriched), and Day 25–Day 60 (adolescence/adulthood-enriched). Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty acids-like 3

  2. Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

    PubMed

    Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2016-06-01

    Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells. PMID:27074779

  3. Adhesion of Shewanella oneidensis MR-1 to Goethite: A Two-Dimensional Correlation Spectroscopic Study.

    PubMed

    Yan, Wei; Wang, Hongbo; Jing, Chuanyong

    2016-04-19

    Bacterial adhesion to mineral surfaces is an important but underappreciated process. To decipher the molecular level process and mechanism, the adhesion of Shewanella oneidensis MR-1 cells to goethite was investigated using flow-cell attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy coupled with two-dimensional correlation spectroscopy (2D-COS) analysis. The FTIR results indicate that bacterial phosphate-moieties play an important role in the formation of mono- and bidentate inner-sphere complexes, whereas carboxylic groups on cell surface only have a minor contribution to its adhesion. The 2D-COS analysis in short-term (0-120 min) and long-term (2-18 h) stages reveal that the adhesion process was in the following sequence: change in H-bonds of proteins on cell surfaces > formation of monodentate inner-sphere surface complexes > formation of outer-sphere surface complexes > transformation of protein secondary structure on cell surfaces > formation of additional bridging bidentate surface complexes. In addition, the adhesion of MR-1 cells on goethite was pH dependent due to pH impacts on the cell structure and the interface charge. The in situ ATR-FTIR integrated with 2D-COS analysis highlights its great potential in exploring complex surface reactions with microbes involved. These results improve our understanding of microbe-mineral interactions at the molecular level and have significant implications for a series of biogeochemical processes. PMID:27029565

  4. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  5. Metabolomics Reveals Dynamic Metabolic Changes Associated with Age in Early Childhood

    PubMed Central

    Chiu, Chih-Yung; Yeh, Kuo-Wei; Lin, Gigin; Chiang, Meng-Han; Yang, Shu-Chen; Chao, Wei-Ju; Yao, Tsung-Chieh; Tsai, Ming-Han; Hua, Man-Chin; Liao, Sui-Ling; Lai, Shen-Hao

    2016-01-01

    Objectives A detailed understanding of the metabolic processes governing rapid growth in early life is still lacking. The aim of this study was to investigate the age-related metabolic changes in healthy children throughout early childhood. Methods Healthy children from a birth cohort were enrolled in this study from birth through 4 years of age. Urinary metabolites were assessed at 6 months, and 1, 2, 3, and 4 yr of age by using 1H-nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical analysis including principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Metabolic pathway analysis was performed using the MetPA web tool. Results A total of 105 urine samples from 30 healthy children were collected and analyzed. Metabolites contributing to the discrimination between age groups were identified by using supervised PLS-DA (Q2 = 0.60; R2 = 0.66). A significantly higher urinary trimethylamine N-oxide (TMAO) and betaine level was found in children aged 6 months. Urinary glycine and glutamine levels declined significantly after 6 months of age and there was a concomitant compensatory increase in urinary creatine and creatinine. Metabolic pathway analysis using MetPA revealed similar nitrogen metabolism associated energy production across all ages assessed. Pathways associated with amino acid metabolism were significantly different between infants aged 6 months and 1 year, whereas pathways associated with carbohydrate metabolism were significantly different between children at ages 2 and 3 years. Conclusions Urine metabolomics ideally represents dynamic metabolic changes across age. Urinary metabolic profiles change significantly within the first year of life, which can potentially provide crucial information about infant nutrition and growth. PMID:26914934

  6. An integrated multi-omics study revealed metabolic alterations underlying the effects of coffee consumption.

    PubMed

    Takahashi, Shoko; Saito, Kenji; Jia, Huijuan; Kato, Hisanori

    2014-01-01

    Many epidemiological studies have indicated that coffee consumption may reduce the risks of developing obesity and diabetes, but the underlying mechanisms of these effects are poorly understood. Our previous study revealed the changes on gene expression profiles in the livers of C57BL/6J mice fed a high-fat diet containing three types of coffee (caffeinated, decaffeinated and green unroasted coffee), using DNA microarrays. The results revealed remarkable alterations in lipid metabolism-related molecules which may be involved in the anti-obesity effects of coffee. We conducted the present study to further elucidate the metabolic alterations underlying the effects of coffee consumption through comprehensive proteomic and metabolomic analyses. Proteomics revealed an up-regulation of isocitrate dehydrogenase (a key enzyme in the TCA cycle) and its related proteins, suggesting increased energy generation. The metabolomics showed an up-regulation of metabolites involved in the urea cycle, with which the transcriptome data were highly consistent, indicating accelerated energy expenditure. The TCA cycle and the urea cycle are likely be accelerated in a concerted manner, since they are directly connected by mutually providing each other's intermediates. The up-regulation of these pathways might result in a metabolic shift causing increased ATP turnover, which is related to the alterations of lipid metabolism. This mechanism may play an important part in the suppressive effects of coffee consumption on obesity, inflammation, and hepatosteatosis. This study newly revealed global metabolic alterations induced by coffee intake, providing significant insights into the association between coffee intake and the prevention of type 2 diabetes, utilizing the benefits of multi-omics analyses. PMID:24618914

  7. Global Profiling of Shewanella oneidensis MR-1: Expression of Hypothetical Genes and Improved functional annotations

    SciTech Connect

    Kolker, Eugene; Picone, Alessandro F.; Galperin, Michael Y.; Romine, Margaret F.; Higdon, Roger; Makarova, Kira S.; Kolker, Natali; Anderson, Gordon A.; Qiu, Xiaoyun; Auberry, Kenneth J.; Babnigg, Gyorgy; Beliaev, Alex S.; Edlefsen, Paul; Elias, Dwayne A.; Gorby, Yuri A.; Holzman, Ted; Klappenbach, Joel; Konstantinidis, Kostas; Land, Miriam L.; Lipton, Mary S.; McCue, Lee-Ann; Monroe, Matthew E.; Pasa-Tolic, Liljiana; Pinchuk, Grigoriy E.; Purvine, Samuel O.; Serres, Margaret; Tsapin, Sasha; Zakrajsek, Brian A.; Zhu, Wenhong; Zhou, Jizhong; Larimer, Frank; Lawrence, Charles; Riley, Monica; Collart, Frank R.; Yates, III, John R.; Smith, Richard D.; Giometti, Carol S.; Nealson, Kenneth; Fredrickson, Jim K.; Tiedje, James M.

    2005-02-08

    The y-proteobacterium Shewanella oneidensis strain MR-1 is a respiratory versatile organism that can reduce a wide range of organics, metals, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the MR-1 genome were annotated as uncharacterized ''hypothetical'' genes. We implemented an integrative approach using experimental and computational analyses to provide more detailed insight into their function. Global expression studies were conducted using RNA and protein expression profiling of cells cultivated under aerobic, suboxic, and fumarate reducing conditions, phosphate limitation and UV irradiation. transcriptomic and proteomic analyses confidently identified 538 ''hypothetical'' genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all ''hypothetical'' proteins). Publicly available analysis tools and databases and our own expression data were applied to improve the annotation of these genes. The annotation results were scored using a seven-category schema that ranked both confidence and precision of the functional assignment. We identified homologs for nearly all of these ''hypothetical'' proteins (96%), thus allowing us to minimally classify them as ''conserved proteins''. Computational and/or experimental evidence provided more precise functional assignments for 297 genes (categories 1-4; 55%). These improved functional annotations will significantly widen our understanding of vital cellular processes including signal transduction, ion transport, secondary metabolism, and transcription, as well as structural elements, such as cellular membranes. We propose that this integrative approach offers a viable means to undertake the enormous challenge of characterizing the rapidly growing number of ''hypothetical'' proteins with each newly sequenced genome.

  8. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response

    PubMed Central

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R.; Fox, Paul L.; Gerson, Stanton L.; Hoppel, Charles L.; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. DOI: http://dx.doi.org/10.7554/eLife.10067.001 PMID:26595448

  9. Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes.

    PubMed

    Lu, Shuhuan; Wang, Jiangxin; Niu, Yanhong; Yang, Jie; Zhou, Jian; Yuan, Yingjin

    2012-07-01

    Inoculum size strongly affects cell growth and lipid accumulation of microalgae, one of the most potential biodiesel feedstock, however, the metabolic mechanism(s) of the lipid biosynthesis upon inoculum size has not been fully explored yet. The effects of inoculum size on cell growth, lipid accumulation, and metabolic changes of a green microalga Chlorella sorokiniana were investigated. In our experimental range of inoculum size, the productivity and the cetane number (CN) of fatty acid methyl esters (FAME) increased with increasing initial cell density, and the inoculum of 1 × 10(7) cells mL(-1) processed much higher productivity (up to 2.02-fold) and CN (up to 1.19-fold) of the FAME than the others. A significant correlation between the metabolic profile and quantity and quality of lipid production was revealed by partial least-squares to latent structures (PLS) analysis, and 15 key metabolites were identified. Most of those metabolites were involved in the photosynthetically fixed carbon metabolism. Furthermore, light intensity as one of the vital limitation factors for the high inoculum size cultivation was evaluated by illumination assay and the results revealed that increasing light intensity could improve the polyunsaturated fatty acids composition and lipid accumulation of C. sorokiniana. The lipid productivity of the culture was improved by 71.21% with the light intensity of 110 µmol m(-2) s(-1), compared to that under the irradiance of 65 µmol m(-2) s(-1). PMID:22252441

  10. A Balanced Tissue Composition Reveals New Metabolic and Gene Expression Markers in Prostate Cancer

    PubMed Central

    Tessem, May-Britt; Bertilsson, Helena; Angelsen, Anders; Bathen, Tone F.; Drabløs, Finn; Rye, Morten Beck

    2016-01-01

    Molecular analysis of patient tissue samples is essential to characterize the in vivo variability in human cancers which are not accessible in cell-lines or animal models. This applies particularly to studies of tumor metabolism. The challenge is, however, the complex mixture of various tissue types within each sample, such as benign epithelium, stroma and cancer tissue, which can introduce systematic biases when cancers are compared to normal samples. In this study we apply a simple strategy to remove such biases using sample selections where the average content of stroma tissue is balanced between the sample groups. The strategy is applied to a prostate cancer patient cohort where data from MR spectroscopy and gene expression have been collected from and integrated on the exact same tissue samples. We reveal in vivo changes in cancer-relevant metabolic pathways which are otherwise hidden in the data due to tissue confounding. In particular, lowered levels of putrescine are connected to increased expression of SRM, reduced levels of citrate are attributed to upregulation of genes promoting fatty acid synthesis, and increased succinate levels coincide with reduced expression of SUCLA2 and SDHD. In addition, the strategy also highlights important metabolic differences between the stroma, epithelium and prostate cancer. These results show that important in vivo metabolic features of cancer can be revealed from patient data only if the heterogeneous tissue composition is properly accounted for in the analysis. PMID:27100877

  11. A Balanced Tissue Composition Reveals New Metabolic and Gene Expression Markers in Prostate Cancer.

    PubMed

    Tessem, May-Britt; Bertilsson, Helena; Angelsen, Anders; Bathen, Tone F; Drabløs, Finn; Rye, Morten Beck

    2016-01-01

    Molecular analysis of patient tissue samples is essential to characterize the in vivo variability in human cancers which are not accessible in cell-lines or animal models. This applies particularly to studies of tumor metabolism. The challenge is, however, the complex mixture of various tissue types within each sample, such as benign epithelium, stroma and cancer tissue, which can introduce systematic biases when cancers are compared to normal samples. In this study we apply a simple strategy to remove such biases using sample selections where the average content of stroma tissue is balanced between the sample groups. The strategy is applied to a prostate cancer patient cohort where data from MR spectroscopy and gene expression have been collected from and integrated on the exact same tissue samples. We reveal in vivo changes in cancer-relevant metabolic pathways which are otherwise hidden in the data due to tissue confounding. In particular, lowered levels of putrescine are connected to increased expression of SRM, reduced levels of citrate are attributed to upregulation of genes promoting fatty acid synthesis, and increased succinate levels coincide with reduced expression of SUCLA2 and SDHD. In addition, the strategy also highlights important metabolic differences between the stroma, epithelium and prostate cancer. These results show that important in vivo metabolic features of cancer can be revealed from patient data only if the heterogeneous tissue composition is properly accounted for in the analysis. PMID:27100877

  12. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

    PubMed Central

    Wang, Lijie; Zhang, Tong; Watson, David G.; Silva, Ana Marta; Coombs, Graham H.

    2015-01-01

    Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts. PMID:26368322

  13. Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

    SciTech Connect

    Picone, Alex F.; Galperin, Michael Y.; Romine, Margaret; Higdon, Roger; Makarova, Kira S.; Kolker, Natali; Anderson, Gordon A; Qiu, Xiaoyun; Babnigg, Gyorgy; Beliaev, Alexander S; Edlefsen, Paul; Elias, Dwayne A.; Gorby, Dr. Yuri A.; Holzman, Ted; Klappenbach, Joel; Konstantinidis, Konstantinos T; Land, Miriam L; Lipton, Mary S.; McCue, Lee Ann; Monroe, Matthew; Pasa-Tolic, Ljiljana; Pinchuk, Grigoriy; Purvine, Samuel; Serres, Margrethe H.; Tsapin, Sasha; Zakrajsek, Brian A.; Zhu, Wenguang; Zhou, Jizhong; Larimer, Frank W; Lawrence, Charles E.; Riley, Monica; Collart, Frank; YatesIII, John R.; Smith, Richard D.; Nealson, Kenneth H.; Fredrickson, James K; Tiedje, James M.

    2005-01-01

    The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximate to40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.

  14. Integrated metabolomics and transcriptomics reveal enhanced specialized metabolism in Medicago truncatula root border cells.

    PubMed

    Watson, Bonnie S; Bedair, Mohamed F; Urbanczyk-Wochniak, Ewa; Huhman, David V; Yang, Dong Sik; Allen, Stacy N; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W

    2015-04-01

    Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4'-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4'-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

  15. Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago truncatula Root Border Cells1[OPEN

    PubMed Central

    Watson, Bonnie S.; Bedair, Mohamed F.; Urbanczyk-Wochniak, Ewa; Huhman, David V.; Yang, Dong Sik; Allen, Stacy N.; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W.

    2015-01-01

    Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4′-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4′-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

  16. Metabolic profiling of a range of peach fruit varieties reveals high metabolic diversity and commonalities and differences during ripening.

    PubMed

    Monti, Laura L; Bustamante, Claudia A; Osorio, Sonia; Gabilondo, Julieta; Borsani, Julia; Lauxmann, Martin A; Maulión, Evangelina; Valentini, Gabriel; Budde, Claudio O; Fernie, Alisdair R; Lara, María V; Drincovich, María F

    2016-01-01

    Peach (Prunus persica) fruits from different varieties display differential organoleptic and nutritional properties, characteristics related to their chemical composition. Here, chemical biodiversity of peach fruits from fifteen varieties, at harvest and after post-harvest ripening, was explored by gas chromatography-mass spectrometry. Metabolic profiling revealed that metabolites involved in organoleptic properties (sugars, organic and amino acids), stress tolerance (raffinose, galactinol, maltitol), and with nutritional properties (amino, caffeoylquinic and dehydroascorbic acids) displayed variety-dependent levels. Peach varieties clustered into four groups: two groups of early-harvest varieties with higher amino acid levels; two groups of mid- and late-harvest varieties with higher maltose levels. Further separation was mostly dependent on organic acids/raffinose levels. Variety-dependent and independent metabolic changes associated with ripening were detected; which contribute to chemical diversity or can be used as ripening markers, respectively. The great variety-dependent diversity in the content of metabolites that define fruit quality reinforces metabolomics usage as a tool to assist fruit quality improvement in peach. PMID:26213052

  17. Autofluorescence Imaging of Living Pancreatic Islets Reveals Fibroblast Growth Factor-21 (FGF21)-Induced Metabolism

    PubMed Central

    Sun, Mark Y.; Yoo, Eunjong; Green, Brenda J.; Altamentova, Svetlana M.; Kilkenny, Dawn M.; Rocheleau, Jonathan V.

    2012-01-01

    Fibroblast growth factor-21 (FGF21) has therapeutic potential for metabolic syndrome due to positive effects on fatty acid metabolism in liver and white adipose tissue. FGF21 also improves pancreatic islet survival in excess palmitate; however, much less is known about FGF21-induced metabolism in this tissue. We first confirmed FGF21-dependent activity in islets by identifying expression of the cognate coreceptor Klothoβ, and by measuring a ligand-stimulated decrease in acetyl-CoA carboxylase expression. To further reveal the effect of FGF21 on metabolism, we employed a unique combination of two-photon and confocal autofluorescence imaging of the NAD(P)H and mitochondrial NADH responses while holding living islets stationary in a microfluidic device. These responses were further correlated to mitochondrial membrane potential and insulin secretion. Glucose-stimulated responses were relatively unchanged by FGF21. In contrast, responses to glucose in the presence of palmitate were significantly reduced compared to controls showing diminished NAD(P)H, mitochondrial NADH, mitochondrial membrane potential, and insulin secretion. Consistent with the glucose-stimulated responses being smaller due to continued fatty acid oxidation, mitochondrial membrane potential was increased in FGF21-treated islets by using the fatty acid transport inhibitor etomoxir. Citrate-stimulated NADPH responses were also significantly larger in FGF21-treated islets suggesting preference for citrate cycling rather than acetyl-CoA carboxylase-dependent fatty acid synthesis. Overall, these data show a reduction in palmitate-induced potentiation of glucose-stimulated metabolism and insulin secretion in FGF21-treated islets, and establish the use of autofluorescence imaging and microfluidic devices to investigate cell metabolism in a limited amount of living tissue. PMID:23283237

  18. 'Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity.

    PubMed

    McIlroy, Simon J; Albertsen, Mads; Andresen, Eva K; Saunders, Aaron M; Kristiansen, Rikke; Stokholm-Bjerregaard, Mikkel; Nielsen, Kåre L; Nielsen, Per H

    2014-03-01

    The glycogen-accumulating organism (GAO) 'Candidatus Competibacter' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-'feast': aerobic-'famine' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, 'Candidatus Competibacter denitrificans' and 'Candidatus Contendobacter odensis', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden-Meyerhof-Parnas and Entner-Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes--identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems. PMID:24173461

  19. Metabolic characteristics of a glycogen-accumulating organism in Defluviicoccus cluster II revealed by comparative genomics.

    PubMed

    Wang, Zhiping; Guo, Feng; Mao, Yanping; Xia, Yu; Zhang, Tong

    2014-11-01

    Glycogen-accumulating organisms (GAOs) may compete with phosphate-accumulating organisms (PAOs) for short-chain fatty acids (VFAs) in anaerobic polyhydroxyalkanoates (PHA) synthesis, but no consequently aerobic polyphosphate accumulation in enhanced biological phosphorus removal (EBPR) process, thus deteriorating the EBPR process. They are detected frequently in the deteriorated EBPR process, but their metabolisms are still far from our comprehensions for there is seldom pure culture. In this study, a nearly complete draft genome of a GAOs in Defluviicoccus cluster II, GAO-HK, is recruited from the metagenome of activated sludge in a full-scale industrial anoxic/aerobic wastewater plant. Comparative genomics reveal similar metabolisms of PHA and glycogen in GAOs of GAO-HK, Defluviicoccus tetraformis TFO71 (TFO71) and Competibacter phosphatis clade IIA (CPIIA), and PAOs of Accumulibacter clade IIA UW-1 (UW-1) and Tetrasphaera elongata Lp2 (Lp2). Although there are similar gene cassettes related with polyphosphate metabolism in these GAOs and PAOs, especially for Defluviicoccus-relative bacteria and UW-1, ppk1 in GAOs are diverse from those in the identified PAOs, implying the difference of polyphosphate metabolism in GAOs and PAOs. Additionally, genes related to the dissimilatory denitrification are absent in TFO71 and GAO-HK, implying that additional nitrate or nitrite may favor PAOs over Defluviicoccus-relative GAOs. Therefore, PAOs suffering from competition of Defluviicoccus-relative GAOs might be rescued with the additional nitrate/nitrite, which is important to improve the stability of EBPR processes. PMID:24889288

  20. Metabolic Multianalyte Microphysiometry Reveals Extracellular Acidosis is an Essential Mediator of Neuronal Preconditioning

    PubMed Central

    2012-01-01

    Metabolic adaptation to stress is a crucial yet poorly understood phenomenon, particularly in the central nervous system (CNS). The ability to identify essential metabolic events which predict neuronal fate in response to injury is critical to developing predictive markers of outcome, for interpreting CNS spectroscopic imaging, and for providing a richer understanding of the relevance of clinical indices of stress which are routinely collected. In this work, real-time multianalyte microphysiometry was used to dynamically assess multiple markers of aerobic and anaerobic respiration through simultaneous electrochemical measurement of extracellular glucose, lactate, oxygen, and acid. Pure neuronal cultures and mixed cultures of neurons and glia were compared following a 90 min exposure to aglycemia. This stress was cytotoxic to neurons yet resulted in no appreciable increase in cell death in age-matched mixed cultures. The metabolic profile of the cultures was similar in that aglycemia resulted in decreases in extracellular acidification and lactate release in both pure neurons and mixed cultures. However, oxygen consumption was only diminished in the neuron enriched cultures. The differences became more pronounced when cells were returned to glucose-containing media upon which extracellular acidification and oxygen consumption never returned to baseline in cells fated to die. Taken together, these data suggest that lactate release is not predictive of neuronal survival. Moreover, they reveal a previously unappreciated relationship of astrocytes in maintaining oxygen uptake and a correlation between metabolic recovery of neurons and extracellular acidification. PMID:22860220

  1. Link between Phosphate Starvation and Glycogen Metabolism in Corynebacterium glutamicum, Revealed by Metabolomics▿ †

    PubMed Central

    Woo, Han Min; Noack, Stephan; Seibold, Gerd M.; Willbold, Sabine; Eikmanns, Bernhard J.; Bott, Michael

    2010-01-01

    In this study, we analyzed the influence of phosphate (Pi) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under Pi limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under Pi excess, the glycogen level in Pi-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under Pi excess, did so under Pi limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under Pi limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited Pi concentrations. PMID:20802079

  2. ‘Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

    PubMed Central

    McIlroy, Simon J; Albertsen, Mads; Andresen, Eva K; Saunders, Aaron M; Kristiansen, Rikke; Stokholm-Bjerregaard, Mikkel; Nielsen, Kåre L; Nielsen, Per H

    2014-01-01

    The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast': aerobic-‘famine' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans' and ‘Candidatus Contendobacter odensis', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems. PMID:24173461

  3. Metabolic multianalyte microphysiometry reveals extracellular acidosis is an essential mediator of neuronal preconditioning.

    PubMed

    McKenzie, Jennifer R; Palubinsky, Amy M; Brown, Jacquelynn E; McLaughlin, Bethann; Cliffel, David E

    2012-07-18

    Metabolic adaptation to stress is a crucial yet poorly understood phenomenon, particularly in the central nervous system (CNS). The ability to identify essential metabolic events which predict neuronal fate in response to injury is critical to developing predictive markers of outcome, for interpreting CNS spectroscopic imaging, and for providing a richer understanding of the relevance of clinical indices of stress which are routinely collected. In this work, real-time multianalyte microphysiometry was used to dynamically assess multiple markers of aerobic and anaerobic respiration through simultaneous electrochemical measurement of extracellular glucose, lactate, oxygen, and acid. Pure neuronal cultures and mixed cultures of neurons and glia were compared following a 90 min exposure to aglycemia. This stress was cytotoxic to neurons yet resulted in no appreciable increase in cell death in age-matched mixed cultures. The metabolic profile of the cultures was similar in that aglycemia resulted in decreases in extracellular acidification and lactate release in both pure neurons and mixed cultures. However, oxygen consumption was only diminished in the neuron enriched cultures. The differences became more pronounced when cells were returned to glucose-containing media upon which extracellular acidification and oxygen consumption never returned to baseline in cells fated to die. Taken together, these data suggest that lactate release is not predictive of neuronal survival. Moreover, they reveal a previously unappreciated relationship of astrocytes in maintaining oxygen uptake and a correlation between metabolic recovery of neurons and extracellular acidification. PMID:22860220

  4. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  5. Integrated Analysis of Metabolite and Transcript Levels Reveals the Metabolic Shifts That Underlie Tomato Fruit Development and Highlight Regulatory Aspects of Metabolic Network Behavior1[W

    PubMed Central

    Carrari, Fernando; Baxter, Charles; Usadel, Björn; Urbanczyk-Wochniak, Ewa; Zanor, Maria-Ines; Nunes-Nesi, Adriano; Nikiforova, Victoria; Centero, Danilo; Ratzka, Antje; Pauly, Markus; Sweetlove, Lee J.; Fernie, Alisdair R.

    2006-01-01

    Tomato (Solanum lycopersicum) is a well-studied model of fleshy fruit development and ripening. Tomato fruit development is well understood from a hormonal-regulatory perspective, and developmental changes in pigment and cell wall metabolism are also well characterized. However, more general aspects of metabolic change during fruit development have not been studied despite the importance of metabolism in the context of final composition of the ripe fruit. In this study, we quantified the abundance of a broad range of metabolites by gas chromatography-mass spectrometry, analyzed a number of the principal metabolic fluxes, and in parallel analyzed transcriptomic changes during tomato fruit development. Metabolic profiling revealed pronounced shifts in the abundance of metabolites of both primary and secondary metabolism during development. The metabolite changes were reflected in the flux analysis that revealed a general decrease in metabolic activity during ripening. However, there were several distinct patterns of metabolite profile, and statistical analysis demonstrated that metabolites in the same (or closely related) pathways changed in abundance in a coordinated manner, indicating a tight regulation of metabolic activity. The metabolite data alone allowed investigations of likely routes through the metabolic network, and, as an example, we analyze the operational feasibility of different pathways of ascorbate synthesis. When combined with the transcriptomic data, several aspects of the regulation of metabolism during fruit ripening were revealed. First, it was apparent that transcript abundance was less strictly coordinated by functional group than metabolite abundance, suggesting that posttranslational mechanisms dominate metabolic regulation. Nevertheless, there were some correlations between specific transcripts and metabolites, and several novel associations were identified that could provide potential targets for manipulation of fruit compositional traits

  6. Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis.

    PubMed

    Xiang, Daoquan; Venglat, Prakash; Tibiche, Chabane; Yang, Hui; Risseeuw, Eddy; Cao, Yongguo; Babic, Vivijan; Cloutier, Mathieu; Keller, Wilf; Wang, Edwin; Selvaraj, Gopalan; Datla, Raju

    2011-05-01

    Embryogenesis is central to the life cycle of most plant species. Despite its importance, because of the difficulty associated with embryo isolation, global gene expression programs involved in plant embryogenesis, especially the early events following fertilization, are largely unknown. To address this gap, we have developed methods to isolate whole live Arabidopsis (Arabidopsis thaliana) embryos as young as zygote and performed genome-wide profiling of gene expression. These studies revealed insights into patterns of gene expression relating to: maternal and paternal contributions to zygote development, chromosomal level clustering of temporal expression in embryogenesis, and embryo-specific functions. Functional analysis of some of the modulated transcription factor encoding genes from our data sets confirmed that they are critical for embryogenesis. Furthermore, we constructed stage-specific metabolic networks mapped with differentially regulated genes by combining the microarray data with the available Kyoto Encyclopedia of Genes and Genomes metabolic data sets. Comparative analysis of these networks revealed the network-associated structural and topological features, pathway interactions, and gene expression with reference to the metabolic activities during embryogenesis. Together, these studies have generated comprehensive gene expression data sets for embryo development in Arabidopsis and may serve as an important foundational resource for other seed plants. PMID:21402797

  7. [Complex metabolic disorders revealing a gastric ulcer of the bulb. A case report].

    PubMed

    Neffati, F; Hellara, I; Jelizi, M A; Bahri, J; Douki, W; Amor, A Ben; Najjar, M F

    2009-01-01

    We report the case of a 54-year-old man, without particular pathological antecedents admitted to the emergency of the university hospital of Monastir, for right renal colic. Radiography of the urinary tract without preparation and renal echography showed bilateral renal lithiasis and a right ureteral lithiasis. The interrogation revealed concept of vomiting after which the patient felt relieved. The biological assessment objectified an hypochloremic metabolic alcalosis, an increase in the anion gap, a severe impaired renal function of obstructive origin and an hypokaliemia. The presence of the lithiasis did not explain on its own the metabolic disorders of this patient. The other investigations showed that initial pathology was an evolutionary bulb ulcer into pre-stenosis justifying treatment by omeprazole and explaining the biological disorders. PMID:19654086

  8. Metatranscriptome Analysis of Aquifer Samples Reveals Unexpected Metabolic Lifestyles Relevant to Active Biogeochemical Cycling

    NASA Astrophysics Data System (ADS)

    Beller, H. R.; Jewell, T. N. M.; Karaoz, U.; Banfield, J. F.; Brodie, E.; Williams, K. H.

    2015-12-01

    Modern molecular ecology techniques are revealing the metabolic potential of uncultivated microorganisms, but there is still much to be learned about the actual biogeochemical roles of microbes that have cultivated relatives. Here, we present metatranscriptomic and metagenomic data from a field study that provides evidence of coupled redox processes that have not been documented in cultivated relatives and, indeed, represent strains with metabolic traits that are novel with respect to closely related isolates. The data come from omics analysis of groundwater samples collected during an experiment in which nitrate (a native electron acceptor) was injected into a perennially suboxic aquifer in Rifle (CO). Transcriptional data indicated that just two groups of chemolithoautotrophic bacteria accounted for a very large portion (~80%) of overall community gene expression: (1) members of the Fe(II)-oxidizing Gallionellaceae family and (2) strains of the S-oxidizing species, Sulfurimonas denitrificans. Metabolic lifestyles for Gallionellaceae strains that were novel compared to cultivated representatives included nitrate-dependent Fe(II) oxidation and S oxidation. Evidence for these metabolisms included highly correlated temporal expression in binned data of nitrate reductase (e.g., narGHI) genes (which have never been reported in Gallionellaceae genomes) and Fe(II) oxidation genes (e.g., mtoA) or S oxidation genes (e.g., dsrE, aprA). Of the two most active strains of S. denitrificans, only one showed strong expression of S oxidation genes, whereas the other was apparently using an unexpected (as-yet unidentified) primary electron donor. Transcriptional data added considerable interpretive value to this study, as (1) metagenomic data would not have highlighted these organisms, which had a disproportionately large role in community metabolism relative to their populations, and (2) co-expression of coupled pathway genes could not be predicted based solely on metagenomic data.

  9. Global transcriptomic analysis of Cyanothece 51142 reveals robust diurnal oscillation of central metabolic processes

    SciTech Connect

    Stockel, Jana; Welsh, Eric A.; Liberton, Michelle L.; Kunnavakkam, Rangesh V.; Aurora, Rajeev; Pakrasi, Himadri B.

    2008-04-22

    Cyanobacteria are oxygenic photosynthetic organisms, and the only prokaryotes known to have a circadian cycle. Unicellular diazotrophic cyanobacteria such as Cyanothece 51142 can fix atmospheric nitrogen, a process exquisitely sensitive to oxygen. Thus, the intracellular environment of Cyanothece oscillates between aerobic and anaerobic conditions during a day-night cycle. This is accomplished by temporal separation of two processes: photosynthesis during the day, and nitrogen fixation at night. While previous studies have examined periodic changes transcript levels for a limited number of genes in Cyanothece and other unicellular diazotrophic cyanobacteria, a comprehensive study of transcriptional activity in a nitrogen-fixing cyanobacterium is necessary to understand the impact of the temporal separation of photosynthesis and nitrogen fixation on global gene regulation and cellular metabolism. We have examined the expression patterns of nearly 5000 genes in Cyanothece 51142 during two consecutive diurnal periods. We found that ~30% of these genes exhibited robust oscillating expression profiles. Interestingly, this set included genes for almost all central metabolic processes in Cyanothece. A transcriptional network of all genes with significantly oscillating transcript levels revealed that the majority of genes in numerous individual pathways, such as glycolysis, pentose phosphate pathway and glycogen metabolism, were co-regulated and maximally expressed at distinct phases during the diurnal cycle. Our analyses suggest that the demands of nitrogen fixation greatly influence major metabolic activities inside Cyanothece cells and thus drive various cellular activities. These studies provide a comprehensive picture of how a physiologically relevant diurnal light-dark cycle influences the metabolism in a photosynthetic bacterium

  10. Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis*

    PubMed Central

    Yang, Mingkun; Wang, Yan; Chen, Ying; Cheng, Zhongyi; Gu, Jing; Deng, Jiaoyu; Bi, Lijun; Chen, Chuangbin; Mo, Ran; Wang, Xude; Ge, Feng

    2015-01-01

    Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen. PMID:25605462

  11. Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis *

    PubMed Central

    Chopra, Tarun; Hamelin, Romain; Armand, Florence; Chiappe, Diego; Moniatte, Marc; McKinney, John D.

    2014-01-01

    Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism. PMID:24997995

  12. MHC class I-related molecule, MR1, and mucosal-associated invariant T cells.

    PubMed

    Franciszkiewicz, Katarzyna; Salou, Marion; Legoux, Francois; Zhou, Qian; Cui, Yue; Bessoles, Stéphanie; Lantz, Olivier

    2016-07-01

    The MHC-related 1, MR1, molecule presents a new class of microbial antigens (derivatives of the riboflavin [Vitamin B2] biosynthesis pathway) to mucosal-associated invariant T (MAIT) cells. This raises many questions regarding antigens loading and intracellular trafficking of the MR1/ligand complexes. The MR1/MAIT field is also important because MAIT cells are very abundant in humans and their frequency is modified in many infectious and non-infectious diseases. Both MR1 and the invariant TCRα chain expressed by MAIT cells are strikingly conserved among species, indicating important functions. Riboflavin is synthesized by plants and most bacteria and yeasts but not animals, and its precursor derivatives activating MAIT cells are short-lived unless bound to MR1. The recognition of MR1 loaded with these compounds is therefore an exquisite manner to detect invasive bacteria. Herein, we provide an historical perspective of the field before describing the main characteristics of MR1, its ligands, and the few available data regarding its cellular biology. We then summarize the current knowledge of MAIT cell differentiation and discuss the definition of MAIT cells in comparison to related subsets. Finally, we describe the phenotype and effector activities of MAIT cells. PMID:27319347

  13. Urocanate reductase: identification of a novel anaerobic respiratory pathway in Shewanella oneidensis MR-1.

    PubMed

    Bogachev, Alexander V; Bertsova, Yulia V; Bloch, Dmitry A; Verkhovsky, Michael I

    2012-12-01

    Interpretation of the constantly expanding body of genomic information requires that the function of each gene be established. Here we report the genomic analysis and structural modelling of a previously uncharacterized redox-metabolism protein UrdA (SO_4620) of Shewanella oneidensis MR-1, which led to a discovery of the novel enzymatic activity, urocanate reductase. Further cloning and expression of urdA, as well as purification and biochemical study of the gene's product UrdA and redox titration of its prosthetic groups confirmed that the latter is indeed a flavin-containing enzyme catalysing the unidirectional reaction of two-electron reduction of urocanic acid to deamino-histidine, an activity not reported earlier. UrdA exhibits both high substrate affinity and high turnover rate (K(m)  < 10 μM, k(cat)  = 360 s(-1) ) and strong specificity in favour of urocanic acid. UrdA homologues are present in various bacterial genera, such as Shewanella, Fusobacterium and Clostridium, the latter including the human pathogen Clostridium tetani. The UrdA activity in S. oneidensis is induced by its substrate under anaerobic conditions and it enables anaerobic growth with urocanic acid as a sole terminal electron acceptor. The latter capability can provide the cells of UrdA-containing bacteria with a niche where no other bacteria can compete and survive. PMID:23078170

  14. Exhaustive Analysis of a Genotype Space Comprising 1015 Central Carbon Metabolisms Reveals an Organization Conducive to Metabolic Innovation

    PubMed Central

    Hosseini, Sayed-Rzgar; Barve, Aditya; Wagner, Andreas

    2015-01-01

    All biological evolution takes place in a space of possible genotypes and their phenotypes. The structure of this space defines the evolutionary potential and limitations of an evolving system. Metabolism is one of the most ancient and fundamental evolving systems, sustaining life by extracting energy from extracellular nutrients. Here we study metabolism’s potential for innovation by analyzing an exhaustive genotype-phenotype map for a space of 1015 metabolisms that encodes all possible subsets of 51 reactions in central carbon metabolism. Using flux balance analysis, we predict the viability of these metabolisms on 10 different carbon sources which give rise to 1024 potential metabolic phenotypes. Although viable metabolisms with any one phenotype comprise a tiny fraction of genotype space, their absolute numbers exceed 109 for some phenotypes. Metabolisms with any one phenotype typically form a single network of genotypes that extends far or all the way through metabolic genotype space, where any two genotypes can be reached from each other through a series of single reaction changes. The minimal distance of genotype networks associated with different phenotypes is small, such that one can reach metabolisms with novel phenotypes – viable on new carbon sources – through one or few genotypic changes. Exceptions to these principles exist for those metabolisms whose complexity (number of reactions) is close to the minimum needed for viability. Increasing metabolic complexity enhances the potential for both evolutionary conservation and evolutionary innovation. PMID:26252881

  15. Metatranscriptomics reveal differences in in situ energy and nitrogen metabolism among hydrothermal vent snail symbionts

    PubMed Central

    Sanders, J G; Beinart, R A; Stewart, F J; Delong, E F; Girguis, P R

    2013-01-01

    Despite the ubiquity of chemoautotrophic symbioses at hydrothermal vents, our understanding of the influence of environmental chemistry on symbiont metabolism is limited. Transcriptomic analyses are useful for linking physiological poise to environmental conditions, but recovering samples from the deep sea is challenging, as the long recovery times can change expression profiles before preservation. Here, we present a novel, in situ RNA sampling and preservation device, which we used to compare the symbiont metatranscriptomes associated with Alviniconcha, a genus of vent snail, in which specific host–symbiont combinations are predictably distributed across a regional geochemical gradient. Metatranscriptomes of these symbionts reveal key differences in energy and nitrogen metabolism relating to both environmental chemistry (that is, the relative expression of genes) and symbiont phylogeny (that is, the specific pathways employed). Unexpectedly, dramatic differences in expression of transposases and flagellar genes suggest that different symbiont types may also have distinct life histories. These data further our understanding of these symbionts' metabolic capabilities and their expression in situ, and suggest an important role for symbionts in mediating their hosts' interaction with regional-scale differences in geochemistry. PMID:23619306

  16. System-wide assembly of pathways and modules hierarchically reveal metabolic mechanism of cerebral ischemia

    PubMed Central

    Zhu, Yan; Guo, Zhili; Zhang, Liangxiao; Zhang, Yingying; Chen, Yinying; Nan, Jingyi; Zhao, Buchang; Xiao, Hongbin; Wang, Zhong; Wang, Yongyan

    2015-01-01

    The relationship between cerebral ischemia and metabolic disorders is poorly understood, which is partly due to the lack of comparative fusing data for larger complete systems and to the complexity of metabolic cascade reactions. Based on the fusing maps of comprehensive serum metabolome, fatty acid and amino acid profiling, we identified 35 potential metabolic biomarkers for ischemic stroke. Our analyses revealed 8 significantly altered pathways by MetPA (Metabolomics Pathway Analysis, impact score >0.10) and 15 significantly rewired modules in a complex ischemic network using the Markov clustering (MCL) method; all of these pathways became more homologous as the number of overlapping nodes was increased. We then detected 24 extensive pathways based on the total modular nodes from the network analysis, 12 of which were new discovery pathways. We provided a new perspective from the viewpoint of abnormal metabolites for the overall study of ischemic stroke as well as a new method to simplify the network analysis by selecting the more closely connected edges and nodes to build a module map of stroke. PMID:26621314

  17. Diurnal Changes in Mitochondrial Function Reveal Daily Optimization of Light and Dark Respiratory Metabolism in Arabidopsis*

    PubMed Central

    Lee, Chun Pong; Eubel, Holger; Millar, A. Harvey

    2010-01-01

    Biomass production by plants is often negatively correlated with respiratory rate, but the value of this rate changes dramatically during diurnal cycles, and hence, biomass is the cumulative result of complex environment-dependent metabolic processes. Mitochondria in photosynthetic plant tissues undertake substantially different metabolic roles during light and dark periods that are dictated by substrate availability and the functional capacity of mitochondria defined by their protein composition. We surveyed the heterogeneity of the mitochondrial proteome and its function during a typical night and day cycle in Arabidopsis shoots. This used a staged, quantitative analysis of the proteome across 10 time points covering 24 h of the life of 3-week-old Arabidopsis shoots grown under 12-h dark and 12-h light conditions. Detailed analysis of enzyme capacities and substrate-dependent respiratory processes of isolated mitochondria were also undertaken during the same time course. Together these data reveal a range of dynamic changes in mitochondrial capacity and uncover day- and night-enhanced protein components. Clear diurnal changes were evident in mitochondrial capacities to drive the TCA cycle and to undertake functions associated with nitrogen and sulfur metabolism, redox poise, and mitochondrial antioxidant defense. These data quantify the nature and nuances of a daily rhythm in Arabidopsis mitochondrial respiratory capacity. PMID:20601493

  18. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus.

    PubMed

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  19. Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice

    PubMed Central

    Zhang, Limin; Hatzakis, Emmanuel; Nichols, Robert G.; Hao, Ruixin; Correll, Jared; Smith, Philip B.; Chiaro, Christopher R.; Perdew, Gary H.; Patterson, Andrew D.

    2016-01-01

    Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) were assessed using global 1H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolic profiling of extracts obtained from serum and liver. 1H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was also observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure. PMID:26023891

  20. Metabolic connectivity mapping reveals effective connectivity in the resting human brain.

    PubMed

    Riedl, Valentin; Utz, Lukas; Castrillón, Gabriel; Grimmer, Timo; Rauschecker, Josef P; Ploner, Markus; Friston, Karl J; Drzezga, Alexander; Sorg, Christian

    2016-01-12

    Directionality of signaling among brain regions provides essential information about human cognition and disease states. Assessing such effective connectivity (EC) across brain states using functional magnetic resonance imaging (fMRI) alone has proven difficult, however. We propose a novel measure of EC, termed metabolic connectivity mapping (MCM), that integrates undirected functional connectivity (FC) with local energy metabolism from fMRI and positron emission tomography (PET) data acquired simultaneously. This method is based on the concept that most energy required for neuronal communication is consumed postsynaptically, i.e., at the target neurons. We investigated MCM and possible changes in EC within the physiological range using "eyes open" versus "eyes closed" conditions in healthy subjects. Independent of condition, MCM reliably detected stable and bidirectional communication between early and higher visual regions. Moreover, we found stable top-down signaling from a frontoparietal network including frontal eye fields. In contrast, we found additional top-down signaling from all major clusters of the salience network to early visual cortex only in the eyes open condition. MCM revealed consistent bidirectional and unidirectional signaling across the entire cortex, along with prominent changes in network interactions across two simple brain states. We propose MCM as a novel approach for inferring EC from neuronal energy metabolism that is ideally suited to study signaling hierarchies in the brain and their defects in brain disorders. PMID:26712010

  1. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus

    PubMed Central

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S.

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  2. Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling

    PubMed Central

    Mobberley, J. M.; Khodadad, C. L. M.; Visscher, P. T.; Reid, R. P.; Hagan, P.; Foster, J. S.

    2015-01-01

    Microbialites are sedimentary deposits formed by the metabolic interactions of microbes and their environment. These lithifying microbial communities represent one of the oldest ecosystems on Earth, yet the molecular mechanisms underlying the function of these communities are poorly understood. In this study, we used comparative metagenomic and metatranscriptomic analyses to characterize the spatial organization of the thrombolites of Highborne Cay, The Bahamas, an actively forming microbialite system. At midday, there were differences in gene expression throughout the spatial profile of the thrombolitic mat with a high abundance of transcripts encoding genes required for photosynthesis, nitrogen fixation and exopolymeric substance production in the upper three mm of the mat. Transcripts associated with denitrification and sulfate reduction were in low abundance throughout the depth profile, suggesting these metabolisms were less active during midday. Comparative metagenomics of the Bahamian thrombolites with other known microbialite ecosystems from across the globe revealed that, despite many shared core pathways, the thrombolites represented genetically distinct communities. This study represents the first time the metatranscriptome of living microbialite has been characterized and offers a new molecular perspective on those microbial metabolisms, and their underlying genetic pathways, that influence the mechanisms of carbonate precipitation in lithifying microbial mat ecosystems. PMID:26213359

  3. New Insights into Sulfur Metabolism in Yeasts as Revealed by Studies of Yarrowia lipolytica

    PubMed Central

    Hébert, Agnès; Forquin-Gomez, Marie-Pierre; Roux, Aurélie; Aubert, Julie; Junot, Christophe; Heilier, Jean-François; Landaud, Sophie; Bonnarme, Pascal

    2013-01-01

    Yarrowia lipolytica, located at the frontier of hemiascomycetous yeasts and fungi, is an excellent candidate for studies of metabolism evolution. This yeast, widely recognized for its technological applications, in particular produces volatile sulfur compounds (VSCs) that fully contribute to the flavor of smear cheese. We report here a relevant global vision of sulfur metabolism in Y. lipolytica based on a comparison between high- and low-sulfur source supplies (sulfate, methionine, or cystine) by combined approaches (transcriptomics, metabolite profiling, and VSC analysis). The strongest repression of the sulfate assimilation pathway was observed in the case of high methionine supply, together with a large accumulation of sulfur intermediates. A high sulfate supply seems to provoke considerable cellular stress via sulfite production, resulting in a decrease of the availability of the glutathione pathway's sulfur intermediates. The most limited effect was observed for the cystine supply, suggesting that the intracellular cysteine level is more controlled than that of methionine and sulfate. Using a combination of metabolomic profiling and genetic experiments, we revealed taurine and hypotaurine metabolism in yeast for the first time. On the basis of a phylogenetic study, we then demonstrated that this pathway was lost by some of the hemiascomycetous yeasts during evolution. PMID:23220962

  4. Defining the human adipose tissue proteome to reveal metabolic alterations in obesity.

    PubMed

    Mardinoglu, Adil; Kampf, Caroline; Asplund, Anna; Fagerberg, Linn; Hallström, Björn M; Edlund, Karolina; Blüher, Matthias; Pontén, Fredrik; Uhlen, Mathias; Nielsen, Jens

    2014-11-01

    White adipose tissue (WAT) has a major role in the progression of obesity. Here, we combined data from RNA-Seq and antibody-based immunohistochemistry to describe the normal physiology of human WAT obtained from three female subjects and explored WAT-specific genes by comparing WAT to 26 other major human tissues. Using the protein evidence in WAT, we validated the content of a genome-scale metabolic model for adipocytes. We employed this high-quality model for the analysis of subcutaneous adipose tissue (SAT) gene expression data obtained from subjects included in the Swedish Obese Subjects Sib Pair study to reveal molecular differences between lean and obese individuals. We integrated SAT gene expression and plasma metabolomics data, investigated the contribution of the metabolic differences in the mitochondria of SAT to the occurrence of obesity, and eventually identified cytosolic branched-chain amino acid (BCAA) transaminase 1 as a potential target that can be used for drug development. We observed decreased glutaminolysis and alterations in the BCAAs metabolism in SAT of obese subjects compared to lean subjects. We also provided mechanistic explanations for the changes in the plasma level of BCAAs, glutamate, pyruvate, and α-ketoglutarate in obese subjects. Finally, we validated a subset of our model-based predictions in 20 SAT samples obtained from 10 lean and 10 obese male and female subjects. PMID:25219818

  5. Singularity analysis of the AKT signaling pathway reveals connections between cancer and metabolic diseases

    NASA Astrophysics Data System (ADS)

    Wang, Guanyu

    2010-12-01

    Connections between cancer and metabolic diseases may consist in the complex network of interactions among a common set of biomolecules. By applying singularity and bifurcation analysis, the phenotypes constrained by the AKT signaling pathway are identified and mapped onto the parameter space, which include cancer and certain metabolic diseases. By considering physiologic properties (sensitivity, robustness and adaptivity) the AKT pathway must possess in order to efficiently sense growth factors and nutrients, the region of normal responses is located. To optimize these properties, the intracellular concentration of the AKT protein must be sufficiently high to saturate its enzymes; the strength of the positive feedback must be stronger than that of the negative feedback. The analysis illuminates the parameter space and reveals system-level mechanisms in regulating biological functions (cell growth, survival, proliferation and metabolism) and how their deregulation may lead to the development of diseases. The analytical expressions summarize the synergistic interactions among many molecules, which provides valuable insights into therapeutic interventions. In particular, a strategy for overcoming the limitations of mTOR inhibition is proposed for cancer therapy.

  6. Metabolic connectivity mapping reveals effective connectivity in the resting human brain

    PubMed Central

    Riedl, Valentin; Utz, Lukas; Castrillón, Gabriel; Grimmer, Timo; Rauschecker, Josef P.; Drzezga, Alexander; Sorg, Christian

    2016-01-01

    Directionality of signaling among brain regions provides essential information about human cognition and disease states. Assessing such effective connectivity (EC) across brain states using functional magnetic resonance imaging (fMRI) alone has proven difficult, however. We propose a novel measure of EC, termed metabolic connectivity mapping (MCM), that integrates undirected functional connectivity (FC) with local energy metabolism from fMRI and positron emission tomography (PET) data acquired simultaneously. This method is based on the concept that most energy required for neuronal communication is consumed postsynaptically, i.e., at the target neurons. We investigated MCM and possible changes in EC within the physiological range using “eyes open” versus “eyes closed” conditions in healthy subjects. Independent of condition, MCM reliably detected stable and bidirectional communication between early and higher visual regions. Moreover, we found stable top-down signaling from a frontoparietal network including frontal eye fields. In contrast, we found additional top-down signaling from all major clusters of the salience network to early visual cortex only in the eyes open condition. MCM revealed consistent bidirectional and unidirectional signaling across the entire cortex, along with prominent changes in network interactions across two simple brain states. We propose MCM as a novel approach for inferring EC from neuronal energy metabolism that is ideally suited to study signaling hierarchies in the brain and their defects in brain disorders. PMID:26712010

  7. Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling

    NASA Astrophysics Data System (ADS)

    Mobberley, J. M.; Khodadad, C. L. M.; Visscher, P. T.; Reid, R. P.; Hagan, P.; Foster, J. S.

    2015-07-01

    Microbialites are sedimentary deposits formed by the metabolic interactions of microbes and their environment. These lithifying microbial communities represent one of the oldest ecosystems on Earth, yet the molecular mechanisms underlying the function of these communities are poorly understood. In this study, we used comparative metagenomic and metatranscriptomic analyses to characterize the spatial organization of the thrombolites of Highborne Cay, The Bahamas, an actively forming microbialite system. At midday, there were differences in gene expression throughout the spatial profile of the thrombolitic mat with a high abundance of transcripts encoding genes required for photosynthesis, nitrogen fixation and exopolymeric substance production in the upper three mm of the mat. Transcripts associated with denitrification and sulfate reduction were in low abundance throughout the depth profile, suggesting these metabolisms were less active during midday. Comparative metagenomics of the Bahamian thrombolites with other known microbialite ecosystems from across the globe revealed that, despite many shared core pathways, the thrombolites represented genetically distinct communities. This study represents the first time the metatranscriptome of living microbialite has been characterized and offers a new molecular perspective on those microbial metabolisms, and their underlying genetic pathways, that influence the mechanisms of carbonate precipitation in lithifying microbial mat ecosystems.

  8. Neurochemical Metabolomics Reveals Disruption to Sphingolipid Metabolism Following Chronic Haloperidol Administration.

    PubMed

    McClay, Joseph L; Vunck, Sarah A; Batman, Angela M; Crowley, James J; Vann, Robert E; Beardsley, Patrick M; van den Oord, Edwin J

    2015-09-01

    Haloperidol is an effective antipsychotic drug for treatment of schizophrenia, but prolonged use can lead to debilitating side effects. To better understand the effects of long-term administration, we measured global metabolic changes in mouse brain following 3 mg/kg/day haloperidol for 28 days. These conditions lead to movement-related side effects in mice akin to those observed in patients after prolonged use. Brain tissue was collected following microwave tissue fixation to arrest metabolism and extracted metabolites were assessed using both liquid and gas chromatography mass spectrometry (MS). Over 300 unique compounds were identified across MS platforms. Haloperidol was found to be present in all test samples and not in controls, indicating experimental validity. Twenty-one compounds differed significantly between test and control groups at the p < 0.05 level. Top compounds were robust to analytical method, also being identified via partial least squares discriminant analysis. Four compounds (sphinganine, N-acetylornithine, leucine and adenosine diphosphate) survived correction for multiple testing in a non-parametric analysis using false discovery rate threshold < 0.1. Pathway analysis of nominally significant compounds (p < 0.05) revealed significant findings for sphingolipid metabolism (p = 0.015) and protein biosynthesis (p = 0.024). Altered sphingolipid metabolism is suggestive of disruptions to myelin. This interpretation is supported by our observation of elevated N-acetyl-aspartyl-glutamate in the haloperidol-treated mice (p = 0.004), a marker previously associated with demyelination. This study further demonstrates the utility of murine neurochemical metabolomics as a method to advance understanding of CNS drug effects. PMID:25850894

  9. Untargeted Metabolomics Reveals Predominant Alterations in Lipid Metabolism Following Light Exposure in Broccoli Sprouts

    PubMed Central

    Maldini, Mariateresa; Natella, Fausta; Baima, Simona; Morelli, Giorgio; Scaccini, Cristina; Langridge, James; Astarita, Giuseppe

    2015-01-01

    The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower) is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird’s-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle). We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant’s developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the potential of an

  10. A Systems Biology Approach Reveals Converging Molecular Mechanisms that Link Different POPs to Common Metabolic Diseases

    PubMed Central

    Ruiz, Patricia; Perlina, Ally; Mumtaz, Moiz; Fowler, Bruce A.

    2015-01-01

    Background: A number of epidemiological studies have identified statistical associations between persistent organic pollutants (POPs) and metabolic diseases, but testable hypotheses regarding underlying molecular mechanisms to explain these linkages have not been published. Objectives: We assessed the underlying mechanisms of POPs that have been associated with metabolic diseases; three well-known POPs [2,3,7,8-tetrachlorodibenzodioxin (TCDD), 2,2´,4,4´,5,5´-hexachlorobiphenyl (PCB 153), and 4,4´-dichlorodiphenyldichloroethylene (p,p´-DDE)] were studied. We used advanced database search tools to delineate testable hypotheses and to guide laboratory-based research studies into underlying mechanisms by which this POP mixture could produce or exacerbate metabolic diseases. Methods: For our searches, we used proprietary systems biology software (MetaCore™/MetaDrug™) to conduct advanced search queries for the underlying interactions database, followed by directional network construction to identify common mechanisms for these POPs within two or fewer interaction steps downstream of their primary targets. These common downstream pathways belong to various cytokine and chemokine families with experimentally well-documented causal associations with type 2 diabetes. Conclusions: Our systems biology approach allowed identification of converging pathways leading to activation of common downstream targets. To our knowledge, this is the first study to propose an integrated global set of step-by-step molecular mechanisms for a combination of three common POPs using a systems biology approach, which may link POP exposure to diseases. Experimental evaluation of the proposed pathways may lead to development of predictive biomarkers of the effects of POPs, which could translate into disease prevention and effective clinical treatment strategies. Citation: Ruiz P, Perlina A, Mumtaz M, Fowler BA. 2016. A systems biology approach reveals converging molecular mechanisms that

  11. Untargeted Metabolomics Reveals Predominant Alterations in Lipid Metabolism Following Light Exposure in Broccoli Sprouts.

    PubMed

    Maldini, Mariateresa; Natella, Fausta; Baima, Simona; Morelli, Giorgio; Scaccini, Cristina; Langridge, James; Astarita, Giuseppe

    2015-01-01

    The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower) is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird's-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle). We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant's developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the potential of an

  12. Hypothalamic metabolic compartmentation during appetite regulation as revealed by magnetic resonance imaging and spectroscopy methods

    PubMed Central

    Lizarbe, Blanca; Benitez, Ania; Peláez Brioso, Gerardo A.; Sánchez-Montañés, Manuel; López-Larrubia, Pilar; Ballesteros, Paloma; Cerdán, Sebastián

    2013-01-01

    We review the role of neuroglial compartmentation and transcellular neurotransmitter cycling during hypothalamic appetite regulation as detected by Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) methods. We address first the neurochemical basis of neuroendocrine regulation in the hypothalamus and the orexigenic and anorexigenic feed-back loops that control appetite. Then we examine the main MRI and MRS strategies that have been used to investigate appetite regulation. Manganese-enhanced magnetic resonance imaging (MEMRI), Blood oxygenation level-dependent contrast (BOLD), and Diffusion-weighted magnetic resonance imaging (DWI) have revealed Mn2+ accumulations, augmented oxygen consumptions, and astrocytic swelling in the hypothalamus under fasting conditions, respectively. High field 1H magnetic resonance in vivo, showed increased hypothalamic myo-inositol concentrations as compared to other cerebral structures. 1H and 13C high resolution magic angle spinning (HRMAS) revealed increased neuroglial oxidative and glycolytic metabolism, as well as increased hypothalamic glutamatergic and GABAergic neurotransmissions under orexigenic stimulation. We propose here an integrative interpretation of all these findings suggesting that the neuroendocrine regulation of appetite is supported by important ionic and metabolic transcellular fluxes which begin at the tripartite orexigenic clefts and become extended spatially in the hypothalamus through astrocytic networks becoming eventually MRI and MRS detectable. PMID:23781199

  13. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    PubMed

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  14. Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit.

    PubMed

    Colombié, Sophie; Nazaret, Christine; Bénard, Camille; Biais, Benoît; Mengin, Virginie; Solé, Marion; Fouillen, Laëtitia; Dieuaide-Noubhani, Martine; Mazat, Jean-Pierre; Beauvoit, Bertrand; Gibon, Yves

    2015-01-01

    Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium-scale stoichiometric model, including the balance of co-factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO2 release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits. PMID:25279440

  15. Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit

    PubMed Central

    Colombié, Sophie; Nazaret, Christine; Bénard, Camille; Biais, Benoît; Mengin, Virginie; Solé, Marion; Fouillen, Laëtitia; Dieuaide-Noubhani, Martine; Mazat, Jean-Pierre; Beauvoit, Bertrand; Gibon, Yves

    2015-01-01

    Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium-scale stoichiometric model, including the balance of co–factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO2 release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits. PMID:25279440

  16. Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism

    PubMed Central

    Mannan, Ahmad A.; Toya, Yoshihiro; Shimizu, Kazuyuki; McFadden, Johnjoe; Kierzek, Andrzej M.; Rocco, Andrea

    2015-01-01

    An understanding of the dynamics of the metabolic profile of a bacterial cell is sought from a dynamical systems analysis of kinetic models. This modelling formalism relies on a deterministic mathematical description of enzyme kinetics and their metabolite regulation. However, it is severely impeded by the lack of available kinetic information, limiting the size of the system that can be modelled. Furthermore, the subsystem of the metabolic network whose dynamics can be modelled is faced with three problems: how to parameterize the model with mostly incomplete steady state data, how to close what is now an inherently open system, and how to account for the impact on growth. In this study we address these challenges of kinetic modelling by capitalizing on multi-‘omics’ steady state data and a genome-scale metabolic network model. We use these to generate parameters that integrate knowledge embedded in the genome-scale metabolic network model, into the most comprehensive kinetic model of the central carbon metabolism of E. coli realized to date. As an application, we performed a dynamical systems analysis of the resulting enriched model. This revealed bistability of the central carbon metabolism and thus its potential to express two distinct metabolic states. Furthermore, since our model-informing technique ensures both stable states are constrained by the same thermodynamically feasible steady state growth rate, the ensuing bistability represents a temporal coexistence of the two states, and by extension, reveals the emergence of a phenotypically heterogeneous population. PMID:26469081

  17. Silencing MR-1 attenuates inflammatory damage in mice heart induced by AngII

    SciTech Connect

    Dai, Wenjian; Chen, Haiyang; Jiang, Jiandong; Kong, Weijia; Wang, Yiguang

    2010-01-15

    Myofibrillogenesis regulator-1(MR-1) can aggravate cardiac hypertrophy induced by angiotensin(Ang) II in mice through activation of NF-{kappa}B signaling pathway, and nuclear transcription factor (NF)-{kappa}B and activator protein-1(AP-1) regulate inflammatory and immune responses by increasing the expression of specific inflammatory genes in various tissues including heart. Whether inhibition of MR-1 expression will attenuate AngII-induced inflammatory injury in mice heart has not been explored. Herein, we monitored the activation of NF-{kappa}B and AP-1, together with expression of pro-inflammatory of interleukin(IL)-6, tumor necrosis factor(TNF)-{alpha}, vascular-cell adhesion molecule (VCAM)-1, platelet endothelial cell adhesion molecule (PECAM), and inflammatory cell infiltration in heart of mice which are induced firstly by AngII (PBS),then received MR-1-siRNA or control-siRNA injecting. We found that the activation of NF-{kappa}B and AP-1 was inhibited significantly, together with the decreased expression of IL-6, TNF-{alpha}, VCAM-1, and PECAM in AngII-induced mice myocardium in MR-1-siRNA injection groups compared with control-siRNA injecting groups. However, the expression level of MR-1 was not an apparent change in PBS-infused groups than in unoperation groups, and MR-1-siRNA do not affect the expression of MR-1 in PBS-infused mice. Our findings suggest that silencing MR-1 protected mice myocardium against inflammatory injury induced by AngII by suppression of pro-inflammatory transcription factors NF-{kappa}B and AP-1 signaling pathway.

  18. Metabolism and development – integration of micro computed tomography data and metabolite profiling reveals metabolic reprogramming from floral initiation to silique development

    PubMed Central

    Bellaire, Anke; Ischebeck, Till; Staedler, Yannick; Weinhaeuser, Isabell; Mair, Andrea; Parameswaran, Sriram; Ito, Toshiro; Schönenberger, Jürg; Weckwerth, Wolfram

    2014-01-01

    The interrelationship of morphogenesis and metabolism is a poorly studied phenomenon. The main paradigm is that development is controlled by gene expression. The aim of the present study was to correlate metabolism to early and late stages of flower and fruit development in order to provide the basis for the identification of metabolic adjustment and limitations. A highly detailed picture of morphogenesis is achieved using nondestructive micro computed tomography. This technique was used to quantify morphometric parameters of early and late flower development in an Arabidopsis thaliana mutant with synchronized flower initiation. The synchronized flower phenotype made it possible to sample enough early floral tissue otherwise not accessible for metabolomic analysis. The integration of metabolomic and morphometric data enabled the correlation of metabolic signatures with the process of flower morphogenesis. These signatures changed significantly during development, indicating a pronounced metabolic reprogramming in the tissue. Distinct sets of metabolites involved in these processes were identified and were linked to the findings of previous gene expression studies of flower development. High correlations with basic leucine zipper (bZIP) transcription factors and nitrogen metabolism genes involved in the control of metabolic carbon : nitrogen partitioning were revealed. Based on these observations a model for metabolic adjustment during flower development is proposed. PMID:24350948

  19. A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

    PubMed Central

    Solis-Escalante, Daniel; Kuijpers, Niels G. A.; Barrajon-Simancas, Nuria; van den Broek, Marcel; Pronk, Jack T.; Daran, Jean-Marc

    2015-01-01

    As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community. PMID:26071034

  20. Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism.

    PubMed

    Chen, Walter W; Freinkman, Elizaveta; Wang, Tim; Birsoy, Kıvanç; Sabatini, David M

    2016-08-25

    Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles. PMID:27565352

  1. Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

    PubMed Central

    Yee, Rebecca; Cui, Peng; Shi, Wanliang; Feng, Jie; Zhang, Ying

    2015-01-01

    Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus. PMID:27025643

  2. Sequence-based Network Completion Reveals the Integrality of Missing Reactions in Metabolic Networks*

    PubMed Central

    Krumholz, Elias W.; Libourel, Igor G. L.

    2015-01-01

    Genome-scale metabolic models are central in connecting genotypes to metabolic phenotypes. However, even for well studied organisms, such as Escherichia coli, draft networks do not contain a complete biochemical network. Missing reactions are referred to as gaps. These gaps need to be filled to enable functional analysis, and gap-filling choices influence model predictions. To investigate whether functional networks existed where all gap-filling reactions were supported by sequence similarity to annotated enzymes, four draft networks were supplemented with all reactions from the Model SEED database for which minimal sequence similarity was found in their genomes. Quadratic programming revealed that the number of reactions that could partake in a gap-filling solution was vast: 3,270 in the case of E. coli, where 72% of the metabolites in the draft network could connect a gap-filling solution. Nonetheless, no network could be completed without the inclusion of orphaned enzymes, suggesting that parts of the biochemistry integral to biomass precursor formation are uncharacterized. However, many gap-filling reactions were well determined, and the resulting networks showed improved prediction of gene essentiality compared with networks generated through canonical gap filling. In addition, gene essentiality predictions that were sensitive to poorly determined gap-filling reactions were of poor quality, suggesting that damage to the network structure resulting from the inclusion of erroneous gap-filling reactions may be predictable. PMID:26041773

  3. Laboratory investigation of high pressure survival in Shewanella oneidensis MR-1 into the gigapascal pressure range

    PubMed Central

    Hazael, Rachael; Foglia, Fabrizia; Kardzhaliyska, Liya; Daniel, Isabelle; Meersman, Filip; McMillan, Paul

    2014-01-01

    The survival of Shewanella oneidensis MR-1 at up to 1500 MPa was investigated by laboratory studies involving exposure to high pressure followed by evaluation of survivors as the number (N) of colony forming units (CFU) that could be cultured following recovery to ambient conditions. Exposing the wild type (WT) bacteria to 250 MPa resulted in only a minor (0.7 log N units) drop in survival compared with the initial concentration of 108 cells/ml. Raising the pressure to above 500 MPa caused a large reduction in the number of viable cells observed following recovery to ambient pressure. Additional pressure increase caused a further decrease in survivability, with approximately 102 CFU/ml recorded following exposure to 1000 MPa (1 GPa) and 1.5 GPa. Pressurizing samples from colonies resuscitated from survivors that had been previously exposed to high pressure resulted in substantially greater survivor counts. Experiments were carried out to examine potential interactions between pressure and temperature variables in determining bacterial survival. One generation of survivors previously exposed to 1 GPa was compared with WT samples to investigate survival between 37 and 8°C. The results did not reveal any coupling between acquired high pressure resistance and temperature effects on growth. PMID:25452750

  4. Laboratory investigation of high pressure survival in Shewanella oneidensis MR-1 into the gigapascal pressure range.

    PubMed

    Hazael, Rachael; Foglia, Fabrizia; Kardzhaliyska, Liya; Daniel, Isabelle; Meersman, Filip; McMillan, Paul

    2014-01-01

    The survival of Shewanella oneidensis MR-1 at up to 1500 MPa was investigated by laboratory studies involving exposure to high pressure followed by evaluation of survivors as the number (N) of colony forming units (CFU) that could be cultured following recovery to ambient conditions. Exposing the wild type (WT) bacteria to 250 MPa resulted in only a minor (0.7 log N units) drop in survival compared with the initial concentration of 10(8) cells/ml. Raising the pressure to above 500 MPa caused a large reduction in the number of viable cells observed following recovery to ambient pressure. Additional pressure increase caused a further decrease in survivability, with approximately 10(2) CFU/ml recorded following exposure to 1000 MPa (1 GPa) and 1.5 GPa. Pressurizing samples from colonies resuscitated from survivors that had been previously exposed to high pressure resulted in substantially greater survivor counts. Experiments were carried out to examine potential interactions between pressure and temperature variables in determining bacterial survival. One generation of survivors previously exposed to 1 GPa was compared with WT samples to investigate survival between 37 and 8°C. The results did not reveal any coupling between acquired high pressure resistance and temperature effects on growth. PMID:25452750

  5. Highly efficient in vitro biosynthesis of silver nanoparticles using Lysinibacillus sphaericus MR-1 and their characterization

    NASA Astrophysics Data System (ADS)

    Gou, Yujun; Zhou, Rongying; Ye, Xiujuan; Gao, Shanshan; Li, Xiangqian

    2015-02-01

    Silver nanoparticles (AgNPs) have been widely used in diverse fields due to their superior properties. Currently the biosynthesis of AgNPs is in the limelight of modern nanotechnology because of its green properties. However, relatively low yield and inefficiency diminish the prospect of applying these biosynthesized AgNPs. In this work, a rapid mass AgNP biosynthesis method using the cell-free extract of a novel bacterial strain, Lysinibacillus sphaericus MR-1, which has been isolated from a chemical fertilizer plant, is reported. In addition, the optimum synthesis conditions of AgNPs were investigated. The optimum pH, temperature, dosage, and reaction time were 12, 70 °C, 20 mM AgNO3, and 75 min, respectively. Finally, AgNPs were characterized by optical absorption spectroscopy, zeta potential and size distribution analysis, x-ray diffraction, electron microscopy, and energy-dispersive x-ray spectroscopy. The results revealed that these biosynthesized AgNPs were bimolecular covered, stable, well-dispersed face centered cubic (fcc) spherical crystalline particles with diameters in the range 5-20 nm. The advantages of this approach are its simplicity, high efficiency, and eco-friendly and cost-effective features.

  6. Spatiotemporal activity of the mshA gene system in Shewanella oneidensis MR-1 biofilms.

    PubMed

    Saville, Renée M; Dieckmann, Nele; Spormann, Alfred M

    2010-07-01

    Type IV pili and a putative EPS biosynthetic gene cluster (mxdABCD) have been implicated previously in biofilm formation in Shewanella oneidensis MR-1. Here, we report that the mannose-sensitive hemagglutinin (MSHA) pilus mediates a reversible, d-mannose-sensitive association of cells to the substratum surface or to other cells that is critical within the first 5 microm of the biofilm from the substratum. The presence of the MSHA pilus alone is insufficient to confer biofilm-forming capacity; its activity, as mediated by the putative pilus retraction motor protein, PilT, is also required. Deletion of pilD, encoding the type IV pili prepilin peptidase, revealed that additional PilD substrate(s) may be involved in biofilm formation beyond the major structural pilin of the MSHA pilus. We also present data showing that the MSHA pilus and mxd genes encode for a complementary set of molecular machineries that constitute the dominant mechanisms enabling biofilm formation in this microorganism under hydrodynamic conditions. PMID:20487019

  7. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

    PubMed

    McWilliam, Hamish E G; Eckle, Sidonia B G; Theodossis, Alex; Liu, Ligong; Chen, Zhenjun; Wubben, Jacinta M; Fairlie, David P; Strugnell, Richard A; Mintern, Justine D; McCluskey, James; Rossjohn, Jamie; Villadangos, Jose A

    2016-05-01

    The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability. PMID:27043408

  8. Roles of 3,3′,4′,5-tetrachlorosalicylanilide in regulating extracellular electron transfer of Shewanella oneidensis MR-1

    PubMed Central

    Wang, Yong-Peng; Yu, Sheng-Song; Zhang, Hai-Ling; Li, Wen-Wei; Cheng, Yuan-Yuan; Yu, Han-Qing

    2015-01-01

    Microbial extracellular electron transfer (EET) is critically involved in many pollutant conversion processes in both natural environment and engineered bioelectrochemical systems (BES), but typically with limited efficiency and poor controllability. In this study, we discover an important role of uncouplers in affecting the microbial energy metabolism and EET. Dose of lower-concentration 3,3′,4′,5-tetrachlorosalicylanilide (TCS) in the anolyte promoted the current generation and substrate degradation of an MFC inoculated with Shewanella oneidensis MR-1. However, higher TCS dosage caused obvious microbial inhibition. Our results suggest a previously unknown role of uncouplers in regulating the microbial EET. In addition, the underlying mechanisms of such processes are investigated. This work broadens our view about the EET behaviors of microorganisms in real water environment where uncouplers are usually present, and suggests a possible new approach to regulate microbial EET in BES. PMID:25612888

  9. Roles of 3,3',4',5-tetrachlorosalicylanilide in regulating extracellular electron transfer of Shewanella oneidensis MR-1.

    PubMed

    Wang, Yong-Peng; Yu, Sheng-Song; Zhang, Hai-Ling; Li, Wen-Wei; Cheng, Yuan-Yuan; Yu, Han-Qing

    2015-01-01

    Microbial extracellular electron transfer (EET) is critically involved in many pollutant conversion processes in both natural environment and engineered bioelectrochemical systems (BES), but typically with limited efficiency and poor controllability. In this study, we discover an important role of uncouplers in affecting the microbial energy metabolism and EET. Dose of lower-concentration 3,3',4',5-tetrachlorosalicylanilide (TCS) in the anolyte promoted the current generation and substrate degradation of an MFC inoculated with Shewanella oneidensis MR-1. However, higher TCS dosage caused obvious microbial inhibition. Our results suggest a previously unknown role of uncouplers in regulating the microbial EET. In addition, the underlying mechanisms of such processes are investigated. This work broadens our view about the EET behaviors of microorganisms in real water environment where uncouplers are usually present, and suggests a possible new approach to regulate microbial EET in BES. PMID:25612888

  10. The Carbohydrate Metabolism Signature of Lactococcus lactis Strain A12 Reveals Its Sourdough Ecosystem Origin

    PubMed Central

    Passerini, Delphine; Coddeville, Michèle; Le Bourgeois, Pascal; Loubière, Pascal; Ritzenthaler, Paul; Fontagné-Faucher, Catherine; Cocaign-Bousquet, Muriel

    2013-01-01

    Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci. PMID:23872564

  11. Metagenomic signatures of a tropical mining-impacted stream reveal complex microbial and metabolic networks.

    PubMed

    Reis, Mariana P; Dias, Marcela F; Costa, Patrícia S; Ávila, Marcelo P; Leite, Laura R; de Araújo, Flávio M G; Salim, Anna C M; Bucciarelli-Rodriguez, Mônica; Oliveira, Guilherme; Chartone-Souza, Edmar; Nascimento, Andréa M A

    2016-10-01

    Bacteria from aquatic ecosystems significantly contribute to biogeochemical cycles, but details of their community structure in tropical mining-impacted environments remain unexplored. In this study, we analyzed a bacterial community from circumneutral-pH tropical stream sediment by 16S rRNA and shotgun deep sequencing. Carrapatos stream sediment, which has been exposed to metal stress due to gold and iron mining (21 [g Fe]/kg), revealed a diverse community, with predominance of Proteobacteria (39.4%), Bacteroidetes (12.2%), and Parcubacteria (11.4%). Among Proteobacteria, the most abundant reads were assigned to neutrophilic iron-oxidizing taxa, such as Gallionella, Sideroxydans, and Mariprofundus, which are involved in Fe cycling and harbor several metal resistance genes. Functional analysis revealed a large number of genes participating in nitrogen and methane metabolic pathways despite the low concentrations of inorganic nitrogen in the Carrapatos stream. Our findings provide important insights into bacterial community interactions in a mining-impacted environment. PMID:27441985

  12. Metabolomic and Proteomic Profiles Reveal the Dynamics of Primary Metabolism during Seed Development of Lotus (Nelumbo nucifera)

    PubMed Central

    Wang, Lei; Fu, Jinlei; Li, Ming; Fragner, Lena; Weckwerth, Wolfram; Yang, Pingfang

    2016-01-01

    Sacred lotus (Nelumbo nucifera) belongs to the Nelumbonaceae family. Its seeds are widely consumed in Asian countries as snacks or even medicine. Besides the market value, lotus seed also plays a crucial role in the lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation, and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP) which corresponded to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal metabolic adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide a reference data set for the evaluation of primary metabolism during lotus seed development. PMID:27375629

  13. Metabolomic and Proteomic Profiles Reveal the Dynamics of Primary Metabolism during Seed Development of Lotus (Nelumbo nucifera).

    PubMed

    Wang, Lei; Fu, Jinlei; Li, Ming; Fragner, Lena; Weckwerth, Wolfram; Yang, Pingfang

    2016-01-01

    Sacred lotus (Nelumbo nucifera) belongs to the Nelumbonaceae family. Its seeds are widely consumed in Asian countries as snacks or even medicine. Besides the market value, lotus seed also plays a crucial role in the lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation, and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP) which corresponded to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal metabolic adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide a reference data set for the evaluation of primary metabolism during lotus seed development. PMID:27375629

  14. Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

    PubMed Central

    2010-01-01

    Background Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary

  15. Charge-associated effects of fullerene derivatives on microbialstructural integrity and central metabolism

    SciTech Connect

    Tang, Yinjie J.; Ashcroft, Jared M.; Chen, Ding; Min, Guangwei; Kim, Chul; Murkhejee, Bipasha; Larabell, Carolyn; Keasling, Jay D.; Chen,Fanqing Frank

    2007-01-23

    The effects of four types of fullerene compounds (C60,C60-OH, C60-COOH, C60-NH2) were examined on two model microorganisms(Escherichia coli W3110 and Shewanella oneidensis MR-1). Positivelycharged C60-NH2 at concentrations as low as 10 mg/L inhibited growth andreduced substrate uptake for both microorganisms. Scanning ElectronMicroscopy (SEM) revealed damage to cellular structures.Neutrally-charged C60 and C60-OH had mild negative effects on S.oneidensis MR-1, whereas the negatively-charged C60-COOH did not affecteither microorganism s growth. The effect of fullerene compounds onglobal metabolism was further investigated using [3-13C]L-lactateisotopic labeling, which tracks perturbations to metabolic reaction ratesin bacteria by examining the change in the isotopic labeling pattern inthe resulting metabolites (often amino acids).1-3 The 13C isotopomeranalysis from all fullerene-exposed cultures revealed no significantdifferences in isotopomer distributions from unstressed cells. Thisresult indicates that microbial central metabolism is robust toenvironmental stress inflicted by fullerene nanoparticles. In addition,although C60-NH2 compounds caused mechanical stress on the cell wall ormembrane, both S. oneidensis MR-1 and E. coli W3110 can efficientlyalleviate such stress by cell aggregation and precipitation of the toxicnanoparticles. The results presented here favor the hypothesis thatfullerenes cause more membrane stress4, 5, 6 than perturbation to energymetabolism7

  16. Physiological and genomic characterization of Arcobacter anaerophilus IR-1 reveals new metabolic features in Epsilonproteobacteria

    PubMed Central

    Roalkvam, Irene; Drønen, Karine; Stokke, Runar; Daae, Frida L.; Dahle, Håkon; Steen, Ida H.

    2015-01-01

    In this study we characterized and sequenced the genome of Arcobacter anaerophilus strain IR-1 isolated from enrichment cultures used in nitrate-amended corrosion experiments. A. anaerophilus IR-1 could grow lithoautotrophically on hydrogen and hydrogen sulfide and lithoheterothrophically on thiosulfate and elemental sulfur. In addition, the strain grew organoheterotrophically on yeast extract, peptone, and various organic acids. We show for the first time that Arcobacter could grow on the complex organic substrate tryptone and oxidize acetate with elemental sulfur as electron acceptor. Electron acceptors utilized by most Epsilonproteobacteria, such as oxygen, nitrate, and sulfur, were also used by A. anaerophilus IR-1. Strain IR-1 was also uniquely able to use iron citrate as electron acceptor. Comparative genomics of the Arcobacter strains A. butzleri RM4018, A. nitrofigilis CI and A. anaerophilus IR-1 revealed that the free-living strains had a wider metabolic range and more genes in common compared to the pathogen strain. The presence of genes for NAD+-reducing hydrogenase (hox) and dissimilatory iron reduction (fre) were unique for A. anaerophilus IR-1 among Epsilonproteobacteria. Finally, the new strain had an incomplete denitrification pathway where the end product was nitrite, which is different from other Arcobacter strains where the end product is ammonia. Altogether, our study shows that traditional characterization in combination with a modern genomics approach can expand our knowledge on free-living Arcobacter, and that this complementary approach could also provide invaluable knowledge about the physiology and metabolic pathways in other Epsilonproteobacteria from various environments. PMID:26441916

  17. Comparative Transcriptomics Reveals Jasmonic Acid-Associated Metabolism Related to Cotton Fiber Initiation.

    PubMed

    Wang, Liman; Zhu, Youmin; Hu, Wenjing; Zhang, Xueying; Cai, Caiping; Guo, Wangzhen

    2015-01-01

    Analysis of mutants and gene expression patterns provides a powerful approach for investigating genes involved in key stages of plant fiber development. In this study, lintless-fuzzless XinWX and linted-fuzzless XinFLM with a single genetic locus difference for lint were used to identify differentially expressed genes. Scanning electron microscopy showed fiber initiation in XinFLM at 0 days post anthesis (DPA). Fiber transcriptional profiling of the lines at three initiation developmental stages (-1, 0, 1 DPA) was performed using an oligonucleotide microarray. Loop comparisons of the differentially expressed genes within and between the lines was carried out, and functional classification and enrichment analysis showed that gene expression patterns during fiber initiation were heavily associated with hormone metabolism, transcription factor regulation, lipid transport, and asparagine biosynthetic processes, as previously reported. Further, four members of the allene-oxide cyclase (AOC) family that function in jasmonate biosynthesis were parallel up-regulation in fiber initiation, especially at -1 DPA, compared to other tissues and organs in linted-fuzzed TM-1. Real time-quantitative PCR (RT-qPCR) analysis in different fiber mutant lines revealed that AOCs were up-regulated higher at -1 DPA in lintless-fuzzless than that in linted-fuzzless and linted-fuzzed materials, and transcription of the AOCs was increased under jasmonic acid (JA) treatment. Expression analysis of JA biosynthesis-associated genes between XinWX and XinFLM showed that they were up-regulated during fiber initiation in the fuzzless-lintless mutant. Taken together, jasmonic acid-associated metabolism was related to cotton fiber initiation. Parallel up-regulation of AOCs expression may be important for normal fiber initiation development, while overproduction of AOCs might disrupt normal fiber development. PMID:26079621

  18. Long-term monitoring reveals carbon–nitrogen metabolism key to microcystin production in eutrophic lakes

    PubMed Central

    Beversdorf, Lucas J.; Miller, Todd R.; McMahon, Katherine D.

    2015-01-01

    The environmental drivers contributing to cyanobacterial dominance in aquatic systems have been extensively studied. However, understanding of toxic vs. non-toxic cyanobacterial population dynamics and the mechanisms regulating cyanotoxin production remain elusive, both physiologically and ecologically. One reason is the disconnect between laboratory and field-based studies. Here, we combined 3 years of temporal data, including microcystin (MC) concentrations, 16 years of long-term ecological research, and 10 years of molecular data to investigate the potential factors leading to the selection of toxic Microcystis and MC production. Our analysis revealed that nitrogen (N) speciation and inorganic carbon (C) availability might be important drivers of Microcystis population dynamics and that an imbalance in cellular C: N ratios may trigger MC production. More specifically, precipitous declines in ammonium concentrations lead to a transitional period of N stress, even in the presence of high nitrate concentrations, that we call the “toxic phase.” Following the toxic phase, temperature and cyanobacterial abundance remained elevated but MC concentrations drastically declined. Increases in ammonium due to lake turnover may have led to down regulation of MC synthesis or a shift in the community from toxic to non-toxic species. While total phosphorus (P) to total N ratios were relatively low over the time-series, MC concentrations were highest when total N to total P ratios were also highest. Similarly, high C: N ratios were also strongly correlated to the toxic phase. We propose a metabolic model that corroborates molecular studies and reflects our ecological observations that C and N metabolism may regulate MC production physiologically and ecologically. In particular, we hypothesize that an imbalance between 2-oxoglutarate and ammonium in the cell regulates MC synthesis in the environment. PMID:26029192

  19. Preferential Use of Central Metabolism In Vivo Reveals a Nutritional Basis for Polymicrobial Infection

    PubMed Central

    Alteri, Christopher J.; Himpsl, Stephanie D.; Mobley, Harry L. T.

    2015-01-01

    The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche. PMID:25568946

  20. Comparative Transcriptomics Reveals Jasmonic Acid-Associated Metabolism Related to Cotton Fiber Initiation

    PubMed Central

    Wang, Liman; Zhu, Youmin; Hu, Wenjing; Zhang, Xueying; Cai, Caiping; Guo, Wangzhen

    2015-01-01

    Analysis of mutants and gene expression patterns provides a powerful approach for investigating genes involved in key stages of plant fiber development. In this study, lintless-fuzzless XinWX and linted-fuzzless XinFLM with a single genetic locus difference for lint were used to identify differentially expressed genes. Scanning electron microscopy showed fiber initiation in XinFLM at 0 days post anthesis (DPA). Fiber transcriptional profiling of the lines at three initiation developmental stages (-1, 0, 1 DPA) was performed using an oligonucleotide microarray. Loop comparisons of the differentially expressed genes within and between the lines was carried out, and functional classification and enrichment analysis showed that gene expression patterns during fiber initiation were heavily associated with hormone metabolism, transcription factor regulation, lipid transport, and asparagine biosynthetic processes, as previously reported. Further, four members of the allene-oxide cyclase (AOC) family that function in jasmonate biosynthesis were parallel up-regulation in fiber initiation, especially at -1 DPA, compared to other tissues and organs in linted-fuzzed TM-1. Real time-quantitative PCR (RT-qPCR) analysis in different fiber mutant lines revealed that AOCs were up-regulated higher at -1 DPA in lintless-fuzzless than that in linted-fuzzless and linted-fuzzed materials, and transcription of the AOCs was increased under jasmonic acid (JA) treatment. Expression analysis of JA biosynthesis-associated genes between XinWX and XinFLM showed that they were up-regulated during fiber initiation in the fuzzless-lintless mutant. Taken together, jasmonic acid-associated metabolism was related to cotton fiber initiation. Parallel up-regulation of AOCs expression may be important for normal fiber initiation development, while overproduction of AOCs might disrupt normal fiber development. PMID:26079621

  1. Catabolism of Exogenous Lactate Reveals It as a Legitimate Metabolic Substrate in Breast Cancer

    PubMed Central

    Kennedy, Kelly M.; Scarbrough, Peter M.; Ribeiro, Anthony; Richardson, Rachel; Yuan, Hong; Sonveaux, Pierre; Landon, Chelsea D.; Chi, Jen-Tsan; Pizzo, Salvatore

    2013-01-01

    Lactate accumulation in tumors has been associated with metastases and poor overall survival in cancer patients. Lactate promotes angiogenesis and metastasis, providing rationale for understanding how it is processed by cells. The concentration of lactate in tumors is a balance between the amount produced, amount carried away by vasculature and if/how it is catabolized by aerobic tumor or stromal cells. We examined lactate metabolism in human normal and breast tumor cell lines and rat breast cancer: 1. at relevant concentrations, 2. under aerobic vs. hypoxic conditions, 3. under conditions of normo vs. hypoglucosis. We also compared the avidity of tumors for lactate vs. glucose and identified key lactate catabolites to reveal how breast cancer cells process it. Lactate was non-toxic at clinically relevant concentrations. It was taken up and catabolized to alanine and glutamate by all cell lines. Kinetic uptake rates of lactate in vivo surpassed that of glucose in R3230Ac mammary carcinomas. The uptake appeared specific to aerobic tumor regions, consistent with the proposed “metabolic symbiont” model; here lactate produced by hypoxic cells is used by aerobic cells. We investigated whether treatment with alpha-cyano-4-hydroxycinnamate (CHC), a MCT1 inhibitor, would kill cells in the presence of high lactate. Both 0.1 mM and 5 mM CHC prevented lactate uptake in R3230Ac cells at lactate concentrations at ≤20 mM but not at 40 mM. 0.1 mM CHC was well-tolerated by R3230Ac and MCF7 cells, but 5 mM CHC killed both cell lines ± lactate, indicating off-target effects. This study showed that breast cancer cells tolerate and use lactate at clinically relevant concentrations in vitro (± glucose) and in vivo. We provided additional support for the metabolic symbiont model and discovered that breast cells prevailingly take up and catabolize lactate, providing rationale for future studies on manipulation of lactate catabolism pathways for therapy. PMID:24069390

  2. Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms

    PubMed Central

    Saka, Hector A.; Thompson, J. Will; Chen, Yi-Shan; Kumar, Yadunanda; Dubois, Laura G.; Moseley, M. Arthur; Valdivia, Raphael H.

    2011-01-01

    Summary Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyze the membranes of the pathogen-containing vacuole (“inclusion”). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of >54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient Type III secretion (T3S) in the RB stage at 18 hpi, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a “per organism” basis. Our results show that EB and RB

  3. GENE EXPRESSION PROFILING IN AGING RATS AND MICE REVEALS CHANGES IN XENOBIOTIC METABOLISM GENES

    EPA Science Inventory

    Detoxification and elimination of xenobiotics are major functions of the liver and is important in maintaining the metabolic homeostasis of the organism. The degree to which aging affects hepatic metabolism is not known. The expression of xenobiotic metabolizing enzymes (XMEs), i...

  4. 13C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells

    PubMed Central

    Luo, Shuai; Guo, Weihua; H. Nealson, Kenneth; Feng, Xueyang; He, Zhen

    2016-01-01

    Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and 13C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the 13C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that 13C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms. PMID:26868848

  5. (13)C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells.

    PubMed

    Luo, Shuai; Guo, Weihua; H Nealson, Kenneth; Feng, Xueyang; He, Zhen

    2016-01-01

    Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and (13)C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the (13)C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that (13)C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms. PMID:26868848

  6. Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells.

    PubMed

    Min, Hae-Ki; Sookoian, Silvia; Pirola, Carlos J; Cheng, Jianfeng; Mirshahi, Faridoddin; Sanyal, Arun J

    2014-07-01

    PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling. PMID:24763554

  7. Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells

    PubMed Central

    Min, Hae-Ki; Sookoian, Silvia; Pirola, Carlos J.; Cheng, Jianfeng; Mirshahi, Faridoddin

    2014-01-01

    PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling. PMID:24763554

  8. The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist

    PubMed Central

    Suen, Garret; Weimer, Paul J.; Stevenson, David M.; Aylward, Frank O.; Boyum, Julie; Deneke, Jan; Drinkwater, Colleen; Ivanova, Natalia N.; Mikhailova, Natalia; Chertkov, Olga; Goodwin, Lynne A.; Currie, Cameron R.; Mead, David; Brumm, Phillip J.

    2011-01-01

    Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation. PMID:21526192

  9. 1H NMR-Based Profiling Reveals Differential Immune-Metabolic Networks during Influenza Virus Infection in Obese Mice

    PubMed Central

    Milner, J. Justin; Wang, Jue; Sheridan, Patricia A.; Ebbels, Tim; Beck, Melinda A.; Saric, Jasmina

    2014-01-01

    Obese individuals are at greater risk for death from influenza virus infection. Paralleling human evidence, obese mice are also more susceptible to influenza infection mortality. However, the underlying mechanisms driving greater influenza severity in the obese remain unclear. Metabolic profiling has been utilized in infectious disease models to enhance prognostic or diagnostic methods, and to gain insight into disease pathogenesis by providing a more global picture of dynamic infection responses. Herein, metabolic profiling was used to develop a deeper understanding of the complex processes contributing to impaired influenza protection in obese mice and to facilitate generation of new explanatory hypotheses. Diet-induced obese and lean mice were infected with influenza A/Puerto Rico/8/34. 1H nuclear magnetic resonance-based metabolic profiling of urine, feces, lung, liver, mesenteric white adipose tissue, bronchoalveolar lavage fluid and serum revealed distinct metabolic signatures in infected obese mice, including perturbations in nucleotide, vitamin, ketone body, amino acid, carbohydrate, choline and lipid metabolic pathways. Further, metabolic data was integrated with immune analyses to obtain a more comprehensive understanding of potential immune-metabolic interactions. Of interest, uncovered metabolic signatures in urine and feces allowed for discrimination of infection status in both lean and obese mice at an early influenza time point, which holds prognostic and diagnostic implications for this methodology. These results confirm that obesity causes distinct metabolic perturbations during influenza infection and provide a basis for generation of new hypotheses and use of this methodology in detection of putative biomarkers and metabolic patterns to predict influenza infection outcome. PMID:24844920

  10. A pangenomic analysis of the Nannochloropsis organellar genomes reveals novel genetic variations in key metabolic genes

    PubMed Central

    2014-01-01

    Background Microalgae in the genus Nannochloropsis are photosynthetic marine Eustigmatophytes of significant interest to the bioenergy and aquaculture sectors due to their ability to efficiently accumulate biomass and lipids for utilization in renewable transportation fuels, aquaculture feed, and other useful bioproducts. To better understand the genetic complement that drives the metabolic processes of these organisms, we present the assembly and comparative pangenomic analysis of the chloroplast and mitochondrial genomes from Nannochloropsis salina CCMP1776. Results The chloroplast and mitochondrial genomes of N. salina are 98.4% and 97% identical to their counterparts in Nannochloropsis gaditana. Comparison of the Nannochloropsis pangenome to other algae within and outside of the same phyla revealed regions of significant genetic divergence in key genes that encode proteins needed for regulation of branched chain amino synthesis (acetohydroxyacid synthase), carbon fixation (RuBisCO activase), energy conservation (ATP synthase), protein synthesis and homeostasis (Clp protease, ribosome). Conclusions Many organellar gene modifications in Nannochloropsis are unique and deviate from conserved orthologs found across the tree of life. Implementation of secondary and tertiary structure prediction was crucial to functionally characterize many proteins and therefore should be implemented in automated annotation pipelines. The exceptional similarity of the N. salina and N. gaditana organellar genomes suggests that N. gaditana be reclassified as a strain of N. salina. PMID:24646409

  11. Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea Photobacterium phosphoreum ANT-2200.

    PubMed

    Zhang, Sheng-Da; Santini, Claire-Lise; Zhang, Wei-Jia; Barbe, Valérie; Mangenot, Sophie; Guyomar, Charlotte; Garel, Marc; Chen, Hai-Tao; Li, Xue-Gong; Yin, Qun-Jian; Zhao, Yuan; Armengaud, Jean; Gaillard, Jean-Charles; Martini, Séverine; Pradel, Nathalie; Vidaud, Claude; Alberto, François; Médigue, Claudine; Tamburini, Christian; Wu, Long-Fei

    2016-05-01

    Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth. PMID:27039108

  12. Fruit ripening mutants reveal cell metabolism and redox state during ripening.

    PubMed

    Kumar, Vinay; Irfan, Mohammad; Ghosh, Sumit; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-03-01

    Ripening which leads to fruit senescence is an inimitable process characterized by vivid changes in color, texture, flavor, and aroma of the fleshy fruits. Our understanding of the mechanisms underlying the regulation of fruit ripening and senescence is far from complete. Molecular and biochemical studies on tomato (Solanum lycopersicum) ripening mutants such as ripening inhibitor (rin), nonripening (nor), and never ripe (Nr) have been useful in our understanding of fruit development and ripening. The MADS-box transcription factor RIN, a global regulator of fruit ripening, is vital for the broad aspects of ripening, in both ethylene-dependent and independent manners. Here, we have carried out microarray analysis to study the expression profiles of tomato genes during ripening of wild type and rin mutant fruits. Analysis of the differentially expressed genes revealed the role of RIN in regulation of several molecular and biochemical events during fruit ripening including fruit specialized metabolism and cellular redox state. The role of reactive oxygen species (ROS) during fruit ripening and senescence was further examined by determining the changes in ROS level during ripening of wild type and mutant fruits and by analyzing expression profiles of the genes involved in maintaining cellular redox state. Taken together, our findings suggest an important role of ROS during fruit ripening and senescence, and therefore, modulation of ROS level during ripening could be useful in achieving desired fruit quality. PMID:26008650

  13. UV-B mediated metabolic rearrangements in poplar revealed by non-targeted metabolomics.

    PubMed

    Kaling, Moritz; Kanawati, Basem; Ghirardo, Andrea; Albert, Andreas; Winkler, Jana Barbro; Heller, Werner; Barta, Csengele; Loreto, Francesco; Schmitt-Kopplin, Philippe; Schnitzler, Jörg-Peter

    2015-05-01

    Plants have to cope with various abiotic stresses including UV-B radiation (280-315 nm). UV-B radiation is perceived by a photoreceptor, triggers morphological responses and primes plant defence mechanisms such as antioxidant levels, photoreapir or accumulation of UV-B screening pigments. As poplar is an important model system for trees, we elucidated the influence of UV-B on overall metabolite patterns in poplar leaves grown under high UV-B radiation. Combining non-targeted metabolomics with gas exchange analysis and confocal microscopy, we aimed understanding how UV-B radiation triggers metabolome-wide changes, affects isoprene emission, photosynthetic performance, epidermal light attenuation and finally how isoprene-free poplars adjust their metabolome under UV-B radiation. Exposure to UV-B radiation caused a comprehensive rearrangement of the leaf metabolome. Several hundreds of metabolites were up- and down-regulated over various pathways. Our analysis, revealed the up-regulation of flavonoids, anthocyanins and polyphenols and the down-regulation of phenolic precursors in the first 36 h of UV-B treatment. We also observed a down-regulation of steroids after 12 h. The accumulation of phenolic compounds leads to a reduced light transmission in UV-B-exposed plants. However, the accumulation of phenolic compounds was reduced in non-isoprene-emitting plants suggesting a metabolic- or signalling-based interaction between isoprenoid and phenolic pathways. PMID:24738572

  14. Metabolic Profiles Reveal Changes in Wild and Cultivated Soybean Seedling Leaves under Salt Stress

    PubMed Central

    Zhang, Jing; Yang, Dongshuang; Li, Mingxia; Shi, Lianxuan

    2016-01-01

    Clarification of the metabolic mechanisms underlying salt stress responses in plants will allow further optimization of crop breeding and cultivation to obtain high yields in saline-alkali land. Here, we characterized 68 differential metabolites of cultivated soybean (Glycine max) and wild soybean (Glycine soja) under neutral-salt and alkali-salt stresses using gas chromatography-mass spectrometry (GC-MS)-based metabolomics, to reveal the physiological and molecular differences in salt tolerance. According to comparisons of growth parameters under the two kinds of salt stresses, the level of inhibition in wild soybean was lower than in cultivated soybean, especially under alkali-salt stress. Moreover, wild soybean contained significantly higher amounts of phenylalanine, asparagine, citraconic acid, citramalic acid, citric acid and α-ketoglutaric acid under neutral-salt stress, and higher amounts of palmitic acid, lignoceric acid, glucose, citric acid and α-ketoglutaric acid under alkali-salt stress, than cultivated soybean. Further investigations demonstrated that the ability of wild soybean to salt tolerance was mainly based on the synthesis of organic and amino acids, and the more active tricarboxylic acid cycle under neutral-salt stress. In addition, the metabolite profiling analysis suggested that the energy generation from β-oxidation, glycolysis and the citric acid cycle plays important roles under alkali-salt stress. Our results extend the understanding of mechanisms involved in wild soybean salt tolerance and provide an important reference for increasing yields and developing salt-tolerant soybean cultivars. PMID:27442489

  15. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult

    SciTech Connect

    Thompson, Melissa R.; VerBerkmoes, Nathan C.; Chourey, Karuna; Brown, Steven D.; Hettich, Robert L.; Thompson, Dorothea K.

    2006-04-05

    Shewanella oneidensis MR-1 is a gram-negative, facultatively anaerobic bacterium originally isolated from a freshwater lake. S. oneidensis MR-1 has the ability to reduce toxic metal ions [e.g., Cr(VI) and U(VI)] found in industrial and governmental waste sites. Cells were grown and exposed to three different metal concentrations in order to probe the dosage response of S. oneidensis MR-1 to Cr(VI) in the form of chromate. Protein fractions were digested with trypsin and analyzed with a multidimensional HPLC-NanoESIMS/MS protocol. The goal of this work is to identify protein components of pathways/mechanisms responsible for both detoxification and reduction of chromate.

  16. Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production

    PubMed Central

    Leoncikas, Vytautas; Wu, Huihai; Ward, Lara T.; Kierzek, Andrzej M.; Plant, Nick J.

    2016-01-01

    A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy. PMID:26813959

  17. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells

    PubMed Central

    Sharma, Prabhat K; Wong, Emily B; Napier, Ruth J; Bishai, William R; Ndung'u, Thumbi; Kasprowicz, Victoria O; Lewinsohn, Deborah A; Lewinsohn, David M; Gold, Marielle C

    2015-01-01

    Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1–2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1–2+ MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8+ T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8+ subsets we demonstrated that high expression of CD26 on CD8+ TRAV1–2+ cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161hi was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161dim. Using cell surface expression of CD8, TRAV1–2, and CD26hi in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis. PMID:25752900

  18. Metabolic Profiling Reveals PAFAH1B3 as a Critical Driver of Breast Cancer Pathogenicity

    PubMed Central

    Mulvihill, Melinda M.; Benjamin, Daniel I.; Ji, Xiaodan; Le Scolan, Erwan; Louie, Sharon M.; Shieh, Alice; Green, McKenna; Narasimhalu, Tara; Morris, Patrick J.; Luo, Kunxin; Nomura, Daniel K.

    2014-01-01

    Many studies have identified metabolic pathways that underlie cellular transformation, but the metabolic drivers of cancer progression remain less well understood. The Hippo transducer pathway has been shown to confer malignant traits on breast cancer cells. In this study, we used metabolic mapping platforms to identify biochemical drivers of cellular transformation and malignant progression driven through RAS and the Hippo pathway in breast cancer, and identified platelet activating factor acetylhydrolase 1B3 (PAFAH1B3) as a key metabolic driver of breast cancer pathogenicity that is upregulated in primary human breast tumors and correlated with poor prognosis. Metabolomic profiling suggests that PAFAH1B3 inactivation attenuates cancer pathogenicity through enhancing tumor-suppressing signaling lipids. Our studies provide a map of altered metabolism that underlies breast cancer progression and put forth PAFAH1B3 as a critical metabolic node in breast cancer. PMID:24954006

  19. Final Summary of "Interdisciplinary Study of Shewanella oneidensis MR-1's Metabolism & Metal Reduction"

    SciTech Connect

    Kolker, Eugene

    2007-06-26

    Our project focused primarily on analysis of different types of data produced by global high-throughput technologies, data integration of gene annotation, and gene and protein expression information, as well as on getting a better functional annotation of Shewanella genes. Specifically, four of our numerous major activities and achievements include the development of: statistical models for identification and expression proteomics, superior to currently available approaches (including our own earlier ones); approaches to improve gene annotations on the whole-organism scale; standards for annotation, transcriptomics and proteomics approaches; and generalized approaches for data integration of gene annotation, gene and protein expression information.

  20. Comparative metabolomics in primates reveals the effects of diet and gene regulatory variation on metabolic divergence

    PubMed Central

    Blekhman, Ran; Perry, George H.; Shahbaz, Sevini; Fiehn, Oliver; Clark, Andrew G.; Gilad, Yoav

    2014-01-01

    Human diets differ from those of non-human primates. Among few obvious differences, humans consume more meat than most non-human primates and regularly cook their food. It is hypothesized that a dietary shift during human evolution has been accompanied by molecular adaptations in metabolic pathways. Consistent with this notion, comparative studies of gene expression levels in primates have found that the regulation of genes with metabolic functions tend to evolve rapidly in the human lineage. The metabolic consequences of these regulatory differences, however, remained unknown. To address this gap, we performed a comparative study using a combination of gene expression and metabolomic profiling in livers from humans, chimpanzees, and rhesus macaques. We show that dietary differences between species have a strong effect on metabolic concentrations. In addition, we found that differences in metabolic concentration across species are correlated with inter-species differences in the expression of the corresponding enzymes, which control the same metabolic reaction. We identified a number of metabolic compounds with lineage-specific profiles, including examples of human-species metabolic differences that may be directly related to dietary differences. PMID:25069065

  1. Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment

    PubMed Central

    He, Dongli; Damaris, Rebecca N.; Fu, Jinlei; Tu, Jinxing; Fu, Tingdong; Xi, Chen; Yi, Bin; Yang, Pingfang

    2016-01-01

    Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid β-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy. PMID:27471506

  2. Comparative Genome Analysis Reveals Metabolic Versatility and Environmental Adaptations of Sulfobacillus thermosulfidooxidans Strain ST

    PubMed Central

    Guo, Xue; Yin, Huaqun; Liang, Yili; Hu, Qi; Zhou, Xishu; Xiao, Yunhua; Ma, Liyuan; Zhang, Xian; Qiu, Guanzhou; Liu, Xueduan

    2014-01-01

    The genus Sulfobacillus is a cohort of mildly thermophilic or thermotolerant acidophiles within the phylum Firmicutes and requires extremely acidic environments and hypersalinity for optimal growth. However, our understanding of them is still preliminary partly because few genome sequences are available. Here, the draft genome of Sulfobacillus thermosulfidooxidans strain ST was deciphered to obtain a comprehensive insight into the genetic content and to understand the cellular mechanisms necessary for its survival. Furthermore, the expressions of key genes related with iron and sulfur oxidation were verified by semi-quantitative RT-PCR analysis. The draft genome sequence of Sulfobacillus thermosulfidooxidans strain ST, which encodes 3225 predicted coding genes on a total length of 3,333,554 bp and a 48.35% G+C, revealed the high degree of heterogeneity with other Sulfobacillus species. The presence of numerous transposases, genomic islands and complete CRISPR/Cas defence systems testifies to its dynamic evolution consistent with the genome heterogeneity. As expected, S. thermosulfidooxidans encodes a suit of conserved enzymes required for the oxidation of inorganic sulfur compounds (ISCs). The model of sulfur oxidation in S. thermosulfidooxidans was proposed, which showed some different characteristics from the sulfur oxidation of Gram-negative A. ferrooxidans. Sulfur oxygenase reductase and heterodisulfide reductase were suggested to play important roles in the sulfur oxidation. Although the iron oxidation ability was observed, some key proteins cannot be identified in S. thermosulfidooxidans. Unexpectedly, a predicted sulfocyanin is proposed to transfer electrons in the iron oxidation. Furthermore, its carbon metabolism is rather flexible, can perform the transformation of pentose through the oxidative and non-oxidative pentose phosphate pathways and has the ability to take up small organic compounds. It encodes a multitude of heavy metal resistance systems to

  3. Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment.

    PubMed

    He, Dongli; Damaris, Rebecca N; Fu, Jinlei; Tu, Jinxing; Fu, Tingdong; Xi, Chen; Yi, Bin; Yang, Pingfang

    2016-01-01

    Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid β-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy. PMID:27471506

  4. Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

    PubMed Central

    Özdemir, Emre; McKinney, John D.

    2015-01-01

    ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. PMID:25691591

  5. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells.

    PubMed

    Laugel, Bruno; Lloyd, Angharad; Meermeier, Erin W; Crowther, Michael D; Connor, Thomas R; Dolton, Garry; Miles, John J; Burrows, Scott R; Gold, Marielle C; Lewinsohn, David M; Sewell, Andrew K

    2016-08-01

    The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells. PMID:27307560

  6. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells

    PubMed Central

    Lloyd, Angharad; Meermeier, Erin W.; Crowther, Michael D.; Connor, Thomas R.; Dolton, Garry; Miles, John J.; Burrows, Scott R.; Gold, Marielle C.; Lewinsohn, David M.

    2016-01-01

    The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1−/− clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1−/− clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells. PMID:27307560

  7. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

    DOE PAGESBeta

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2014-12-15

    We report that integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass andmore » corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. We found that the result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Lastly, taken together, the metabolic network modeling assisted

  8. Genome-based metabolic mapping and 13C flux analysis reveal systematic properties of an oleaginous microalga Chlorella protothecoides.

    PubMed

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2015-02-01

    Integrated and genome-based flux balance analysis, metabolomics, and (13)C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary (13)C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and (13)C

  9. Dynamic full field optical coherence tomography: subcellular metabolic contrast revealed in tissues by interferometric signals temporal analysis

    PubMed Central

    Apelian, Clement; Harms, Fabrice; Thouvenin, Olivier; Boccara, A. Claude

    2016-01-01

    We developed a new endogenous approach to reveal subcellular metabolic contrast in fresh ex vivo tissues taking advantage of the time dependence of the full field optical coherence tomography interferometric signals. This method reveals signals linked with local activity of the endogenous scattering elements which can reveal cells where other OCT-based techniques fail or need exogenous contrast agents. We benefit from the micrometric transverse resolution of full field OCT to image intracellular features. We used this time dependence to identify different dynamics at the millisecond scale on a wide range of organs in normal or pathological conditions. PMID:27446672

  10. Structure and function of the non-classical major histocompatibility complex molecule MR1.

    PubMed

    Krovi, S Harsha; Gapin, Laurent

    2016-08-01

    Polymorphic major histocompatibility complex (MHC) molecules play a central role in the vertebrate adaptive immune system. By presenting short peptides derived from pathogen-derived proteins, these "classical" MHC molecules can alert the T cell branch of the immune system of infected cells and clear the pathogen. There exist other "non-classical" MHC molecules, which while similar in structure to classical MHC proteins, are contrasted by their limited polymorphism. While the functions of many class Ib MHC molecules have still to be elucidated, the nature and diversity of antigens (if any) that some of them might present to the immune system is expected to be more restricted and might function as another approach to distinguish self from non-self. The MHC-related 1 (MR1) molecule is a member of this family of non-classical MHC proteins. It was recently shown to present unique antigens in the form of vitamin metabolites found in certain microbes. MR1 is strongly conserved genetically, structurally, and functionally through mammalian evolution, indicating its necessity in ensuring an effective immune system for members of this class. Although MR1 will be celebrating 21 years this year since its discovery, most of our understanding of how this molecule functions has only been uncovered in the past decade. Herein, we discuss where MR1 is expressed, how it selectively is able to bind to its appropriate antigens and how it, then, is able to specifically activate a distinct population of T cells. PMID:27448212

  11. Efficiencies of Bio-electrocatalytic Production of Hydrogen from Lactate Using Shewanella oneidensis MR-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shewanella oneidensis MR-1 was grown in a chemostatic, continuously-fed bioelectrochemical cell under slightly aerated conditions. The start-up phase was controlled potentiostatically (0.4 V vs. SHE). When a stable performance was achieved, the reactor was switched to bio-electrocatalytic producti...

  12. Metabolic Profiling during Peach Fruit Development and Ripening Reveals the Metabolic Networks That Underpin Each Developmental Stage1[C][W

    PubMed Central

    Lombardo, Verónica A.; Osorio, Sonia; Borsani, Julia; Lauxmann, Martin A.; Bustamante, Claudia A.; Budde, Claudio O.; Andreo, Carlos S.; Lara, María V.; Fernie, Alisdair R.; Drincovich, María F.

    2011-01-01

    Fruit from rosaceous species collectively display a great variety of flavors and textures as well as a generally high content of nutritionally beneficial metabolites. However, relatively little analysis of metabolic networks in rosaceous fruit has been reported. Among rosaceous species, peach (Prunus persica) has stone fruits composed of a juicy mesocarp and lignified endocarp. Here, peach mesocarp metabolic networks were studied across development using metabolomics and analysis of key regulatory enzymes. Principal component analysis of peach metabolic composition revealed clear metabolic shifts from early through late development stages and subsequently during postharvest ripening. Early developmental stages were characterized by a substantial decrease in protein abundance and high levels of bioactive polyphenols and amino acids, which are substrates for the phenylpropanoid and lignin pathways during stone hardening. Sucrose levels showed a large increase during development, reflecting translocation from the leaf, while the importance of galactinol and raffinose is also inferred. Our study further suggests that posttranscriptional mechanisms are key for metabolic regulation at early stages. In contrast to early developmental stages, a decrease in amino acid levels is coupled to an induction of transcripts encoding amino acid and organic acid catabolic enzymes during ripening. These data are consistent with the mobilization of amino acids to support respiration. In addition, sucrose cycling, suggested by the parallel increase of transcripts encoding sucrose degradative and synthetic enzymes, appears to operate during postharvest ripening. When taken together, these data highlight singular metabolic programs for peach development and may allow the identification of key factors related to agronomic traits of this important crop species. PMID:22021422

  13. Urinary Metabolomics Reveals Alterations of Aromatic Amino Acid Metabolism of Alzheimer's Disease in the Transgenic CRND8 Mice.

    PubMed

    Tang, Zhi; Liu, Liangfeng; Li, Yongle; Dong, Jiyang; Li, Min; Huang, Jiandong; Lin, Shuhai; Cai, Zongwei

    2016-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with amyloid plaques accumulation as the key feature involved in its pathology. To date, however, the biochemical changes in AD have not been clearly characterized. Here, we present that urinary metabolomics based on high resolution mass spectrometry was employed for delineation of metabolic alterations in transgenic CRND8 mice. In this noninvasive approach, urinary metabolome reveals the biochemical changes in early onset of this AD mouse model. In virtue of comprehensive metabolite profiling and multivariate statistical analysis, a total of 73 differential metabolites of urine sample sets was identified in 12-week and 18-week transgenic mice compared to wild-type littermates, covering perturbations of aromatic amino acid metabolism, the Krebs cycle and one-carbon metabolism. Of particular interest is that divergent tryptophan metabolism, such as upregulation of serotonin pathway while downregulation of kynurenine pathway, was observed. Meanwhile, the accumulation of both N-acetylvanilalanine and 3-methoxytyrosine indicated aromatic L-amino acid decarboxylase deficiency. And the microbial metabolites derived from aromatic amino acid metabolism and drug-like phase II metabolic response via the glycine conjugation reactions were also highlighted, indicating that genetic modification in mouse brain not only alters genotype but also perturbs the gut microbiome. Together, our study demonstrated that the integrative approach employing mass spectrometry-based metabolomics and a transgenic mouse model for AD may provide new evidence for distinct metabolic signatures. The perturbations of metabolic pathways may have far-reaching implications for early diagnosis and intervention in AD. PMID:26825095

  14. Metabolic profiling reveals altered pattern of central metabolism in navel orange plants as a result of boron deficiency.

    PubMed

    Liu, Guidong; Dong, Xiaochang; Liu, Leichao; Wu, Lishu; Peng, Shu'ang; Jiang, Cuncang

    2015-04-01

    We focused on the changes of metabolite profiles in navel orange plants under long-term boron (B) deficiency using a gas chromatography-mass spectrometry (GC-MS) approach. Curling of the leaves and leaf chlorosis were observed only in the upper leaves (present before start of the treatment) of B-deficient plants, while the lower leaves (grown during treatment) did not show any visible symptoms. The metabolites with up-accumulation in B-deficient leaves were mainly proline, l-ornithine, lysine, glucoheptonic acid, fucose, fumarate, oxalate, quinate, myo-inositol and allo-inositol, while the metabolites with down-accumulation in B-deficient leaves were mainly serine, asparagine, saccharic acid, citrate, succinate, shikimate and phytol. The levels of glucose and fructose were increased only in the upper leaves by B deficiency, while starch content was increased in all the leaves and in roots. The increased levels of malate, ribitol, gluconic acid and glyceric acid occurred only in the lower leaves of B-deficient plants. The increased levels of phenols only in the upper leaves indicated that the effects of B on phenol metabolism in citrus plants may be a consequence of disruptions in leaf structure. Metabolites with opposite reactions in upper and lower leaves were mainly glutamine, glycine and pyrrole-2-carboxylic acid. To our knowledge, the phenomena of allo-inositol even higher than myo-inositol occurred characterized for the first time in this species. These results suggested that the altered pattern of central metabolism may be either specific or adaptive responses of navel orange plants to B deficiency. PMID:25212059

  15. Roles of Two Shewanella oneidensis MR-1 Extracellular Endonucleases ▿ †

    PubMed Central

    Gödeke, Julia; Heun, Magnus; Bubendorfer, Sebastian; Paul, Kristina; Thormann, Kai M.

    2011-01-01

    The dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated that S. oneidensis MR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of two Shewanella extracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. Both exeM and exeS are highly expressed under phosphate-limited conditions. Mutants lacking exeS and/or exeM exhibit decreased eDNA degradation; however, the capability of S. oneidensis MR-1 to use DNA as the sole source of phosphorus is only affected in mutants lacking exeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion of exeM and/or exeS significantly affects biofilm formation of S. oneidensis MR-1 under static conditions, and expression of exeM and exeS drastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion of exeM leads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM of S. oneidensis MR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment. PMID:21705528

  16. Continuous monitoring reveals multiple controls on ecosystem metabolism in a suburban stream.

    EPA Science Inventory

    Ecosystem metabolism is an important mechanism for nutrient retention in streams, yet few high studies have investigated temporal patterns in gross primary production (GPP) and ecosystem respiration (ER) using high frequency measurements. This is a potentially important oversig...

  17. Systems level analysis of the Chlamydomonas reinhardtii metabolic network reveals variability in evolutionary co-conservation.

    PubMed

    Chaiboonchoe, Amphun; Ghamsari, Lila; Dohai, Bushra; Ng, Patrick; Khraiwesh, Basel; Jaiswal, Ashish; Jijakli, Kenan; Koussa, Joseph; Nelson, David R; Cai, Hong; Yang, Xinping; Chang, Roger L; Papin, Jason; Yu, Haiyuan; Balaji, Santhanam; Salehi-Ashtiani, Kourosh

    2016-07-19

    Metabolic networks, which are mathematical representations of organismal metabolism, are reconstructed to provide computational platforms to guide metabolic engineering experiments and explore fundamental questions on metabolism. Systems level analyses, such as interrogation of phylogenetic relationships within the network, can provide further guidance on the modification of metabolic circuitries. Chlamydomonas reinhardtii, a biofuel relevant green alga that has retained key genes with plant, animal, and protist affinities, serves as an ideal model organism to investigate the interplay between gene function and phylogenetic affinities at multiple organizational levels. Here, using detailed topological and functional analyses, coupled with transcriptomics studies on a metabolic network that we have reconstructed for C. reinhardtii, we show that network connectivity has a significant concordance with the co-conservation of genes; however, a distinction between topological and functional relationships is observable within the network. Dynamic and static modes of co-conservation were defined and observed in a subset of gene-pairs across the network topologically. In contrast, genes with predicted synthetic interactions, or genes involved in coupled reactions, show significant enrichment for both shorter and longer phylogenetic distances. Based on our results, we propose that the metabolic network of C. reinhardtii is assembled with an architecture to minimize phylogenetic profile distances topologically, while it includes an expansion of such distances for functionally interacting genes. This arrangement may increase the robustness of C. reinhardtii's network in dealing with varied environmental challenges that the species may face. The defined evolutionary constraints within the network, which identify important pairings of genes in metabolism, may offer guidance on synthetic biology approaches to optimize the production of desirable metabolites. PMID:27357594

  18. Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis

    PubMed Central

    Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; Bianchi, Cristina; Johnstone, Megan E.; Donohoe, Dallas R.; Trott, Josephine F.; Aboud, Omran Abu; Stirdivant, Steven; Neri, Bruce; Wolfert, Robert; Stewart, Benjamin; Perego, Roberto; Hsieh, James J.; Weiss, Robert H.

    2015-01-01

    Kidney cancer (or renal cell carcinoma [RCC]) is known as “the internist’s tumor” because it has protean systemic manifestations suggesting it utilizes complex, non-physiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the VHL tumor suppressor, in characterizing higher grade tumors we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, while the β-oxidation pathway is inhibited leading to increased fatty acyl-carnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared to other metabolic pathways. Together, our results offer a rationale to evaluate novel anti-metabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC. PMID:25952651

  19. NMR based metabolomics reveals acute hippocampal metabolic fluctuations during cranial irradiation in murine model.

    PubMed

    Rana, Poonam; Gupta, Mamta; Khan, Ahmad Raza; Hemanth Kumar, B S; Roy, Raja; Khushu, Subash

    2014-07-01

    Cranial irradiation is widely used as a treatment modality or prophylactic treatment in cancer patients, but it is frequently related to neurocognitive impairment in cancer survivors. Though most of radiation-induced changes occur during early and late delayed phase of radiation sickness, recent reports have supported the evidence of impaired neurogenesis within 24-48 h of radiation exposure that may implicate changes in acute phase as well. Inspection of these acute changes could be considered important as they may have long lasting effect on cognitive development and functions. In the present study, (1)H NMR spectroscopy based metabolomic approach was used to obtain comprehensive information of hippocampus metabolic physiology during acute phase of radiation sickness in a mouse model for single dose 8 Gy cranial irradiation. The analysis demonstrated reduced metabolic activity in irradiated animals compared to controls, typically evident in citric acid cycle intermediates, glutamine/glutamate and ketone bodies metabolism thus providing strong indication that the hippocampus is metabolically responsive to radiation exposure. The data suggested reduced glucose utilization, altered intermediary and neurotransmitter metabolism in hippocampus tissue extract. To the best of our knowledge this is the first metabolomic study to document cranial irradiation induced acute metabolic changes using in vitro(1)H NMR spectroscopy. PMID:24787771

  20. Combined Noninvasive Imaging and Modeling Approaches Reveal Metabolic Compartmentation in the Barley Endosperm[W][OA

    PubMed Central

    Rolletschek, Hardy; Melkus, Gerd; Grafahrend-Belau, Eva; Fuchs, Johannes; Heinzel, Nicolas; Schreiber, Falk; Jakob, Peter M.; Borisjuk, Ljudmilla

    2011-01-01

    The starchy endosperm of cereals is a priori taken as a metabolically uniform tissue. By applying a noninvasive assay based on 13C/1H-magnetic resonance imaging (MRI) to barley (Hordeum vulgare) grains, we uncovered metabolic compartmentation in the endosperm. 13C-Suc feeding during grain filling showed that the primary site of Ala synthesis was the central region of the endosperm, the part of the caryopsis experiencing the highest level of hypoxia. Region-specific metabolism in the endosperm was characterized by flux balance analysis (FBA) and metabolite profiling. FBA predicts that in the central region of the endosperm, the tricarboxylic acid cycle shifts to a noncyclic mode, accompanied by elevated glycolytic flux and the accumulation of Ala. The metabolic compartmentation within the endosperm is advantageous for the grain's carbon and energy economy, with a prominent role being played by Ala aminotransferase. An investigation of caryopses with a genetically perturbed tissue pattern demonstrated that Ala accumulation is a consequence of oxygen status, rather than being either tissue specific or dependent on the supply of Suc. Hence the 13C-Ala gradient can be used as an in vivo marker for hypoxia. The combination of MRI and metabolic modeling offers opportunities for the noninvasive analysis of metabolic compartmentation in plants. PMID:21856793

  1. A new tool in C. elegans reveals changes in secretory protein metabolism in ire-1-deficient animals.

    PubMed

    Safra, Modi; Henis-Korenblit, Sivan

    2014-01-01

    We recently showed that the ire-1/xbp-1 arm of the UPR plays a crucial role in maintaining basic endoplasmic reticulum (ER) functions required for the metabolism of secreted proteins even during unstressed growth conditions. During these studies we realized that although C. elegans is a powerful system to study the genetics of many cellular processes; it lacks effective tools for tracking the metabolism of secreted proteins at the cell and organism levels. Here, we outline how genetic manipulations and expression analysis of a DAF-28::GFP translational fusion transgene can be combined to infer different steps in the life cycle of secretory proteins. We demonstrate how we have used this tool to reveal folding defects, clearance defects, and secretion defects in ire-1 and xbp-1 mutants. We believe that further studies using this tool will deepen the understanding of secretory protein metabolism. PMID:25191629

  2. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

    PubMed Central

    Zauri, Melania; Berridge, Georgina; Thézénas, Marie-Laëtitia; Pugh, Kathryn M.; Goldin, Robert; Kessler, Benedikt M.; Kriaucionis, Skirmantas

    2015-01-01

    Summary Cells require nucleotides to support DNA replication and to repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Since epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands1-3, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2′deoxycytidine (5mdC) has been investigated before4-6, currently it remains unknown how cells deal with the recently identified oxidised forms of 5mdC – 5-hydroxymethyl-2′deoxycytidine (5hmdC), 5-formy-2′deoxycytidine (5fdC) and 5-carboxyl-2′deoxycytidine (5cadC)7-10. Here we demonstrate that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Interestingly, by screening cancer cell lines for growth defects following exposure to 5hmdC, we unexpectedly identify a subset of cell lines where 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches we discover that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidised epigenetic bases, and suggest a therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment

  3. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells.

    PubMed

    Harriff, Melanie J; Karamooz, Elham; Burr, Ansen; Grant, Wilmon F; Canfield, Elizabeth T; Sorensen, Michelle L; Moita, Luis F; Lewinsohn, David M

    2016-03-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  4. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

    PubMed Central

    Burr, Ansen; Grant, Wilmon F.; Canfield, Elizabeth T.; Sorensen, Michelle L.; Moita, Luis F.; Lewinsohn, David M.

    2016-01-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  5. Draft Genome Sequence of Burkholderia sp. MR1, a Methylarsenate-Reducing Bacterial Isolate from Florida Golf Course Soil.

    PubMed

    Pawitwar, Shashank S; Utturkar, Sagar M; Brown, Steven D; Yoshinaga, Masafumi; Rosen, Barry P

    2015-01-01

    To elucidate the environmental organoarsenical biocycle, we isolated a soil organism, Burkholderia sp. MR1, which reduces relatively nontoxic pentavalent methylarsenate to the more toxic trivalent methylarsenite, with the goal of identifying the gene for the reductase. Here, we report the draft genome sequence of Burkholderia sp. MR1. PMID:26044439

  6. Subsurface Cycling of Nitrogen and Anaerobic Aromatic Hydrocarbon Biodegradation Revealed by Nucleic Acid and Metabolic Biomarkers▿ †

    PubMed Central

    Yagi, Jane M.; Suflita, Joseph M.; Gieg, Lisa M.; DeRito, Christopher M.; Jeon, Che-Ok; Madsen, Eugene L.

    2010-01-01

    Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore in situ field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome c nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred in situ. These data link formation (via DNRA) and destruction (via nitrification) of ammonia to in situ cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane). PMID:20348302

  7. Molecular profiling of cetuximab and bevacizumab treatment of colorectal tumours reveals perturbations in metabolic and hypoxic response pathways

    PubMed Central

    Ji, Hong; Simpson, Richard J.; Rigopoulos, Angela; Murone, Carmel; Fang, Catherine; Gong, Sylvia; O'Keefe, Graeme

    2015-01-01

    Angiogenesis and epidermal growth factor receptor (EGFR) inhibition has been shown to have anti-tumour efficacy, and enhance the therapeutic effects of cytotoxic chemotherapy in metastatic colorectal cancer. The interplay of signalling alterations and changes in metabolism and hypoxia in tumours following anti-VEGF and anti-EGFR treatment is not well understood. We aimed to explore the pharmacodynamics of cetuximab and bevacizumab treatment in human colon carcinoma tumour cells in vitro and xenograft models through proteomic profiling, molecular imaging of metabolism and hypoxia, and evaluation of therapy-induced changes in tumour cells and the tumour microenvironment. Both cetuximab and bevacizumab inhibited tumour growth in vivo, and this effect was associated with selectively perturbed glucose metabolism and reduced hypoxic volumes based on PET/MRI imaging. Global proteomic profiling of xenograft tumours (in presence of cetuximab, bevacizumab, and combination treatments) revealed alterations in proteins involved in glucose, lipid and fatty acid metabolism (e.g., GPD2, ATP5B, STAT3, FASN), as well as hypoxic regulators and vasculogenesis (e.g., ATP5B, THBS1, HSPG2). These findings correlated with western immunoblotting (xenograft lysates) and histological examination by immunohistochemistry. These results define important mechanistic insight into the dynamic changes in metabolic and hypoxic response pathways in colorectal tumours following treatment with cetuximab and bevacizumab, and highlight the ability of these therapies to selectively impact on tumour cells and extracellular microenvironment. PMID:26517691

  8. Systemic analysis of inducible target of rapamycin mutants reveal a general metabolic switch controlling growth in Arabidopsis thaliana.

    PubMed

    Caldana, Camila; Li, Yan; Leisse, Andrea; Zhang, Yi; Bartholomaeus, Lisa; Fernie, Alisdair R; Willmitzer, Lothar; Giavalisco, Patrick

    2013-03-01

    The target of rapamycin (TOR) pathway is a major regulator of growth in all eukaryotes, integrating energy, nutrient and stress signals into molecular decisions. By using large-scale MS-based metabolite profiling of primary, secondary and lipid compounds in combination with array-based transcript profiling, we show that the TOR protein not only regulates growth but also influences nutrient partitioning and central energy metabolism. The study was performed on plants exhibiting conditional down-regulation of AtTOR expression, revealing strong regulation of genes involved in pathways such as the cell cycle, cell-wall modifications and senescence, together with major changes in transcripts and metabolites of the primary and secondary metabolism. In agreement with these results, our morphological and metabolic analyses disclosed major metabolic changes leading to massive accumulations of storage lipids and starch. The implications of these data in the context of the general role of TOR in eukaryotic systems are discussed in parallel with the plant-specific aspects of TOR function. Finally, we propose a role for harnessing the plant TOR pathway by utilizing it as a potent metabolic switch, offering a possible route for biotechnological optimization of plant energy content and carbon partitioning for the production of bioenergy. PMID:23173928

  9. Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory

    PubMed Central

    Tadi, Monika; Allaman, Igor; Lengacher, Sylvain; Grenningloh, Gabriele; Magistretti, Pierre J.

    2015-01-01

    We examined the expression of genes related to brain energy metabolism and particularly those encoding glia (astrocyte)-specific functions in the dorsal hippocampus subsequent to learning. Context-dependent avoidance behavior was tested in mice using the step-through Inhibitory Avoidance (IA) paradigm. Animals were sacrificed 3, 9, 24, or 72 hours after training or 3 hours after retention testing. The quantitative determination of mRNA levels revealed learning-induced changes in the expression of genes thought to be involved in astrocyte-neuron metabolic coupling in a time dependent manner. Twenty four hours following IA training, an enhanced gene expression was seen, particularly for genes encoding monocarboxylate transporters 1 and 4 (MCT1, MCT4), alpha2 subunit of the Na/K-ATPase and glucose transporter type 1. To assess the functional role for one of these genes in learning, we studied MCT1 deficient mice and found that they exhibit impaired memory in the inhibitory avoidance task. Together, these observations indicate that neuron-glia metabolic coupling undergoes metabolic adaptations following learning as indicated by the change in expression of key metabolic genes. PMID:26513352

  10. Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina

    PubMed Central

    Lindsay, Ken J.; Du, Jianhai; Sloat, Stephanie R.; Contreras, Laura; Linton, Jonathan D.; Turner, Sally J.; Sadilek, Martin; Hurley, James B.

    2014-01-01

    Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell’s ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1. PMID:25313047

  11. Insights into Mycoplasma genitalium Metabolism Revealed by the Structure of MG289, an Extracytoplasmic Thiamine Binding Lipoprotein

    PubMed Central

    Sippel, Katherine H.; Venkatakrishnan, Balasubramanian; Boehlein, Susan K.; Sankaran, Banumathi; Quirit, Jeanne G.; Govindasamy, Lakshamanan; Agbandje-McKenna, Mavis; Goodison, Steve; Rosser, Charles J.; McKenna, Robert

    2010-01-01

    Summary Mycoplasma genitalium is one of the smallest organisms capable of self-replication and its sequence is considered a starting point for understanding the minimal genome required for life. MG289, a putative phosphonate substrate binding protein, is considered to be one of these essential genes. The crystal structure of MG289 has been solved at 1.95 Å resolution. The structurally identified thiamine binding region reveals possible mechanisms for ligand promiscuity. MG289 was determined to be an extracytoplasmic thiamine binding lipoprotein. Computational analysis, size exclusion chromatography, and small angle X-ray scattering indicates that MG289 homodimerizes in a concentration-dependant manner. Comparisons to the thiamine pyrophosphate binding homolog Cypl reveal insights into the metabolic differences between mycoplasmal species including identifying possible kinases for cofactor phosphorylation and describing the mechanism of thiamine transport into the cell. These results provide a baseline to build our understanding of the minimal metabolic requirements of a living organism. PMID:21117240

  12. Transcriptome and selected metabolite analyses reveal points of sugar metabolism in jackfruit (Artocarpus heterophyllus Lam.).

    PubMed

    Hu, Lisong; Wu, Gang; Hao, Chaoyun; Yu, Huan; Tan, Lehe

    2016-07-01

    Artocarpus heterophyllus Lam., commonly known as jackfruit, produces the largest tree-borne fruit known thus far. The edible part of the fruit develops from the perianths, and contains many sugar-derived compounds. However, its sugar metabolism is poorly understood. A fruit perianth transcriptome was sequenced on an Illumina HiSeq 2500 platform, producing 32,459 unigenes with an average length of 1345nt. Sugar metabolism was characterized by comparing expression patterns of genes related to sugar metabolism and evaluating correlations with enzyme activity and sugar accumulation during fruit perianth development. During early development, high expression levels of acid invertases and corresponding enzyme activities were responsible for the rapid utilization of imported sucrose for fruit growth. The differential expression of starch metabolism-related genes and corresponding enzyme activities were responsible for starch accumulated before fruit ripening but decreased during ripening. Sucrose accumulated during ripening, when the expression levels of genes for sucrose synthesis were elevated and high enzyme activity was observed. The comprehensive transcriptome analysis presents fundamental information on sugar metabolism and will be a useful reference for further research on fruit perianth development in jackfruit. PMID:27181946

  13. Desorption Electrospray Ionization Mass Spectrometry Reveals Lipid Metabolism of Individual Oocytes and Embryos

    PubMed Central

    González-Serrano, Andrés Felipe; Pirro, Valentina; Ferreira, Christina R.; Oliveri, Paolo; Eberlin, Livia S.; Heinzmann, Julia; Lucas-Hahn, Andrea; Niemann, Heiner; Cooks, Robert Graham

    2013-01-01

    Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (qRT-PCR) analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG). Principal component analysis (PCA) and linear discriminant analysis (LDA), performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism. PMID:24073231

  14. Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis.

    PubMed

    Lee, Sunjae; Mardinoglu, Adil; Zhang, Cheng; Lee, Doheon; Nielsen, Jens

    2016-07-01

    Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies. PMID:27216817

  15. Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis

    PubMed Central

    Lee, Sunjae; Mardinoglu, Adil; Zhang, Cheng; Lee, Doheon; Nielsen, Jens

    2016-01-01

    Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies. PMID:27216817

  16. Coordinating Environmental Genomics and Geochemistry Reveals Metabolic Transitions in a Hot Spring Ecosystem

    PubMed Central

    Swingley, Wesley D.; Meyer-Dombard, D’Arcy R.; Shock, Everett L.; Alsop, Eric B.; Falenski, Heinz D.; Havig, Jeff R.; Raymond, Jason

    2012-01-01

    We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the “Bison Pool” (BP) Environmental Genome and a complementary contextual geochemical dataset of ∼75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92°C chemotrophic streamer biofilm community in the BP source pool to a 56°C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic “transition” community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring

  17. Coordinating environmental genomics and geochemistry reveals metabolic transitions in a hot spring ecosystem.

    PubMed

    Swingley, Wesley D; Meyer-Dombard, D'Arcy R; Shock, Everett L; Alsop, Eric B; Falenski, Heinz D; Havig, Jeff R; Raymond, Jason

    2012-01-01

    We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the "Bison Pool" (BP) Environmental Genome and a complementary contextual geochemical dataset of ~75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92 °C chemotrophic streamer biofilm community in the BP source pool to a 56 °C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic "transition" community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing

  18. Glucolytic fingerprinting reveals metabolic groups within the genus Bifidobacterium: an exploratory study.

    PubMed

    Rios-Covián, D; Sánchez, B; Cuesta, I; Cueto-Díaz, S; Hernández-Barranco, A M; Gueimonde, M; De Los Reyes-Gavilán, C G

    2016-03-11

    Microorganisms of the genus Bifidobacterium are inhabitants of diverse niches including the digestive tract of humans and animals. The species Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum have qualified presumption of safety status granted by EFSA and several strains are considered probiotic, and are being included in functional dairy fermented products. In the present work we carried out a preliminary exploration of general metabolic characteristics and organic acid production profiles of a reduced number of strains selected from these and other species of the genus Bifidobacterium. The use of resting cells allowed obtaining metabolic fingerprints without interference of metabolites accumulated during growth in culture media. Acetic acid was the most abundant organic acid formed per mol of glucose consumed (from 1.07±0.03 to 1.71±0.22 mol) followed by lactic acid (from 0.34±0.06 to 0.90±0.12 mol), with moderate differences in production among strains; pyruvic, succinic and formic acids were also produced at considerably lower proportions, with variability among strains. The acetic to lactic acid ratio showed lower values in stationary phase as regard to the exponential phase for most, but not all, the microorganisms; this was due to a decrease in acetic acid molar proportions together with increases of lactic acid proportions in stationary phase. A linear discriminant analysis allowed to cluster strains into species with 51-100% probability, evidencing different metabolic profiles, according to the relative production of organic acids from glucose by resting cells, of microorganisms collected at the exponential phase of growth. Looking for a single metabolic marker that could adequately discriminate metabolic groups, we found that groups established by the acetic to lactic acid ratio fit well with differences previously evidenced by the discriminant analysis. The proper

  19. Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death

    PubMed Central

    2016-01-01

    Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death. PMID:25965523

  20. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

    PubMed Central

    2015-01-01

    Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

  1. Correlation network analysis reveals relationships between diet-induced changes in human gut microbiota and metabolic health

    PubMed Central

    Kelder, T; Stroeve, J H M; Bijlsma, S; Radonjic, M; Roeselers, G

    2014-01-01

    Background: Recent evidence suggests that the gut microbiota plays an important role in human metabolism and energy homeostasis and is therefore a relevant factor in the assessment of metabolic health and flexibility. Understanding of these host–microbiome interactions aids the design of nutritional strategies that act via modulation of the microbiota. Nevertheless, relating gut microbiota composition to host health states remains challenging because of the sheer complexity of these ecosystems and the large degrees of interindividual variation in human microbiota composition. Methods: We assessed fecal microbiota composition and host response patterns of metabolic and inflammatory markers in 10 apparently healthy men subjected to a high-fat high-caloric diet (HFHC, 1300 kcal/day extra) for 4 weeks. DNA was isolated from stool and barcoded 16S rRNA gene amplicons were sequenced. Metabolic health parameters, including anthropomorphic and blood parameters, where determined at t=0 and t=4 weeks. Results: A correlation network approach revealed diet-induced changes in Bacteroides levels related to changes in carbohydrate oxidation rates, whereas the change in Firmicutes correlates with changes in fat oxidation. These results were confirmed by multivariate models. We identified correlations between microbial diversity indices and several inflammation-related host parameters that suggest a relation between diet-induced changes in gut microbiota diversity and inflammatory processes. Conclusions: This approach allowed us to identify significant correlations between abundances of microbial taxa and diet-induced shifts in several metabolic health parameters. Constructed correlation networks provide an overview of these relations, revealing groups of correlations that are of particular interest for explaining host health aspects through changes in the gut microbiota. PMID:24979151

  2. Metabolic Brain Covariant Networks as Revealed by FDG-PET with Reference to Resting-State fMRI Networks

    PubMed Central

    Di, Xin

    2012-01-01

    Abstract The human brain is inherently organized as separate networks, as has been widely revealed by resting-state functional magnetic resonance imaging (fMRI). Although the large-scale functional connectivity can be partially explained by the underlying white-matter structural connectivity, the question of whether the underlying functional connectivity is related to brain metabolic factors is still largely unanswered. The present study investigated the presence of metabolic covariant networks across subjects using a set of fluorodeoxyglucose (18F, FDG) positron-emission tomography (PET) images. Spatial-independent component analysis was performed on the subject series of FDG-PET images. A number of networks that were mainly homotopic regions could be identified, including visual, auditory, motor, cerebellar, and subcortical networks. However, the anterior-posterior networks such as the default-mode and left frontoparietal networks could not be observed. Region-of-interest-based correlation analysis confirmed that the intersubject metabolic covariances within the default-mode and left frontoparietal networks were reduced as compared with corresponding time-series correlations using resting-state fMRI from an independent sample. In contrast, homotopic intersubject metabolic covariances observed using PET were comparable to the corresponding fMRI resting-state time-series correlations. The current study provides preliminary illustration, suggesting that the human brain metabolism pertains to organized covariance patterns that might partially reflect functional connectivity as revealed by resting-state blood oxygen level dependent (BOLD). The discrepancy between the PET covariance and BOLD functional connectivity might reflect the differences of energy consumption coupling and ongoing neural synchronization within these brain networks. PMID:23025619

  3. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    SciTech Connect

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

    2015-10-22

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Furthermore, glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.

  4. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    NASA Astrophysics Data System (ADS)

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

    2015-10-01

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.

  5. Genome-scale model reveals metabolic basis of biomass partitioning in a model diatom

    DOE PAGESBeta

    Levering, Jennifer; Broddrick, Jared; Dupont, Christopher L.; Peers, Graham; Beeri, Karen; Mayers, Joshua; Gallina, Alessandra A.; Allen, Andrew E.; Palsson, Bernhard O.; Zengler, Karsten; et al

    2016-05-06

    Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curatedmore » reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. Furthermore, the model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications.« less

  6. Genome-Scale Model Reveals Metabolic Basis of Biomass Partitioning in a Model Diatom.

    PubMed

    Levering, Jennifer; Broddrick, Jared; Dupont, Christopher L; Peers, Graham; Beeri, Karen; Mayers, Joshua; Gallina, Alessandra A; Allen, Andrew E; Palsson, Bernhard O; Zengler, Karsten

    2016-01-01

    Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications. PMID:27152931

  7. Resistance to Aerobic Exercise Training Causes Metabolic Dysfunction and Reveals Novel Exercise-Regulated Signaling Networks

    PubMed Central

    Lessard, Sarah J.; Rivas, Donato A.; Alves-Wagner, Ana B.; Hirshman, Michael F.; Gallagher, Iain J.; Constantin-Teodosiu, Dumitru; Atkins, Ryan; Greenhaff, Paul L.; Qi, Nathan R.; Gustafsson, Thomas; Fielding, Roger A.; Timmons, James A.; Britton, Steven L.; Koch, Lauren G.; Goodyear, Laurie J.

    2013-01-01

    Low aerobic exercise capacity is a risk factor for diabetes and a strong predictor of mortality, yet some individuals are “exercise-resistant” and unable to improve exercise capacity through exercise training. To test the hypothesis that resistance to aerobic exercise training underlies metabolic disease risk, we used selective breeding for 15 generations to develop rat models of low and high aerobic response to training. Before exercise training, rats selected as low and high responders had similar exercise capacities. However, after 8 weeks of treadmill training, low responders failed to improve their exercise capacity, whereas high responders improved by 54%. Remarkably, low responders to aerobic training exhibited pronounced metabolic dysfunction characterized by insulin resistance and increased adiposity, demonstrating that the exercise-resistant phenotype segregates with disease risk. Low responders had impaired exercise-induced angiogenesis in muscle; however, mitochondrial capacity was intact and increased normally with exercise training, demonstrating that mitochondria are not limiting for aerobic adaptation or responsible for metabolic dysfunction in low responders. Low responders had increased stress/inflammatory signaling and altered transforming growth factor-β signaling, characterized by hyperphosphorylation of a novel exercise-regulated phosphorylation site on SMAD2. Using this powerful biological model system, we have discovered key pathways for low exercise training response that may represent novel targets for the treatment of metabolic disease. PMID:23610057

  8. Metabonomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits

    PubMed Central

    Zhao, Xinjie; Fritsche, Jens; Wang, Jiangshan; Chen, Jing; Rittig, Kilian; Schmitt-Kopplin, Philippe; Fritsche, Andreas; Häring, Hans-Ulrich; Schleicher, Erwin D.

    2010-01-01

    Impaired glucose tolerance (IGT) which precedes overt type 2 diabetes (T2DM) for decades is associated with multiple metabolic alterations in insulin sensitive tissues. In an UPLC-qTOF-mass spectrometry-driven non-targeted metabonomics approach we investigated plasma as well as spot urine of 51 non-diabetic, overnight fasted individuals aiming to separate subjects with IGT from controls thereby identify pathways affected by the pre-diabetic metabolic state. We could clearly demonstrate that normal glucose tolerant (NGT) and IGT subjects clustered in two distinct groups independent of the investigated metabonome. These findings reflect considerable differences in individual metabolite fingerprints, both in plasma and urine. Pre-diabetes associated alterations in fatty acid-, tryptophan-, uric acid-, bile acid-, and lysophosphatidylcholine-metabolism, as well as the TCA cycle were identified. Of note, individuals with IGT also showed decreased levels of gut flora-associated metabolites namely hippuric acid, methylxanthine, methyluric acid, and 3-hydroxyhippuric acid. The findings of our non-targeted UPLC-qTOF-MS metabonomics analysis in plasma and spot urine of individuals with IGT vs NGT offers novel insights into the metabolic alterations occurring in the long, asymptomatic period preceding the manifestation of T2DM thereby giving prospects for new intervention targets. Electronic supplementary material The online version of this article (doi:10.1007/s11306-010-0203-1) contains supplementary material, which is available to authorized users. PMID:20676218

  9. Microbial structures, functions, and metabolic pathways in wastewater treatment bioreactors revealed using high-throughput sequencing.

    PubMed

    Ye, Lin; Zhang, Tong; Wang, Taitao; Fang, Zhiwei

    2012-12-18

    The objective of this study was to explore microbial community structures, functional profiles, and metabolic pathways in a lab-scale and a full-scale wastewater treatment bioreactors. In order to do this, over 12 gigabases of metagenomic sequence data and 600,000 paired-end sequences of bacterial 16S rRNA gene were generated with the Illumina HiSeq 2000 platform, using DNA extracted from activated sludge in the two bioreactors. Three kinds of sequences (16S rRNA gene amplicons, 16S rRNA gene sequences obtained from metagenomic sequencing, and predicted proteins) were used to conduct taxonomic assignments. Specially, relative abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed. Compared with quantitative real-time PCR (qPCR), metagenomic sequencing was demonstrated to be a better approach to quantify AOA and AOB in activated sludge samples. It was found that AOB were more abundant than AOA in both reactors. Furthermore, the analysis of the metabolic profiles indicated that the overall patterns of metabolic pathways in the two reactors were quite similar (73.3% of functions shared). However, for some pathways (such as carbohydrate metabolism and membrane transport), the two reactors differed in the number of pathway-specific genes. PMID:23151157

  10. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    PubMed Central

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

    2015-01-01

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines. PMID:26489853

  11. Genome-Scale Model Reveals Metabolic Basis of Biomass Partitioning in a Model Diatom

    PubMed Central

    Broddrick, Jared; Dupont, Christopher L.; Peers, Graham; Beeri, Karen; Mayers, Joshua; Gallina, Alessandra A.; Allen, Andrew E.; Palsson, Bernhard O.; Zengler, Karsten

    2016-01-01

    Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications. PMID:27152931

  12. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    DOE PAGESBeta

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; et al

    2015-10-22

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cellmore » cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Furthermore, glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.« less

  13. Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain.

    PubMed

    Patassini, Stefano; Begley, Paul; Xu, Jingshu; Church, Stephanie J; Reid, Suzanne J; Kim, Eric H; Curtis, Maurice A; Dragunow, Mike; Waldvogel, Henry J; Snell, Russell G; Unwin, Richard D; Faull, Richard L M; Cooper, Garth J S

    2016-09-01

    Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain. PMID:27267344

  14. Plasma metabolic signatures reveal the regulatory effect of exercise training in db/db mice.

    PubMed

    Xiang, L; Cheang, W S; Lin, S H; Wang, L; Li, Y L; Huang, Y; Cai, Z W

    2015-09-01

    Type 2 diabetes (T2DM) is caused by a complex set of interactions between genetic modifications and life styles. This complexity creates challenges for a full understanding of the altered metabolic pathways that contribute to the development of T2DM, which needs a comprehensive metabolic analysis. Exercise training is a common therapeutic approach known to antagonize the metabolic consequences of T2DM. However, the metabolic phenotypes of exercise effected in T2DM have not been clearly characterized. Here, we present the effect of physical activity on biochemical changes in diabetic db/db mice. An untargeted metabolomics study based on liquid chromatography coupled with high resolution mass spectrometry was carried out to delineate the plasma metabolic signatures in conjunction with a multivariate statistical analysis. As a result, a total of 24 differential metabolites were identified, covering amino acids, organic acids and lipids. Three biomarkers, including lysine, creatine and uridine, were significantly reversed by exercise training in db/db diabetic mice groups compared to lean db/m+ groups. Of note, pantothenic acid and palmitoylcarnitine, which are involved in fatty acid β-oxidation (FAO), were promoted by exercise training in diabetic mice rather than in lean mice. These findings indicated that diabetic mice might be more susceptible to exercise for energy expenditure. Together, the results might demonstrate that exercise could mitigate insulin resistance in T2DM through improving FAO and that uridine in blood might be an important indicator to reflect insulin sensitivity promoted by exercise training in T2DM mice. PMID:26227879

  15. Genome-resolved metagenomics reveals that sulfur metabolism dominates the microbial ecology of rising hydrothermal plumes

    NASA Astrophysics Data System (ADS)

    Anantharaman, K.; Breier, J. A., Jr.; Jain, S.; Reed, D. C.; Dick, G.

    2015-12-01

    Deep-sea hydrothermal plumes occur when hot fluids from hydrothermal vents replete with chemically reduced elements and compounds like sulfide, methane, hydrogen, ammonia, iron and manganese mix with cold, oxic seawater. Chemosynthetic microbes use these reduced chemicals to power primary production and are pervasive throughout the deep sea, even at sites far removed from hydrothermal vents. Although neutrally-buoyant hydrothermal plumes have been well-studied, rising hydrothermal plumes have received little attention even though they represent an important interface in the deep-sea where microbial metabolism and particle formation processes control the transformation of important elements and impact global biogeochemical cycles. In this study, we used genome-resolved metagenomic analyses and thermodynamic-bioenergetic modeling to study the microbial ecology of rising hydrothermal plumes at five different hydrothermal vents spanning a range of geochemical gradients at the Eastern Lau Spreading Center (ELSC) in the Western Pacific Ocean. Our analyses show that differences in the geochemistry of hydrothermal vents do not manifest in microbial diversity and community composition, both of which display only minor variance across ELSC hydrothermal plumes. Microbial metabolism is dominated by oxidation of reduced sulfur species and supports a diversity of bacteria, archaea and viruses that provide intriguing insights into metabolic plasticity and virus-mediated horizontal gene transfer in the microbial community. The manifestation of sulfur oxidation genes in hydrogen and methane oxidizing organisms hints at metabolic opportunism in deep-sea microbes that would enable them to respond to varying redox conditions in hydrothermal plumes. Finally, we infer that the abundance, diversity and metabolic versatility of microbes associated with sulfur oxidation impart functional redundancy that could allow it to persist in the dynamic settings of hydrothermal plumes.

  16. Metabolic profiling reveals altered nitrogen nutrient regimes have diverse effects on the metabolism of hydroponically-grown tomato (Solanum lycopersicum) plants.

    PubMed

    Urbanczyk-Wochniak, Ewa; Fernie, Alisdair R

    2005-01-01

    The role of inorganic nitrogen assimilation in the production of amino acids is one of the most important biochemical processes in plants. For this reason, a detailed broad-range characterization of the metabolic response of tomato (Solanum lycopersicum) leaves to the alteration of nitrate level was performed. Tomato plants were grown hydroponically in liquid culture under three different nitrate regimes: saturated (8 mM NO3-), replete (4 mM NO3-) and deficient (0.4 mM NO3-). All treatments were performed under varied light intensity, with leaf samples being collected after 7, 14, and 21 d. In addition, the short-term response (after 1, 24, 48, and 94 h) to varying nutrient status was evaluated at the higher light intensity. GC-MS analysis of the levels of amino acids, tricarboxylic acid cycle intermediates, sugars, sugar alcohols, and representative compounds of secondary metabolism revealed substantial changes under the various growth regimes applied. The data presented here suggest that nitrate nutrition has wide-ranging effects on plant leaf metabolism with nitrate deficiency resulting in decreases in many amino and organic acids and increases in the level of several carbohydrates and phosphoesters, as well as a handful of secondary metabolites. These results are compared with previously reported transcript profiles of altered nitrogen regimes and discussed within the context of current models of carbon nitrogen interaction. PMID:15596475

  17. The octaheme SirA catalyses dissimilatory sulfite reduction in Shewanella oneidensis MR-1

    SciTech Connect

    Shirodkar, Sheetal; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad

    2011-01-01

    Shewanella oneidensis MR-1 is a metal reducer that uses a large number of electron acceptors that include thiosulfate, polysulfide, and sulfite. The enzyme required for thiosulfate and polysulfide respiration has been recently identified, but the mechanisms of sulfite reduction remained unexplored. Analysis of MR-1 cultures grown anaerobically with sulfite suggested that the dissimilatory sulfite reductase catalyzes six-electron reduction of sulfite to sulfide. Reduction of sulfite required menaquinones and c cytochromes but appeared to be independent of the intermediate electron carrier CymA. Furthermore, the terminal sulfite reductase, SirA, was identified as an octaheme c cytochrome with an atypical heme binding site that represents a new class of sulfite reductases. The sirA locus was identified in the genomes of several sequenced Shewanella genomes, and its presence appears to be linked to the ability of these organisms to reduce sulfite under anaerobic conditions.

  18. Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Wu, Chao; Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Wen-Wei; Li, Dao-Bo; Yu, Han-Qing

    2013-05-01

    Shewanella oneidensis MR-1 is an extensively studied dissimilatory metal-reducing bacterium with a great potential for bioremediation and electricity generation. It secretes flavins as electron shuttles which play an important role in extracellular electron transfer. However, the influence of various environmental factors on the secretion of flavins is largely unknown. Here, the effects of electron acceptors, including fumarate, ferrihydrite, Fe(III)-nitrilotriacetic acid (NTA), nitrate and trimethylamine oxide (TMAO), on the secretion of flavins were investigated. The level of riboflavin and riboflavin-5'-phosphate (FMN) secreted by S. oneidensis MR-1 varied considerably with different electron acceptors. While nitrate and ferrihydrite suppressed the secretion of flavins in relative to fumarate, Fe(III)-NTA and TMAO promoted such a secretion and greatly enhanced ferrihydrite reduction and electricity generation. This work clearly demonstrates that electron acceptors could considerably affect the secretion of flavins and consequent microbial EET. Such impacts of electron acceptors in the environment deserve more attention. PMID:23558182

  19. MR1-restricted mucosal associated invariant T (MAIT) cells in the immune response to Mycobacterium tuberculosis

    PubMed Central

    Gold, Marielle C.; Napier, Ruth J.; Lewinsohn, David M.

    2014-01-01

    Summary The intracellular pathogen Mycobacterium tuberculosis (Mtb) and its human host have long co-evolved. Although the host cellular immune response is critical to the control of the bacterium information on the specific contribution of different immune cell subsets in humans is incomplete. Mucosal associated invariant T (MAIT) cells are a prevalent and unique T-cell population in humans with the capacity to detect intracellular infection with bacteria including Mtb. MAIT cells detect bacterially derived metabolites presented by the evolutionarily conserved major histocompatibility complex-like molecule MR1. Here we review recent advances in our understanding of this T-cell subset and address the potential roles for MR1-restricted T cells in the control, diagnosis, and therapy of tuberculosis. PMID:25703558

  20. Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production

    PubMed Central

    2013-01-01

    Background Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. Results Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category “carbohydrate metabolic process” and in “fatty acid biosynthetic process” in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. Conclusions The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the

  1. Influence of riboflavin on the reduction of radionuclides by Shewanella oneidenis MR-1.

    PubMed

    Cherkouk, Andrea; Law, Gareth T W; Rizoulis, Athanasios; Law, Katie; Renshaw, Joanna C; Morris, Katherine; Livens, Francis R; Lloyd, Jonathan R

    2016-03-28

    Uranium (as UO2(2+)), technetium (as TcO4(-)) and neptunium (as NpO2(+)) are highly mobile radionuclides that can be reduced enzymatically by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble species. The redox chemistry of Pu is more complicated, but the dominant oxidation state in most environments is highly insoluble Pu(IV), which can be reduced to Pu(III) which has a potentially increased solubility which could enhance migration of Pu in the environment. Recently it was shown that flavins (riboflavin and flavin mononucleotide (FMN)) secreted by Shewanella oneidensis MR-1 can act as electron shuttles, promoting anoxic growth coupled to the accelerated reduction of poorly-crystalline Fe(III) oxides. Here, we studied the role of riboflavin in mediating the reduction of radionuclides in cultures of Shewanella oneidensis MR-1. Our results demonstrate that the addition of 10 μM riboflavin enhances the reduction rate of Tc(VII) to Tc(IV), Pu(IV) to Pu(III) and to a lesser extent, Np(V) to Np(IV), but has no significant influence on the reduction rate of U(VI) by Shewanella oneidensis MR-1. Thus riboflavin can act as an extracellular electron shuttle to enhance rates of Tc(VII), Np(V) and Pu(IV) reduction, and may therefore play a role in controlling the oxidation state of key redox active actinides and fission products in natural and engineered environments. These results also suggest that the addition of riboflavin could be used to accelerate the bioremediation of radionuclide-contaminated environments. PMID:26632613

  2. Metabolism

    MedlinePlus

    Metabolism refers to all the physical and chemical processes in the body that convert or use energy, ... Tortora GJ, Derrickson BH. Metabolism. In: Tortora GJ, Derrickson BH. Principles of Anatomy and Physiology . 14th ed. Hoboken, NJ: John H Wiley and Sons; 2013: ...

  3. Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

    PubMed

    Paulick, Anja; Delalez, Nicolas J; Brenzinger, Susanne; Steel, Bradley C; Berry, Richard M; Armitage, Judith P; Thormann, Kai M

    2015-06-01

    The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels. PMID:25727785

  4. Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics.

    PubMed

    Kumar, Yashwant; Zhang, Limin; Panigrahi, Priyabrata; Dholakia, Bhushan B; Dewangan, Veena; Chavan, Sachin G; Kunjir, Shrikant M; Wu, Xiangyu; Li, Ning; Rajmohanan, Pattuparambil R; Kadoo, Narendra Y; Giri, Ashok P; Tang, Huiru; Gupta, Vidya S

    2016-07-01

    Molecular changes elicited by plants in response to fungal attack and how this affects plant-pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label-free proteomics and NMR-based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis-related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea-Foc interactions. PMID:26801007

  5. Visualization of in vivo metabolic flows reveals accelerated utilization of glucose and lactate in penumbra of ischemic heart.

    PubMed

    Sugiura, Yuki; Katsumata, Yoshinori; Sano, Motoaki; Honda, Kurara; Kajimura, Mayumi; Fukuda, Keiichi; Suematsu, Makoto

    2016-01-01

    Acute ischemia produces dynamic changes in labile metabolites. To capture snapshots of such acute metabolic changes, we utilized focused microwave treatment to fix metabolic flow in vivo in hearts of mice 10 min after ligation of the left anterior descending artery. The left ventricle was subdivided into short-axis serial slices and the metabolites were analyzed by capillary electrophoresis mass spectrometry and matrix-assisted laser desorption/ionization imaging mass spectrometry. These techniques allowed us to determine the fate of exogenously administered (13)C6-glucose and (13)C3-lactate. The penumbra regions, which are adjacent to the ischemic core, exhibited the greatest adenine nucleotide energy charge and an adenosine overflow extending from the ischemic core, which can cause ischemic hyperemia. Imaging analysis of metabolic pathway flows revealed that the penumbra executes accelerated glucose oxidation, with remaining lactate utilization for tricarboxylic acid cycle for energy compensation, suggesting unexpected metabolic interplays of the penumbra with the ischemic core and normoxic regions. PMID:27581923

  6. Visualization of in vivo metabolic flows reveals accelerated utilization of glucose and lactate in penumbra of ischemic heart

    PubMed Central

    Sugiura, Yuki; Katsumata, Yoshinori; Sano, Motoaki; Honda, Kurara; Kajimura, Mayumi; Fukuda, Keiichi; Suematsu, Makoto

    2016-01-01

    Acute ischemia produces dynamic changes in labile metabolites. To capture snapshots of such acute metabolic changes, we utilized focused microwave treatment to fix metabolic flow in vivo in hearts of mice 10 min after ligation of the left anterior descending artery. The left ventricle was subdivided into short-axis serial slices and the metabolites were analyzed by capillary electrophoresis mass spectrometry and matrix-assisted laser desorption/ionization imaging mass spectrometry. These techniques allowed us to determine the fate of exogenously administered 13C6-glucose and 13C3-lactate. The penumbra regions, which are adjacent to the ischemic core, exhibited the greatest adenine nucleotide energy charge and an adenosine overflow extending from the ischemic core, which can cause ischemic hyperemia. Imaging analysis of metabolic pathway flows revealed that the penumbra executes accelerated glucose oxidation, with remaining lactate utilization for tricarboxylic acid cycle for energy compensation, suggesting unexpected metabolic interplays of the penumbra with the ischemic core and normoxic regions. PMID:27581923

  7. Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis Flowers[W][OPEN

    PubMed Central

    Ginglinger, Jean-François; Boachon, Benoit; Höfer, René; Paetz, Christian; Köllner, Tobias G.; Miesch, Laurence; Lugan, Raphael; Baltenweck, Raymonde; Mutterer, Jérôme; Ullmann, Pascaline; Beran, Franziska; Claudel, Patricia; Verstappen, Francel; Fischer, Marc J.C.; Karst, Francis; Bouwmeester, Harro; Miesch, Michel; Schneider, Bernd; Gershenzon, Jonathan; Ehlting, Jürgen; Werck-Reichhart, Danièle

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (−)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined. PMID:24285789

  8. The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics*

    PubMed Central

    Wang, Jieping; Mei, Han; Zheng, Cao; Qian, Hongliang; Cui, Cui; Fu, Yang; Su, Jianmei; Liu, Ziduo; Yu, Ziniu; He, Jin

    2013-01-01

    Bacillus thuringiensis is a well-known entomopathogenic bacterium used worldwide as an environmentally compatible biopesticide. During sporulation, B. thuringiensis accumulates a large number of parasporal crystals consisting of insecticidal crystal proteins (ICPs) that can account for nearly 20–30% of the cell's dry weight. However, the metabolic regulation mechanisms of ICP synthesis remain to be elucidated. In this study, the combined efforts in transcriptomics and proteomics mainly uncovered the following 6 metabolic regulation mechanisms: (1) proteases and the amino acid metabolism (particularly, the branched-chain amino acids) became more active during sporulation; (2) stored poly-β-hydroxybutyrate and acetoin, together with some low-quality substances provided considerable carbon and energy sources for sporulation and parasporal crystal formation; (3) the pentose phosphate shunt demonstrated an interesting regulation mechanism involving gluconate when CT-43 cells were grown in GYS medium; (4) the tricarboxylic acid cycle was significantly modified during sporulation; (5) an obvious increase in the quantitative levels of enzymes and cytochromes involved in energy production via the electron transport system was observed; (6) most F0F1-ATPase subunits were remarkably up-regulated during sporulation. This study, for the first time, systematically reveals the metabolic regulation mechanisms involved in the supply of amino acids, carbon substances, and energy for B. thuringiensis spore and parasporal crystal formation at both the transcriptional and translational levels. PMID:23408684

  9. High-field proton magnetic resonance spectroscopy reveals metabolic effects of normal brain aging

    PubMed Central

    Harris, Janna L.; Yeh, Hung-Wen; Swerdlow, Russell H.; Choi, In-Young; Lee, Phil; Brooks, William M.

    2014-01-01

    Altered brain metabolism is likely to be an important contributor to normal cognitive decline and brain pathology in elderly individuals. To characterize the metabolic changes associated with normal brain aging, we used high-field proton magnetic resonance spectroscopy in vivo to quantify 20 neurochemicals in the hippocampus and sensorimotor cortex of young adult and aged rats. We found significant differences in the neurochemical profile of the aged brain when compared with younger adults, including lower aspartate, ascorbate, glutamate, and macromolecules, and higher glucose, myo-inositol, N-acetylaspartylglutamate, total choline, and glutamine. These neurochemical biomarkers point to specific cellular mechanisms that are altered in brain aging, such as bioenergetics, oxidative stress, inflammation, cell membrane turnover, and endogenous neuroprotection. Proton magnetic resonance spectroscopy may be a valuable translational approach for studying mechanisms of brain aging and pathology, and for investigating treatments to preserve or enhance cognitive function in aging. PMID:24559659

  10. Molecular anatomy of the gut-brain axis revealed with transgenic technologies: implications in metabolic research

    PubMed Central

    Udit, Swalpa; Gautron, Laurent

    2013-01-01

    Neurons residing in the gut-brain axis remain understudied despite their important role in coordinating metabolic functions. This lack of knowledge is observed, in part, because labeling gut-brain axis neurons and their connections using conventional neuroanatomical methods is inherently challenging. This article summarizes genetic approaches that enable the labeling of distinct populations of gut-brain axis neurons in living laboratory rodents. In particular, we review the respective strengths and limitations of currently available genetic and viral approaches that permit the marking of gut-brain axis neurons without the need for antibodies or conventional neurotropic tracers. Finally, we discuss how these methodological advances are progressively transforming the study of the healthy and diseased gut-brain axis in the context of its role in chronic metabolic diseases, including diabetes and obesity. PMID:23914153

  11. NMR metabolomics of human lung tumours reveals distinct metabolic signatures for adenocarcinoma and squamous cell carcinoma.

    PubMed

    Rocha, Cláudia M; Barros, António S; Goodfellow, Brian J; Carreira, Isabel M; Gomes, Ana; Sousa, Vitor; Bernardo, João; Carvalho, Lina; Gil, Ana M; Duarte, Iola F

    2015-01-01

    Lung tumour subtyping, particularly the distinction between adenocarcinoma (AdC) and squamous cell carcinoma (SqCC), is a critical diagnostic requirement. In this work, the metabolic signatures of lung carcinomas were investigated through (1)H NMR metabolomics, with a view to provide additional criteria for improved diagnosis and treatment planning. High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (NMR) spectroscopy was used to analyse matched tumour and adjacent control tissues from 56 patients undergoing surgical excision of primary lung carcinomas. Multivariate modeling allowed tumour and control tissues to be discriminated with high accuracy (97% classification rate), mainly due to significant differences in the levels of 13 metabolites. Notably, the magnitude of those differences were clearly distinct for AdC and SqCC: major alterations in AdC were related to phospholipid metabolism (increased phosphocholine, glycerophosphocholine and phosphoethanolamine, together with decreased acetate) and protein catabolism (increased peptide moieties), whereas SqCC had stronger glycolytic and glutaminolytic profiles (negatively correlated variations in glucose and lactate and positively correlated increases in glutamate and alanine). Other tumour metabolic features were increased creatine, glutathione, taurine and uridine nucleotides, the first two being especially prominent in SqCC and the latter in AdC. Furthermore, multivariate analysis of AdC and SqCC profiles allowed their discrimination with a 94% classification rate, thus showing great potential for aiding lung tumours subtyping. Overall, this study has provided new, clear evidence of distinct metabolic signatures for lung AdC and SqCC, which can potentially impact on diagnosis and provide important leads for future research on novel therapeutic targets or imaging tracers. PMID:25368033

  12. Gene expression profiling reveals Nef induced deregulation of lipid metabolism in HIV-1 infected T cells.

    PubMed

    Shrivastava, Surya; Trivedi, Jay; Mitra, Debashis

    2016-03-25

    Human Immunodeficiency Virus-1 (HIV-1) encodes a 27 kDa Negative Factor or Nef protein, which is increasingly proving to be a misnomer. Nef seems to be crucial for AIDS progression as individuals infected with nef-deleted strain of HIV were reported to become Long Term Non Progressors (LTNP). These findings necessitate tracing of Nef's footprint on landscape of cellular transcriptome favoring HIV-1 pathogenesis. We have tried to explore effect of Nef on cellular gene expression profile in conjunction with rest of HIV-1 proteins. Our results show that 237 genes are differentially regulated due to the presence of Nef during infection, which belong to several broad categories like "signaling", "apoptosis", "transcription" and "lipid metabolism" in gene ontology analysis. Furthermore, our results show that Nef causes disruption of lipid content in HIV-1 infected T cells. Molecular inhibitors of lipid metabolism like Atorvastatin and Ranolazine were found to have profound effect on wild type virus as compared to nef-deleted HIV-1. Thus our results suggest that interference in lipid metabolism is a potential mechanism through which Nef contributes in enhancing HIV-1 pathogenesis. PMID:26915805

  13. Systematic Analysis Reveals that Cancer Mutations Converge on Deregulated Metabolism of Arachidonate and Xenobiotics.

    PubMed

    Gatto, Francesco; Schulze, Almut; Nielsen, Jens

    2016-07-19

    Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network. PMID:27396332

  14. Structural Phylogenomics Reveals Gradual Evolutionary Replacement of Abiotic Chemistries by Protein Enzymes in Purine Metabolism

    PubMed Central

    Caetano-Anollés, Kelsey; Caetano-Anollés, Gustavo

    2013-01-01

    The origin of metabolism has been linked to abiotic chemistries that existed in our planet at the beginning of life. While plausible chemical pathways have been proposed, including the synthesis of nucleobases, ribose and ribonucleotides, the cooption of these reactions by modern enzymes remains shrouded in mystery. Here we study the emergence of purine metabolism. The ages of protein domains derived from a census of fold family structure in hundreds of genomes were mapped onto enzymes in metabolic diagrams. We find that the origin of the nucleotide interconversion pathway benefited most parsimoniously from the prebiotic formation of adenine nucleosides. In turn, pathways of nucleotide biosynthesis, catabolism and salvage originated ∼300 million years later by concerted enzymatic recruitments and gradual replacement of abiotic chemistries. Remarkably, this process led to the emergence of the fully enzymatic biosynthetic pathway ∼3 billion years ago, concurrently with the appearance of a functional ribosome. The simultaneous appearance of purine biosynthesis and the ribosome probably fulfilled the expanding matter-energy and processing needs of genomic information. PMID:23516625

  15. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    PubMed

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  16. Metabolic properties in stroked rats revealed by relaxation-enhanced magnetic resonance spectroscopy at ultrahigh fields.

    PubMed

    Shemesh, Noam; Rosenberg, Jens T; Dumez, Jean-Nicolas; Muniz, Jose A; Grant, Samuel C; Frydman, Lucio

    2014-01-01

    (1)H magnetic resonance spectroscopy (MRS) yields site-specific signatures that directly report metabolic concentrations, biochemistry and kinetics-provided spectral sensitivity and quality are sufficient. Here, an enabling relaxation-enhanced (RE) MRS approach is demonstrated that by combining highly selective spectral excitations with operation at very high magnetic fields, delivers spectra exhibiting signal-to-noise ratios >50:1 in under 6 s for ~5 × 5 × 5 (mm)(3) voxels, with flat baselines and no interference from water. With this spectral quality, MRS was used to interrogate a number of metabolic properties in stroked rat models. Metabolic confinements imposed by randomly oriented micro-architectures were detected and found to change upon ischaemia; intensities of downfield resonances were found to be selectively altered in stroked hemispheres; and longitudinal relaxation time of lactic acid was found to increase by over 50% its control value as early as 3-h post ischaemia, paralleling the onset of cytotoxic oedema. These results demonstrate potential of (1)H MRS at ultrahigh fields. PMID:25229942

  17. The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry.

    PubMed

    O'Neill, Ellis C; Trick, Martin; Hill, Lionel; Rejzek, Martin; Dusi, Renata G; Hamilton, Chris J; Zimba, Paul V; Henrissat, Bernard; Field, Robert A

    2015-10-01

    Euglena gracilis is a highly complex alga belonging to the green plant line that shows characteristics of both plants and animals, while in evolutionary terms it is most closely related to the protozoan parasites Trypanosoma and Leishmania. This well-studied organism has long been known as a rich source of vitamins A, C and E, as well as amino acids that are essential for the human diet. Here we present de novo transcriptome sequencing and preliminary analysis, providing a basis for the molecular and functional genomics studies that will be required to direct metabolic engineering efforts aimed at enhancing the quality and quantity of high value products from E. gracilis. The transcriptome contains over 30,000 protein-encoding genes, supporting metabolic pathways for lipids, amino acids, carbohydrates and vitamins, along with capabilities for polyketide and non-ribosomal peptide biosynthesis. The metabolic and environmental robustness of Euglena is supported by a substantial capacity for responding to biotic and abiotic stress: it has the capacity to deploy three separate pathways for vitamin C (ascorbate) production, as well as producing vitamin E (α-tocopherol) and, in addition to glutathione, the redox-active thiols nor-trypanothione and ovothiol. PMID:26289754

  18. Metaproteomics Applied to Activated Sludge for Industrial Wastewater Treatment Revealed a Dominant Methylotrophic Metabolism of Hyphomicrobium zavarzinii.

    PubMed

    Salerno, Carlo; Benndorf, Dirk; Kluge, Sabine; Palese, Luigi Leonardo; Reichl, Udo; Pollice, Alfieri

    2016-07-01

    In biological wastewater treatments, microbial populations of the so-called activated sludge work together in the abatement of pollutants. In this work, the metabolic behavior of the biomass of a lab-scale plant treating industrial pharmaceutical wastewater was investigated through a metaproteomic approach. The complete treatment process included a membrane biological reactor (MBR) coupled with an advanced oxidation process (AOP) for partial breakdown of non-biodegradable molecules. Proteins from biomass samples collected pre- and post-AOP application were investigated by two-dimensional gel electrophoresis (2DE), mass spectrometry (MS), and finally identified by database search. Results showed that most proteins remained constant between pre- and post-AOP. Methanol dehydrogenase (MDH) belonging to Hyphomicrobium zavarzinii appeared as the most constantly expressed protein in the studied consortium. Other identified proteins belonging to Hyphomicrobium spp. revealed a predominant methylotrophic metabolism, and H. zavarzinii appeared as a key actor in the studied microbial community. PMID:27090901

  19. Integrative analyses of genetic variation in enzyme activities of primary carbohydrate metabolism reveal distinct modes of regulation in Arabidopsis thaliana

    PubMed Central

    Keurentjes, Joost JB; Sulpice, Ronan; Gibon, Yves; Steinhauser, Marie-Caroline; Fu, Jingyuan; Koornneef, Maarten; Stitt, Mark; Vreugdenhil, Dick

    2008-01-01

    Background Plant primary carbohydrate metabolism is complex and flexible, and is regulated at many levels. Changes of transcript levels do not always lead to changes in enzyme activities, and these do not always affect metabolite levels and fluxes. To analyze interactions between these three levels of function, we have performed parallel genetic analyses of 15 enzyme activities involved in primary carbohydrate metabolism, transcript levels for their encoding structural genes, and a set of relevant metabolites. Quantitative analyses of each trait were performed in the Arabidopsis thaliana Ler × Cvi recombinant inbred line (RIL) population and subjected to correlation and quantitative trait locus (QTL) analysis. Results Traits affecting primary metabolism were often correlated, possibly due to developmental control affecting multiple genes, enzymes, or metabolites. Moreover, the activity QTLs of several enzymes co-localized with the expression QTLs (eQTLs) of their structural genes, or with metabolite accumulation QTLs of their substrates or products. In addition, many trait-specific QTLs were identified, revealing that there is also specific regulation of individual metabolic traits. Regulation of enzyme activities often occurred through multiple loci, involving both cis- and trans-acting transcriptional or post-transcriptional control of structural genes, as well as independently of the structural genes. Conclusion Future studies of the regulatory processes in primary carbohydrate metabolism will benefit from an integrative genetic analysis of gene transcription, enzyme activity, and metabolite content. The multiparallel QTL analyses of the various interconnected transducers of biological information flow, described here for the first time, can assist in determining the causes and consequences of genetic regulation at different levels of complex biological systems. PMID:18710526

  20. RNA Profiles of Porcine Embryos during Genome Activation Reveal Complex Metabolic Switch Sensitive to In Vitro Conditions

    PubMed Central

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben; Hyttel, Poul; Collas, Philippe; Cabot, Ryan

    2013-01-01

    Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell stage) EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery), protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism), different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos

  1. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    DOE PAGESBeta

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

  2. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    SciTech Connect

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

  3. Changes in cancer cell metabolism revealed by direct sample analysis with MALDI mass spectrometry.

    PubMed

    Pirman, David A; Efuet, Ekem; Ding, Xiao-Ping; Pan, Yong; Tan, Lin; Fischer, Susan M; DuBois, Raymond N; Yang, Peiying

    2013-01-01

    Biomarker discovery using mass spectrometry (MS) has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI), allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3), derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA), inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC) cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC) cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2) in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the metabolism of

  4. iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis

    PubMed Central

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

    2016-01-01

    Background Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. Results The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Conclusions

  5. Dietary rescue of altered metabolism gene reveals unexpected Drosophila mating cues.

    PubMed

    Bousquet, François; Chauvel, Isabelle; Flaven-Pouchon, Justin; Farine, Jean-Pierre; Ferveur, Jean-François

    2016-03-01

    To develop and reproduce, animals need long-chain MUFAs and PUFAs. Although some unsaturated FAs (UFAs) can be synthesized by the organism, others must be provided by the diet. The gene, desat1, involved in Drosophila melanogaster UFA metabolism, is necessary for both larval development and for adult sex pheromone communication. We first characterized desat1 expression in larval tissues. Then, we found that larvae in which desat1 expression was knocked down throughout development died during the larval stages when raised on standard food. By contrast pure MUFAs or PUFAs, but not saturated FAs, added to the larval diet rescued animals to adulthood with the best effect being obtained with oleic acid (C18:1). Male and female mating behavior and fertility were affected very differently by preimaginal UFA-rich diet. Adult diet also strongly influenced several aspects of reproduction: flies raised on a C18:1-rich diet showed increased mating performance compared with flies raised on standard adult diet. Therefore, both larval and adult desat1 expression control sex-specific mating signals. A similar nutrigenetics approach may be useful in other metabolic mutants to uncover cryptic effects otherwise masked by severe developmental defects. PMID:26759364

  6. Novel Loci for Metabolic Networks and Multi-Tissue Expression Studies Reveal Genes for Atherosclerosis

    PubMed Central

    Inouye, Michael; Ripatti, Samuli; Kettunen, Johannes; Lyytikäinen, Leo-Pekka; Oksala, Niku; Laurila, Pirkka-Pekka; Kangas, Antti J.; Soininen, Pasi; Savolainen, Markku J.; Viikari, Jorma; Kähönen, Mika; Perola, Markus; Salomaa, Veikko; Raitakari, Olli; Lehtimäki, Terho; Taskinen, Marja-Riitta; Järvelin, Marjo-Riitta; Ala-Korpela, Mika; Palotie, Aarno; de Bakker, Paul I. W.

    2012-01-01

    Association testing of multiple correlated phenotypes offers better power than univariate analysis of single traits. We analyzed 6,600 individuals from two population-based cohorts with both genome-wide SNP data and serum metabolomic profiles. From the observed correlation structure of 130 metabolites measured by nuclear magnetic resonance, we identified 11 metabolic networks and performed a multivariate genome-wide association analysis. We identified 34 genomic loci at genome-wide significance, of which 7 are novel. In comparison to univariate tests, multivariate association analysis identified nearly twice as many significant associations in total. Multi-tissue gene expression studies identified variants in our top loci, SERPINA1 and AQP9, as eQTLs and showed that SERPINA1 and AQP9 expression in human blood was associated with metabolites from their corresponding metabolic networks. Finally, liver expression of AQP9 was associated with atherosclerotic lesion area in mice, and in human arterial tissue both SERPINA1 and AQP9 were shown to be upregulated (6.3-fold and 4.6-fold, respectively) in atherosclerotic plaques. Our study illustrates the power of multi-phenotype GWAS and highlights candidate genes for atherosclerosis. PMID:22916037

  7. Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability*

    PubMed Central

    Patella, Francesca; Schug, Zachary T.; Persi, Erez; Neilson, Lisa J.; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R.; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-01-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability. PMID:25573745

  8. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti.

    PubMed

    diCenzo, George C; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  9. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes

    SciTech Connect

    Hawley, Alyse K.; Brewer, Heather M.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Hallam, Steven J.

    2014-08-05

    Oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified marine waters. Currently OMZs are expanding due to global climate change. This expansion alters marine ecosystem function and the productivity of fisheries due to habitat compression and changes in biogeochemical cycling leading to fixed nitrogen loss and greenhouse gas production. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally anoxic fjord, Saanich Inlet to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification and inorganic carbon fixation predominantly co-varied with abundance and distribution patterns of Thaumarchaeota, Nitrospira, Planctomycetes and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Within these groups, pathways mediating inorganic carbon fixation and nitrogen and sulfur transformations were differentially expressed across the redoxcline. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters and denitrification, sulfur-oxidation and inorganic carbon fixation pathways affiliated with SUP05 dominated suboxic and anoxic waters. Nitrite-oxidation and anammox pathways affiliated with Nitrospina and Planctomycetes respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The differential expression of these pathways under changing water column redox conditions has quantitative implications for coupled biogeochemical cycling linking different modes of inorganic carbon fixation with distributed nitrogen and sulfur-based energy metabolism extensible to coastal and open ocean OMZs.

  10. Key electrophysiological, molecular, and metabolic signatures of sleep and wakefulness revealed in primary cortical cultures.

    PubMed

    Hinard, Valérie; Mikhail, Cyril; Pradervand, Sylvain; Curie, Thomas; Houtkooper, Riekelt H; Auwerx, Johan; Franken, Paul; Tafti, Mehdi

    2012-09-01

    Although sleep is defined as a behavioral state, at the cortical level sleep has local and use-dependent features suggesting that it is a property of neuronal assemblies requiring sleep in function of the activation experienced during prior wakefulness. Here we show that mature cortical cultured neurons display a default state characterized by synchronized burst-pause firing activity reminiscent of sleep. This default sleep-like state can be changed to transient tonic firing reminiscent of wakefulness when cultures are stimulated with a mixture of waking neurotransmitters and spontaneously returns to sleep-like state. In addition to electrophysiological similarities, the transcriptome of stimulated cultures strikingly resembles the cortical transcriptome of sleep-deprived mice, and plastic changes as reflected by AMPA receptors phosphorylation are also similar. We used our in vitro model and sleep-deprived animals to map the metabolic pathways activated by waking. Only a few metabolic pathways were identified, including glycolysis, aminoacid, and lipids. Unexpectedly large increases in lysolipids were found both in vivo after sleep deprivation and in vitro after stimulation, strongly suggesting that sleep might play a major role in reestablishing the neuronal membrane homeostasis. With our in vitro model, the cellular and molecular consequences of sleep and wakefulness can now be investigated in a dish. PMID:22956841

  11. Distinct Metabolic Profile of Primary Focal Segmental Glomerulosclerosis Revealed by NMR-Based Metabolomics

    PubMed Central

    Wang, Weiming; Ren, Hong; Xie, Jingyuan; Shen, Pingyan; Lin, Donghai; Chen, Nan

    2013-01-01

    Background Primary focal segmental glomerulosclerosis (FSGS) is pathological entity which is characterized by idiopathic steroid-resistant nephrotic syndrome (SRNS) and progression to end-stage renal disease (ESRD) in the majority of affected individuals. Currently, there is no practical noninvasive technique to predict different pathological types of glomerulopathies. In this study, the role of urinary metabolomics in the diagnosis and pathogenesis of FSGS was investigated. Methods NMR-based metabolomics was applied for the urinary metabolic profile in the patients with FSGS (n = 25), membranous nephropathy (MN, n = 24), minimal change disease (MCD, n = 14) and IgA nephropathy (IgAN, n = 26), and healthy controls (CON, n = 35). The acquired data were analyzed using principal component analysis (PCA) followed by orthogonal projections to latent structure discriminant analysis (OPLS-DA). Model validity was verified using permutation tests. Results FSGS patients were clearly distinguished from healthy controls and other three types of glomerulopathies with good sensitivity and specificity based on their global urinary metabolic profiles. In FSGS patients, urinary levels of glucose, dimethylamine and trimethylamine increased compared with healthy controls, while pyruvate, valine, hippurate, isoleucine, phenylacetylglycine, citrate, tyrosine, 3-methylhistidine and β-hydroxyisovalerate decreased. Additionally, FSGS patients had lower urine N-methylnicotinamide levels compared with other glomerulopathies. Conclusions NMR-based metabonomic approach is amenable for the noninvasive diagnosis and differential diagnosis of FSGS as well as other glomerulopathies, and it could indicate the possible mechanisms of primary FSGS. PMID:24244321

  12. Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms

    SciTech Connect

    Justice, Nicholas B.; Norman, Anders; Brown, Christopher T.; Singh, Andrea; Thomas, Brian C.; Banfield, Jillian F.

    2014-12-15

    Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood. Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 a strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Lastly, within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes. Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.

  13. Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli

    NASA Astrophysics Data System (ADS)

    Buryakina, Tatyana Yu.; Su, Pin-Tzu; Syu, Wan-Jr; Allen Chang, C.; Fan, Hsiu-Fang; Kao, Fu-Jen

    2012-10-01

    Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

  14. Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

    PubMed

    Patella, Francesca; Schug, Zachary T; Persi, Erez; Neilson, Lisa J; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-03-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability. PMID:25573745

  15. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

    PubMed Central

    diCenzo, George C.; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M.; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  16. Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism.

    PubMed

    Liu, Xing; Lu, Yuanfu; Guan, Xinfu; Dong, Bingning; Chavan, Hemantkumar; Wang, Jin; Zhang, Yiqing; Krishnamurthy, Partha; Li, Feng

    2015-09-01

    Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity. PMID:26212543

  17. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells

    PubMed Central

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-01-01

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs. PMID:26864636

  18. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells

    NASA Astrophysics Data System (ADS)

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-02-01

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

  19. [Investigation of Protein Translocation Sec-System with Heterologous Gene Expression in Shewanella oneidensis MR-1 Bacterium Cells].

    PubMed

    Mordkovich, N N; Okorokova, N A; Veiko, V P

    2015-01-01

    A comparison of the primary structures of the protein translocation Sec-system proteins in the Shewanella oneidensis MR-1 and Escherichia coli bacteria was carried out. The process of translocation of recombinant pro-enteroxins (SEB and SEH) from Staphylococcus aureus and pro-streptavidin (SAV) from Streptomyces avidinii in the S. oneidensis MR-1 and E. coli cell periplasm was studied. It was demonstrated that these marker proteins are transferred into the periplasmic space of the S. oneidensis MR-1 transformant strain cells. The identity of N-terminal amino acid sequences of mature recombinant SEB, SEH, and SAV proteins (generated during post-translation proteolysis of leader peptide by the Sec-system both in E. coli and S. oneidensis MR-1) was established. PMID:26204774

  20. Metabolite Profiling Reveals YihU as a Novel Hydroxybutyrate Dehydrogenase for Alternative Succinic Semialdehyde Metabolism in Escherichia coli*

    PubMed Central

    Saito, Natsumi; Robert, Martin; Kochi, Hayataro; Matsuo, Goh; Kakazu, Yuji; Soga, Tomoyoshi; Tomita, Masaru

    2009-01-01

    The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to γ-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. PMID:19372223

  1. Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis

    PubMed Central

    Huang, Shu; Wang, Jun; Yue, Wucheng; Chen, Jiao; Gaughan, Sarah; Lu, Weiqun; Lu, Guoqing; Wang, Chenghui

    2015-01-01

    Molting is a critical developmental process for crustaceans, yet the underlying molecular mechanism is unknown. In this study, we used RNA-Seq to investigate transcriptomic profiles of the hepatopancreas and identified differentially expressed genes at four molting stages of Chinese mitten crab (Eriocheir sinensis). A total of 97,398 transcripts were assembled, with 31,900 transcripts annotated. Transcriptomic comparison revealed 1,189 genes differentially expressed amongst different molting stages. We observed a pattern associated with energy metabolism and physiological responses during a molting cycle. In specific, differentially expressed genes enriched in postmolt were linked to energy consumption whereas genes enriched in intermolt were related to carbohydrates, lipids metabolic and biosynthetic processes. In premolt, a preparation stage for upcoming molting and energy consumption, highly expressed genes were enriched in response to steroid hormone stimulus and immune system development. The expression profiles of twelve functional genes detected via RNA-Seq were corroborated through real-time RT-PCR assay. Together, our results, including assembled transcriptomes, annotated functional elements and enriched differentially expressed genes amongst different molting stages, provide novel insights into the functions of the hepatopancreas in energy metabolism and biological processes pertaining to molting in crustaceans. PMID:26369734

  2. Single-cell genome and metatranscriptome sequencing reveal metabolic interactions of an alkane-degrading methanogenic community

    PubMed Central

    Embree, Mallory; Nagarajan, Harish; Movahedi, Narjes; Chitsaz, Hamidreza; Zengler, Karsten

    2014-01-01

    Microbial interactions have a key role in global geochemical cycles. Although we possess significant knowledge about the general biochemical processes occurring in microbial communities, we are often unable to decipher key functions of individual microorganisms within the environment in part owing to the inability to cultivate or study them in isolation. Here, we circumvent this shortcoming through the use of single-cell genome sequencing and a novel low-input metatranscriptomics protocol to reveal the intricate metabolic capabilities and microbial interactions of an alkane-degrading methanogenic community. This methanogenic consortium oxidizes saturated hydrocarbons under anoxic conditions through a thus-far-uncharacterized biochemical process. The genome sequence of a dominant bacterial member of this community, belonging to the genus Smithella, was sequenced and served as the basis for subsequent analysis through metabolic reconstruction. Metatranscriptomic data generated from less than 500 pg of mRNA highlighted metabolically active genes during anaerobic alkane oxidation in comparison with growth on fatty acids. These data sets suggest that Smithella is not activating hexadecane by fumarate addition. Differential expression assisted in the identification of hypothetical proteins with no known homology that may be involved in hexadecane activation. Additionally, the combination of 16S rDNA sequence and metatranscriptomic data enabled the study of other prevalent organisms within the consortium and their interactions with Smithella, thus yielding a comprehensive characterization of individual constituents at the genome scale during methanogenic alkane oxidation. PMID:24152715

  3. Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the “pathway redundancy” in metabolic networks

    PubMed Central

    2013-01-01

    Background Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the “pathway redundancy” of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer. Results Here we report the genetic characterization of CPOAA biosynthesis with revealing a “pathway redundancy” in metabolic networks. Independent mutation of the four genes (polL-N and polP) directly resulted in the accumulation of polyoxin I, suggesting their positive roles for CPOAA biosynthesis. Moreover, the individual mutant of polN and polP also partially retains polyoxin production, suggesting the existence of the alternative homologs substituting their functional roles. Conclusions It is unveiled that argA and argB in L-arginine biosynthetic pathway contributed to the “pathway redundancy”, more interestingly, argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide an example for the research on the “pathway redundancy” in metabolic networks, and lay a solid foundation for targeted enhancement of polyoxin production with synthetic biology strategies. PMID:24314013

  4. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    PubMed

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  5. Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity

    PubMed Central

    Burniston, Jatin G.; Kenyani, Jenna; Wastling, Jonathan M.; Burant, Charles F.; Qi, Nathan R.; Koch, Lauren G.; Britton, Steven L.

    2012-01-01

    Selection on running capacity has created rat phenotypes of high capacity runners (HCR) that have enhanced cardiac function and low capacity runners (LCR) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCR and LCR from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCR was 6-fold greater than LCR. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant (P<0.05; false discovery rate <10 %, calculated using q-values) differences between HCR and LCR. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the beta-oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCR also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of alpha B-crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress. PMID:21751351

  6. Untargeted Metabolomics Analysis Reveals a Link between ETHE1-Mediated Disruptive Redox State and Altered Metabolic Regulation.

    PubMed

    Sahebekhtiari, Navid; Nielsen, Camilla Bak; Johannsen, Mogens; Palmfeldt, Johan

    2016-05-01

    Defects in the gene encoding the persulfide dioxygenase ETHE1 are known to cause the severe inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of known clinical characteristics, the molecular mechanisms underlying the ETHE1 deficiency are still obscure. Herein, to further analyze the molecular phenotype of the disease, we applied an untargeted metabolomics approach on cultivated fibroblasts of EE patients for pinpointing alterations in metabolite levels. Metabolites, as direct signatures of biochemical functions, can decipher biochemical pathways involved in the cellular phenotype of patient cells. Using liquid chromatography-mass spectrometry-based untargeted metabolomics, we identified 18 metabolites that have altered levels in fibroblasts from EE patients. Our data demonstrate disrupted redox state in EE patient cells, which is reflected by significantly decreased level of reduced glutathione. Furthermore, the down-regulation of several intermediate metabolites such as the redox cofactors NAD(+) and NADH as well as Krebs cycle intermediates revealed clear alteration in metabolic regulation. Pantothenic acid and several amino acids exhibited decreased levels, whereas the β-citrylglutamate with a putative role in brain development had an increased level in the EE patient cells. These observations indicate the severe impact of ETHE1 deficiency on cellular physiology and redox state, meanwhile suggesting targets for experimental studies on novel treatment options for the devastating metabolic disorder. PMID:27074420

  7. Dissecting lipid metabolism in meibomian glands of humans and mice: An integrative study reveals a network of metabolic reactions not duplicated in other tissues.

    PubMed

    Butovich, Igor A; McMahon, Anne; Wojtowicz, Jadwiga C; Lin, Feng; Mancini, Ronald; Itani, Kamel

    2016-06-01

    Lipids comprise the bulk of the meibomian gland secretion (meibum) which is produced by meibocytes. Complex arrays of lipogenic reactions in meibomian glands, which we collectively call meibogenesis, have not been explored on a molecular level yet. Our goals were to elucidate the possible biosynthetic pathways that underlie the generation of meibum, reveal similarities in, and differences between, lipid metabolism in meibomian glands and other organs and tissues, and integrate meibomian gland studies into the field of general metabolomics. Specifically, we have conducted detailed analyses of human and mouse specimens using genomic, immunohistochemical, and lipidomic approaches. Among equally highly expressed genes found in meibomian glands of both species were those related to fatty acid elongation, branching, desaturation, esterification, reduction of fatty acids to alcohols, and cholesterol biosynthesis. Importantly, corresponding lipid products were detected in meibum of both species using lipidomic approaches. For the first time, a cohesive, unifying biosynthetic scheme that connects genomic, lipidomic, and immunohistochemical observations is outlined and discussed. PMID:27032494

  8. A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis

    PubMed Central

    Liechti, G.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.; VanNieuwenhze, M.; Maurelli, A. T.

    2014-01-01

    Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG. PMID:24336210

  9. Urinary metabolic profiling of rat models revealed protective function of scoparone against alcohol induced hepatotoxicity

    PubMed Central

    Zhang, Aihua; Sun, Hui; Wang, Xijun

    2014-01-01

    Alcohol-induced liver disease (ALD) is a leading cause of non-accident-related deaths in the world. Identification of an early specific signature of ALD would aid in therapeutic intervention. Scoparone is an important constituent of Yinchenhao, and displayed bright prospects in hepatoprotective effect. However, its precise molecular mechanism has not been well explored. The present study was designed to assess the effects and possible mechanisms of scoparone against alcohol-induced liver injury. UPLC/ESI-Q-TOF/MS combined with pattern recognition approaches including PCA, and PLS-DA were integrated to get differentiating metabolites for the pathways and clarify mechanisms of disease, highlight insights into drug discovery. The results indicated four ions in the positive mode were characterized as potential differentiating metabolites which can be regulated by scoparone treatment, and suggested that therapeutic effect of scoparone could regulated the dysfunctions of citrate cycle, sphingolipid metabolism, taurine and hypotaurine. PMID:25341677

  10. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

    PubMed

    Shimada, Kenichi; Skouta, Rachid; Kaplan, Anna; Yang, Wan Seok; Hayano, Miki; Dixon, Scott J; Brown, Lewis M; Valenzuela, Carlos A; Wolpaw, Adam J; Stockwell, Brent R

    2016-07-01

    Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes. PMID:27159577

  11. The Metabolic Enzyme ManA Reveals a Link between Cell Wall Integrity and Chromosome Morphology

    PubMed Central

    Elbaz, Maya; Ben-Yehuda, Sigal

    2010-01-01

    Synchronizing cell growth, division and DNA replication is an essential property of all living cells. Accurate coordination of these cellular events is especially crucial for bacteria, which can grow rapidly and undergo multifork replication. Here we show that the metabolic protein ManA, which is a component of mannose phosphotransferase system, participates in cell wall construction of the rod shaped bacterium Bacillus subtilis. When growing rapidly, cells lacking ManA exhibit aberrant cell wall architecture, polyploidy and abnormal chromosome morphologies. We demonstrate that these cellular defects are derived from the role played by ManA in cell wall formation. Furthermore, we show that ManA is required for maintaining the proper carbohydrate composition of the cell wall, particularly of teichoic acid constituents. This perturbed cell wall synthesis causes asynchrony between cell wall elongation, division and nucleoid segregation. PMID:20862359

  12. Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms

    DOE PAGESBeta

    Justice, Nicholas B.; Norman, Anders; Brown, Christopher T.; Singh, Andrea; Thomas, Brian C.; Banfield, Jillian F.

    2014-12-15

    Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood. Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 amore » strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Lastly, within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes. Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.« less

  13. Proteomic Analysis Reveals That Iron Availability Alters the Metabolic Status of the Pathogenic Fungus Paracoccidioides brasiliensis

    PubMed Central

    Parente, Ana F. A.; Bailão, Alexandre M.; Borges, Clayton L.; Parente, Juliana A.; Magalhães, Adriana D.; Ricart, Carlos A. O.; Soares, Célia M. A.

    2011-01-01

    Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways. PMID:21829521

  14. The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features

    SciTech Connect

    Aklujkar, Muktak; Haveman, Shelley; DiDonatoJr., Raymond; Chertkov, Olga; Han, Cliff; Land, Miriam L; Brown, Peter; Lovley, Derek

    2012-01-01

    Background: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species. Results: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate: ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires. Conclusions: Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.

  15. Adaptive Control Model Reveals Systematic Feedback and Key Molecules in Metabolic Pathway Regulation

    PubMed Central

    Moffitt, Richard A.; Merrill, Alfred H.; Wang, May D.

    2011-01-01

    Abstract Robust behavior in metabolic pathways resembles stabilized performance in systems under autonomous control. This suggests we can apply control theory to study existing regulation in these cellular networks. Here, we use model-reference adaptive control (MRAC) to investigate the dynamics of de novo sphingolipid synthesis regulation in a combined theoretical and experimental case study. The effects of serine palmitoyltransferase over-expression on this pathway are studied in vitro using human embryonic kidney cells. We report two key results from comparing numerical simulations with observed data. First, MRAC simulations of pathway dynamics are comparable to simulations from a standard model using mass action kinetics. The root-sum-square (RSS) between data and simulations in both cases differ by less than 5%. Second, MRAC simulations suggest systematic pathway regulation in terms of adaptive feedback from individual molecules. In response to increased metabolite levels available for de novo sphingolipid synthesis, feedback from molecules along the main artery of the pathway is regulated more frequently and with greater amplitude than from other molecules along the branches. These biological insights are consistent with current knowledge while being new that they may guide future research in sphingolipid biology. In summary, we report a novel approach to study regulation in cellular networks by applying control theory in the context of robust metabolic pathways. We do this to uncover potential insight into the dynamics of regulation and the reverse engineering of cellular networks for systems biology. This new modeling approach and the implementation routines designed for this case study may be extended to other systems. Supplementary Material is available at www.liebertonline.com/cmb. PMID:21314456

  16. Carbon Metabolism of Soil microorganisms at Low Temperatures: Position-Specific 13C Labeled Glucose Reveals the Story

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Bore, E. K.; Halicki, S.; Kuzyakov, Y.; Dippold, M.

    2015-12-01

    Metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze soil metabolism at low temperature, isotopomeres of position-specifically 13C labeled glucose were applied at three temperature levels; +5, -5 -20 oC. In additon, one sterilization treatment with sodium azide at +5 oC was also performed. Soils were incubated for 1, 3 and 10 days while soil samples at -20 oC were additionally sampled after 30 days. The 13C from individual molecule position in respired CO2 was quantifed. Incorporation of 13C in bulk soil, extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of different microbial communities classified by 13C phospholipid fatty acid analysis (PLFA) was carried out. Our 13CO2 data showed a dominance of C-1 respiration at +5 °C for treatments with and without sodium azide, but total respiration for sodium azide inhibited treatments increased by 14%. In contrast, at -5 and -20 oC metabolic behavior showed intermingling of preferential respiration of the glucose C-4 and C-1 positions. Therefore, at +5 °C, pentose phosphate pathway activity is a dominant metabolic pathway used by microorganisms to metabolize glucose. The respiration increase due to NaN3 inhibition was attributed to endoenzymes released from dead organisms that are stabilized at the soil matrix and have access to suitable substrate and co-factors to permit their funtions. Our PLFA analysis showed that incorporation of glucose 13C was higher in Gram negative bacteria than other microbial groups as they are most competitive for LMWOS. Only a limited amount of microbial groups maintained their glucose utilizing activity at -5 and -20 °C and they strongly shifted towards a metabolization of glucose via both glycolysis and pentose phosphate pathways indicating both growth and cellular maintenance. This study revealed a remarkable microbial acitivity

  17. Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

    SciTech Connect

    Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.; Hurst, Gregory B.; Rohlin, Lars; Gunsalus, Robert P.; McInerney, Michael J.

    2015-02-11

    We report that microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. In conclusion, the proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

  18. Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16

    PubMed Central

    2014-01-01

    Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism, CO2 transport, ion transport, and internal pH regulation. Understanding the metabolic role of CAs in the chemolithoautotropic bacterium Ralstonia eutropha is important for the development of high performance fermentation processes based on the bacterium’s capability to fix carbon using the Calvin-Benson-Bassham (CBB) cycle. Analysis of the R. eutropha H16 genome sequence revealed the presence of four CA genes: can, can2, caa and cag. We evaluated the importance of each of the CAs in the metabolism of R. eutropha by examination of growth and enzyme activity in gene deletion, complementation, and overexpression strains. All four purified CAs were capable of performing the interconversion of CO2 and HCO3–, although the equilibrium towards the formation of CO2 or HCO3– differs with each CA. Deletion of can, encoding a β-CA, affected the growth of R. eutropha; however the growth defect could be compensated by adding CO2 to the culture. Deletion of the caa, encoding an α-CA, had the strongest deleterious influence on cell growth. Strains with deletion or overexpression of can2 or cag genes exhibited similar behavior to wild type under most of the conditions tested. In this work, Caa was studied in greater detail using microscopy and complementation experiments, which helped confirm its periplasmic localization and determine its importance for robust growth of R. eutropha. A hypothesis for the coordinated role of these four enzymes in the metabolism of R. eutropha is proposed. PMID:24410804

  19. Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

    PubMed Central

    Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.; Hurst, Gregory B.; Rohlin, Lars; Gunsalus, Robert P.; McInerney, Michael J.

    2015-01-01

    Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status. PMID:25717324

  20. Systems Level Analyses Reveal Multiple Regulatory Activities of CodY Controlling Metabolism, Motility and Virulence in Listeria monocytogenes.

    PubMed

    Lobel, Lior; Herskovits, Anat A

    2016-02-01

    Bacteria sense and respond to many environmental cues, rewiring their regulatory network to facilitate adaptation to new conditions/niches. Global transcription factors that co-regulate multiple pathways simultaneously are essential to this regulatory rewiring. CodY is one such global regulator, controlling expression of both metabolic and virulence genes in Gram-positive bacteria. Branch chained amino acids (BCAAs) serve as a ligand for CodY and modulate its activity. Classically, CodY was considered to function primarily as a repressor under rich growth conditions. However, our previous studies of the bacterial pathogen Listeria monocytogenes revealed that CodY is active also when the bacteria are starved for BCAAs. Under these conditions, CodY loses the ability to repress genes (e.g., metabolic genes) and functions as a direct activator of the master virulence regulator gene, prfA. This observation raised the possibility that CodY possesses multiple functions that allow it to coordinate gene expression across a wide spectrum of metabolic growth conditions, and thus better adapt bacteria to the mammalian niche. To gain a deeper understanding of CodY's regulatory repertoire and identify direct target genes, we performed a genome wide analysis of the CodY regulon and DNA binding under both rich and minimal growth conditions, using RNA-Seq and ChIP-Seq techniques. We demonstrate here that CodY is indeed active (i.e., binds DNA) under both conditions, serving as a repressor and activator of different genes. Further, we identified new genes and pathways that are directly regulated by CodY (e.g., sigB, arg, his, actA, glpF, gadG, gdhA, poxB, glnR and fla genes), integrating metabolism, stress responses, motility and virulence in L. monocytogenes. This study establishes CodY as a multifaceted factor regulating L. monocytogenes physiology in a highly versatile manner. PMID:26895237

  1. Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

    DOE PAGESBeta

    Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.; Hurst, Gregory B.; Rohlin, Lars; Gunsalus, Robert P.; McInerney, Michael J.

    2015-02-11

    We report that microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detectedmore » were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. In conclusion, the proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.« less

  2. Systems Level Analyses Reveal Multiple Regulatory Activities of CodY Controlling Metabolism, Motility and Virulence in Listeria monocytogenes

    PubMed Central

    Lobel, Lior; Herskovits, Anat A.

    2016-01-01

    Bacteria sense and respond to many environmental cues, rewiring their regulatory network to facilitate adaptation to new conditions/niches. Global transcription factors that co-regulate multiple pathways simultaneously are essential to this regulatory rewiring. CodY is one such global regulator, controlling expression of both metabolic and virulence genes in Gram-positive bacteria. Branch chained amino acids (BCAAs) serve as a ligand for CodY and modulate its activity. Classically, CodY was considered to function primarily as a repressor under rich growth conditions. However, our previous studies of the bacterial pathogen Listeria monocytogenes revealed that CodY is active also when the bacteria are starved for BCAAs. Under these conditions, CodY loses the ability to repress genes (e.g., metabolic genes) and functions as a direct activator of the master virulence regulator gene, prfA. This observation raised the possibility that CodY possesses multiple functions that allow it to coordinate gene expression across a wide spectrum of metabolic growth conditions, and thus better adapt bacteria to the mammalian niche. To gain a deeper understanding of CodY’s regulatory repertoire and identify direct target genes, we performed a genome wide analysis of the CodY regulon and DNA binding under both rich and minimal growth conditions, using RNA-Seq and ChIP-Seq techniques. We demonstrate here that CodY is indeed active (i.e., binds DNA) under both conditions, serving as a repressor and activator of different genes. Further, we identified new genes and pathways that are directly regulated by CodY (e.g., sigB, arg, his, actA, glpF, gadG, gdhA, poxB, glnR and fla genes), integrating metabolism, stress responses, motility and virulence in L. monocytogenes. This study establishes CodY as a multifaceted factor regulating L. monocytogenes physiology in a highly versatile manner. PMID:26895237

  3. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  4. Comparative photosynthetic and metabolic analyses reveal mechanism of improved cold stress tolerance in bermudagrass by exogenous melatonin.

    PubMed

    Hu, Zhengrong; Fan, Jibiao; Xie, Yan; Amombo, Erick; Liu, Ao; Gitau, Margaret Mukami; Khaldun, A B M; Chen, Liang; Fu, Jinmin

    2016-03-01

    Melatonin (N-acetyl-5-methoxytryptamine) has been reported to participate in plant development and abiotic stress responses. The main objective of this study was to investigate the role of melatonin in the cold-sensitive (S) and the cold-tolerant (T) bermudagrass genotypes' response to cold stress. The genotypes were treated with 100 μM melatonin and exposed to 4 °C temperature for 3 days. In both genotypes, cold stress increased the endogenous melatonin levels, and more prominently in T than S. Physiological responses indicated that exogenous melatonin triggered antioxidant activities in both genotypes, while it alleviated cell damage in the T genotype response to cold stress. Melatonin treatment under cold stress increased fluorescence curve levels for both genotypes, and higher in T than S genotypes. In both genotypes, the alterations in photosynthetic fluorescence parameters after melatonin treatment highlighted the participation of melatonin in improving photosystem response to cold stress, particularly for the cold-tolerant genotype. The metabolic analyses revealed the alterations of 44 cold-responsive metabolites in the two genotypes, mainly including carbohydrates, organic acids and amino acids. After exogenous melatonin treatment under cold condition, there was high accumulation of metabolites in the cold-tolerant regimes than their cold-sensitive counterparts. Collectively, the present study revealed differential modulations of melatonin between the cold-sensitive and the cold-tolerant genotypes in response to cold stress. This was mainly by impacting antioxidant system, photosystem II, as well as metabolic homeostasis. PMID:26807934

  5. Evolution of E. coli on [U-13C]Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

    PubMed Central

    Sandberg, Troy E.; Long, Christopher P.; Gonzalez, Jacqueline E.; Feist, Adam M.; Antoniewicz, Maciek R.; Palsson, Bernhard O.

    2016-01-01

    13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth. PMID:26964043

  6. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Acute Chromate Challenge

    SciTech Connect

    Thompson, Melissa R; Verberkmoes, Nathan C; Chourey, Karuna; Shah, Manesh B; Thompson, Dorothea K; Hettich, Robert {Bob} L

    2007-01-01

    Proteome alterations in the metal-reducing bacterium Shewanella oneidensis MR-1 in response to different acute dose challenges (0.3, 0.5, or 1 mM) of the toxic metal chromate [Cr(VI)] were characterized with multidimensional HPLC-MS/MS on a linear trapping quadrupole MS. A total of 2,406 functionally diverse proteins were identified, with a subset demonstrating dosage-dependent up- and down-regulated expression, such as proteins involved in detoxification and iron binding and transport.

  7. The Immunology of CD1- and MR1-Restricted T Cells.

    PubMed

    Mori, Lucia; Lepore, Marco; De Libero, Gennaro

    2016-05-20

    CD1- and MHC-related molecule-1 (MR1)-restricted T lymphocytes recognize nonpeptidic antigens, such as lipids and small metabolites, and account for a major fraction of circulating and tissue-resident T cells. They represent a readily activated, long-lasting population of effector cells and contribute to the early phases of immune response, orchestrating the function of other cells. This review addresses the main aspects of their immunological functions, including antigen and T cell receptor repertoires, mechanisms of nonpeptidic antigen presentation, and the current evidence for their participation in human and experimental diseases. PMID:26927205

  8. Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma

    PubMed Central

    Hettmer, Simone; Schinzel, Anna C; Tchessalova, Daria; Schneider, Michaela; Parker, Christina L; Bronson, Roderick T; Richards, Nigel GJ; Hahn, William C; Wagers, Amy J

    2015-01-01

    Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value. DOI: http://dx.doi.org/10.7554/eLife.09436.001 PMID:26499495

  9. Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes

    PubMed Central

    2012-01-01

    Background MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. Results The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. Conclusions We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons. PMID:22494833

  10. Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

    PubMed

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; Winkler, Robert; León-Ramírez, Claudia G; Ruiz-Herrera, José

    2012-03-01

    Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell. PMID:22222500

  11. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes

    PubMed Central

    Hawley, Alyse K.; Brewer, Heather M.; Norbeck, Angela D.; Paša-Tolić, Ljiljana; Hallam, Steven J.

    2014-01-01

    Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand. PMID:25053816

  12. In vivo multiphoton NADH fluorescence reveals depth-dependent keratinocyte metabolism in human skin.

    PubMed

    Balu, Mihaela; Mazhar, Amaan; Hayakawa, Carole K; Mittal, Richa; Krasieva, Tatiana B; König, Karsten; Venugopalan, Vasan; Tromberg, Bruce J

    2013-01-01

    We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 μmoles/10(6) cells/h. PMID:23332078

  13. In Vivo Multiphoton NADH Fluorescence Reveals Depth-Dependent Keratinocyte Metabolism in Human Skin

    PubMed Central

    Balu, Mihaela; Mazhar, Amaan; Hayakawa, Carole K.; Mittal, Richa; Krasieva, Tatiana B.; König, Karsten; Venugopalan, Vasan; Tromberg, Bruce J.

    2013-01-01

    We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 μmoles/106 cells/h. PMID:23332078

  14. Metabolic diversity and ecological niches of Achromatium populations revealed with single-cell genomic sequencing

    PubMed Central

    Mansor, Muammar; Hamilton, Trinity L.; Fantle, Matthew S.; Macalady, Jennifer L.

    2015-01-01

    Large, sulfur-cycling, calcite-precipitating bacteria in the genus Achromatium represent a significant proportion of bacterial communities near sediment-water interfaces at sites throughout the world. Our understanding of their potentially crucial roles in calcium, carbon, sulfur, nitrogen, and iron cycling is limited because they have not been cultured or sequenced using environmental genomics approaches to date. We utilized single-cell genomic sequencing to obtain one incomplete and two nearly complete draft genomes for Achromatium collected at Warm Mineral Springs (WMS), FL. Based on 16S rRNA gene sequences, the three cells represent distinct and relatively distant Achromatium populations (91–92% identity). The draft genomes encode key genes involved in sulfur and hydrogen oxidation; oxygen, nitrogen and polysulfide respiration; carbon and nitrogen fixation; organic carbon assimilation and storage; chemotaxis; twitching motility; antibiotic resistance; and membrane transport. Known genes for iron and manganese energy metabolism were not detected. The presence of pyrophosphatase and vacuolar (V)-type ATPases, which are generally rare in bacterial genomes, suggests a role for these enzymes in calcium transport, proton pumping, and/or energy generation in the membranes of calcite-containing inclusions. PMID:26322031

  15. In vivo NMR metabolic profiling of Fabrea salina reveals sequential defense mechanisms against ultraviolet radiation.

    PubMed

    Marangoni, Roberto; Paris, Debora; Melck, Dominique; Fulgentini, Lorenzo; Colombetti, Giuliano; Motta, Andrea

    2011-01-01

    Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement. PMID:21190674

  16. New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.

    PubMed

    Meng, Fantao; Han, Yong; Srisai, Dollada; Belakhov, Valery; Farias, Monica; Xu, Yong; Palmiter, Richard D; Baasov, Timor; Wu, Qi

    2016-03-29

    Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes. PMID:26976589

  17. Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling.

    PubMed

    Jain, Madhulika; Petzold, Christopher J; Schelle, Michael W; Leavell, Michael D; Mougous, Joseph D; Bertozzi, Carolyn R; Leary, Julie A; Cox, Jeffery S

    2007-03-20

    Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Using a mass spectrometric approach to simultaneously monitor hundreds of lipids, we discovered that the size and abundance of two lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1), are controlled by the availability of a common precursor, methyl malonyl CoA (MMCoA). Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. Furthermore, perturbation of MMCoA metabolism attenuated pathogen replication in mice. Importantly, we detected increased PDIM synthesis in bacteria growing within host tissues and in bacteria grown in culture on odd-chain fatty acids. Because M. tuberculosis catabolizes host lipids to grow during infection, we propose that growth of M. tuberculosis on fatty acids in vivo leads to increased flux of MMCoA through lipid biosynthetic pathways, resulting in increased virulence lipid synthesis. Our results suggest that the shift to host lipid catabolism during infection allows for increased virulence lipid anabolism by the bacterium. PMID:17360366

  18. Expectancy for food or expectancy for chocolate reveals timing systems for metabolism and reward.

    PubMed

    Angeles-Castellanos, M; Salgado-Delgado, R; Rodríguez, K; Buijs, R M; Escobar, C

    2008-07-31

    The clock gene protein Per 1 (PER1) is expressed in several brain structures and oscillates associated with the suprachiasmatic nucleus (SCN). Restricted feeding schedules (RFS) induce anticipatory activity and impose daily oscillations of c-Fos and clock proteins in brain structures. Daily access to a palatable treat (chocolate) also elicits anticipatory activity and induces c-Fos expression mainly in corticolimbic structures. Here the influence of daily access to food or chocolate was explored by the analysis of the oscillatory patterns of PER1 in hypothalamic and corticolimbic structures. Wistar rats were exposed to RFS or to daily access to chocolate for 3 weeks. Persistence of food or chocolate entrained rhythms was determined 8 days after cessation of the feeding protocols. RFS and chocolate induced a phase shift in PER1 rhythmicity in corticolimbic structures with peak values at zeitgeber time 12 and a higher amplitude in the chocolate group. Both RFS and chocolate groups showed an upregulation of PER1 in the SCN. Food and chocolate entrained rhythms persisted for 8 days in behavior and in PER1 expression in the dorsomedial hypothalamic nucleus, accumbens, prefrontal cortex and central amygdala. The present data demonstrate the existence of different oscillatory systems in the brain that can be activated by entrainment to metabolic stimuli or to reward and suggest the participation of PER1 in both entraining pathways. Persistence and amplification of PER1 oscillations in structures associated with reward suggest that this oscillatory process is fundamental to food addictive behavior. PMID:18585440

  19. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes.

    PubMed

    Hawley, Alyse K; Brewer, Heather M; Norbeck, Angela D; Paša-Tolić, Ljiljana; Hallam, Steven J

    2014-08-01

    Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand. PMID:25053816

  20. Transcriptomic Analysis Reveals the Metabolic Mechanism of L-Ascorbic Acid in Ziziphus jujuba Mill.

    PubMed Central

    Zhang, Chunmei; Huang, Jian; Li, Xingang

    2016-01-01

    Chinese jujube (Ziziphus jujuba Mill.) is the most economically important member of the Rhamnaceae family and contains a high concentration of ascorbic acid (AsA). To explore the metabolic mechanism of AsA accumulation, we investigated the abundance of AsA in the fruit development stages, the leaf and flower of Z. jujuba cv Junzao, and the mature fruit of one type of wild jujube (Z. jujuba var. spinosa Hu, Yanchuan sour jujube). And the expression patterns of genes involved in AsA biosynthesis, degradation, and recycling were analyzed. The result showed that AsA biosynthesis during early fruit development (the enlargement stage) is the main reason for jujube high accumulation. The L-galactose pathway plays a predominant role in the biosynthesis of AsA during jujube fruit development, and the genes GMP1, GME1, GGP, and GaLDH involved in the determination of AsA concentration during fruit development and in different genotypes; the myo-inositol pathway along with the genes GME2 and GMP2 in the L-galactose pathway play a compensatory role in maintaining AsA accumulation during the ripening stage. These findings enhance our understanding of the molecular mechanism in regulating AsA accumulation for jujube. PMID:26913041

  1. Comparative c-type cytochrome expression analysis in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C grown with soluble and insoluble oxidised metal electron acceptors

    SciTech Connect

    Nissen, Silke; Liu, Xiaoxin; Chourey, Karuna; Hettich, Robert {Bob} L; Wagner, Darlene D; Pffifner, Susan; Loeffler, Frank E

    2012-01-01

    The genomes of Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C encode 40 and 69 putative c-type cytochrome genes, respectively. Deletion mutant and biochemical studies have assigned specific functions to a few c-type cytochromes involved in electron transfer to oxidised metals in Shewanella oneidensis strain MR-1. Although promising, the genetic approach is limited to gene deletions that produce a distinct phenotype, and organism for which a genetic system is available. To more comprehensively investigate and compare c-type cytochrome expression in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C, proteomic measurements were used to characterise lysates of cells grown with soluble Fe(III) (as ferric citrate) and insoluble Mn(IV) (as MnO2) as electron acceptors. Strain MR-1 expressed 19 and 20, and strain 2CP-C expressed 27 and 25 c-type cytochromes when grown with Fe(III) and Mn(IV), respectively. The majority of c-type cytochromes (77% for strain MR-1 and 63% for strain 2CP-C) were expressed under both growth conditions; however, the analysis also revealed unique c-type cytochromes that were specifically expressed in cells grown with soluble Fe(III) or insoluble Mn(IV). Proteomic characterisation proved to be a promising approach for determining the c-type cytochrome complement expressed under different growth conditions, and will help elucidating the specific functions of more c-type cytochromes that are the basis for Shewanella and Anaeromyxobacter respiratory versatility.

  2. Sequence and Genetic Characterization of etrA, an fnr Analog that Regulates Anaerobic Respiration in Shewanella putrefaciens MR-1

    NASA Technical Reports Server (NTRS)

    Saffarini, Daad A.; Nelson, Kenneth H.

    1993-01-01

    An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-l. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S.putrefaciens etrA is able to complement an fnr mutant of E.coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etr.A mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S.putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.

  3. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  4. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  5. Ferrous Phosphate Surface Precipitates Resulting from the Reduction of Intragrain 6-line Ferrihydrite by Shewanella oneidensis MR-1

    SciTech Connect

    Peretyazhko, Tetyana; Zachara, John M.; Kennedy, David W.; Fredrickson, Jim K.; Arey, Bruce W.; McKinley, James P.; Wang, Chong M.; Dohnalkova, Alice; Xia, Yuanxian

    2010-07-01

    The reductive biotransformation of 6-line ferrihydrite located within porous silica (intragrain ferrihydrite) by Shewanella oneidensis MR-1 was investigated and compared to the behavior of 6-line ferrihydrite in suspension (free ferrihydrite). The effect of buffer type (PIPES and NaHCO3) and phosphate (P) on the extent of reduction and formation of Fe(II) secondary phases was investigated under anoxic conditions. Electron microscopy and micro X-ray diffraction were applied to evaluate the morphology and mineralogy of the biogenic precipitates and to study the distribution of microorganisms on the surface of porous silica after bioreduction. Kinetic reduction experiments with free and intragrain ferrihydrite revealed contrasting behaviour with respect to the buffer and presence of P. The overall amount of intragrain ferrihydrite reduction was less than that of free ferrihydrite [at 5 mmol L-1 Fe(III)T]. In the intragrain ferrihydrite suspensions, 200-300 µmol L-1 dissolved Fe(III) was released during the initial stages of incubation; no Fe(III)aq was detected in the free ferrihydrite suspensions. Reductive mineralization was not observed in the intragrain ferrihydrite incubations without P, and all biogenic Fe(II) concentrated in the aqueous phase. Distinctive surface precipitates of Fe(II) phosphates with spherical morphology were observed on porous silica when P was present. These precipitates were well colonized by microorganisms and fragments of extracellular materials at the end of incubation.

  6. Control of Formation and Cellular Detachment from Shewanella oneidensis MR-1 Biofilms by Cyclic di-GMP

    SciTech Connect

    Thormann, Kai M.; Duttler, Stefanie; Saville, Renee; Hyodo, Mamoru; Shukla, Soni; Hayakawa, Yoshihiro; Spormann, Alfred M.

    2006-04-01

    Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral -reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a Delta mxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

  7. Antibacterial activity of graphene-modified anode on Shewanella oneidensis MR-1 biofilm in microbial fuel cell

    NASA Astrophysics Data System (ADS)

    Chen, Jie; Deng, Feng; Hu, Yongyou; Sun, Jian; Yang, Yonggang

    2015-09-01

    To clearly illustrate the antibacterial activity of graphene on anodic exoelectrogen, the growth of a Shewanella oneidensis MR-1 biofilm on graphene-modified anodes (GMAs) and bare graphite anodes (BGs) were compared. The GMAs with different amounts of graphene were obtained by the cyclic voltammetric electrodeposition of 5, 20 and 40 potential cycles (5-G, 20-G and 40-G). Confocal scanning laser microscopy and cyclic voltammetry results demonstrated that graphene exhibited an obvious antibacterial effect for initial Shewanella MR biofilm growth. After 5 h of inoculation, 40-G, 20-G and 5-G had 6.3, 8.8 and 13.9% lower levels of biofilm viability, respectively, compared to BG, and all three exhibited approximately 70% lower electrochemical activity compared to BG. However, 18 h later, the biofilm on the GMAs exhibited much higher viability than that of the BG, and the electrochemical activity increased to a similar level. This study revealed the dual effect of graphene, including the antibacterial activity on biofilms and the enhancement of bacterial attachment and electron transfer.

  8. Fate of Fe and Cd upon microbial reduction of Cd-loaded polyferric flocs by Shewanella oneidensis MR-1.

    PubMed

    Li, Chenchen; Yi, Xiaoyun; Dang, Zhi; Yu, Hui; Zeng, Tao; Wei, Chaohai; Feng, Chunhua

    2016-02-01

    Polyferric sulphate has been widely used for emergent control on incidental release of heavy metals such as Cd to surface water, causing precipitation of Cd-loaded polyferric flocs to the sediment. To date, little is known about whether the dissolution of the flocs in the presence of dissimilatory iron reducing bacteria (DIRB) can occur and how the dissolution influences the fate of Fe and Cd in the sediment. Here, we demonstrated that Shewanella oneidensis MR-1, as representative DIRB, has the ability to reduce the flocs, resulting in the release of Fe(2+) and Cd(2+) to the solution. Batch experiment results showed that the concentrations of Fe(2+) and Cd(2+)reached the maximum values at 48 h and then decreased over the remaining incubation time. The characterizations on the solid phase by the scanning electron microscopy coupled with energy dispersive spectrometer, X-ray diffraction, and X-ray photoelectron spectroscopy technologies revealed the formation of iron minerals such as goethite and magnetite as a consequence of microbial Fe(III) reduction. The newly formed iron minerals played a significant role in re-immobilizing Cd by sorption. These results imply that microbial reduction of polyferric flocs is an important contributor to the transport and transformation of metals in the sediment-water interface. PMID:26583288

  9. Transcriptome Analysis Reveals that Red and Blue Light Regulate Growth and Phytohormone Metabolism in Norway Spruce [Picea abies (L.) Karst.

    PubMed Central

    OuYang, Fangqun; Mao, Jian-Feng; Wang, Junhui; Zhang, Shougong; Li, Yue

    2015-01-01

    The mechanisms by which different light spectra regulate plant shoot elongation vary, and phytohormones respond differently to such spectrum-associated regulatory effects. Light supplementation can effectively control seedling growth in Norway spruce. However, knowledge of the effective spectrum for promoting growth and phytohormone metabolism in this species is lacking. In this study, 3-year-old Norway spruce clones were illuminated for 12 h after sunset under blue or red light-emitting diode (LED) light for 90 d, and stem increments and other growth traits were determined. Endogenous hormone levels and transcriptome differences in the current needles were assessed to identify genes related to the red and blue light regulatory responses. The results showed that the stem increment and gibberellin (GA) levels of the seedlings illuminated by red light were 8.6% and 29.0% higher, respectively, than those of the seedlings illuminated by blue light. The indoleacetic acid (IAA) level of the seedlings illuminated by red light was 54.6% lower than that of the seedlings illuminated by blue light, and there were no significant differences in abscisic acid (ABA) or zeatin riboside [ZR] between the two groups of seedlings. The transcriptome results revealed 58,736,166 and 60,555,192 clean reads for the blue-light- and red-light-illuminated samples, respectively. Illumina sequencing revealed 21,923 unigenes, and 2744 (approximately 93.8%) out of 2926 differentially expressed genes (DEGs) were found to be upregulated under blue light. The main KEGG classifications of the DEGs were metabolic pathway (29%), biosynthesis of secondary metabolites (20.49%) and hormone signal transduction (8.39%). With regard to hormone signal transduction, AUXIN-RESISTANT1 (AUX1), AUX/IAA genes, auxin-inducible genes, and early auxin-responsive genes [(auxin response factor (ARF) and small auxin-up RNA (SAUR)] were all upregulated under blue light compared with red light, which might have yielded the

  10. Transcriptome Analysis Reveals that Red and Blue Light Regulate Growth and Phytohormone Metabolism in Norway Spruce [Picea abies (L.) Karst].

    PubMed

    OuYang, Fangqun; Mao, Jian-Feng; Wang, Junhui; Zhang, Shougong; Li, Yue

    2015-01-01

    The mechanisms by which different light spectra regulate plant shoot elongation vary, and phytohormones respond differently to such spectrum-associated regulatory effects. Light supplementation can effectively control seedling growth in Norway spruce. However, knowledge of the effective spectrum for promoting growth and phytohormone metabolism in this species is lacking. In this study, 3-year-old Norway spruce clones were illuminated for 12 h after sunset under blue or red light-emitting diode (LED) light for 90 d, and stem increments and other growth traits were determined. Endogenous hormone levels and transcriptome differences in the current needles were assessed to identify genes related to the red and blue light regulatory responses. The results showed that the stem increment and gibberellin (GA) levels of the seedlings illuminated by red light were 8.6% and 29.0% higher, respectively, than those of the seedlings illuminated by blue light. The indoleacetic acid (IAA) level of the seedlings illuminated by red light was 54.6% lower than that of the seedlings illuminated by blue light, and there were no significant differences in abscisic acid (ABA) or zeatin riboside [ZR] between the two groups of seedlings. The transcriptome results revealed 58,736,166 and 60,555,192 clean reads for the blue-light- and red-light-illuminated samples, respectively. Illumina sequencing revealed 21,923 unigenes, and 2744 (approximately 93.8%) out of 2926 differentially expressed genes (DEGs) were found to be upregulated under blue light. The main KEGG classifications of the DEGs were metabolic pathway (29%), biosynthesis of secondary metabolites (20.49%) and hormone signal transduction (8.39%). With regard to hormone signal transduction, AUXIN-RESISTANT1 (AUX1), AUX/IAA genes, auxin-inducible genes, and early auxin-responsive genes [(auxin response factor (ARF) and small auxin-up RNA (SAUR)] were all upregulated under blue light compared with red light, which might have yielded the

  11. De Novo Transcriptome Sequencing Reveals Important Molecular Networks and Metabolic Pathways of the Plant, Chlorophytum borivilianum

    PubMed Central

    Kalra, Shikha; Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Kumar, Sunil; Kaur, Jagdeep; Ramachandran, Srinivasan; Singh, Kashmir

    2013-01-01

    Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum. PMID:24376689

  12. NMR-Based Metabolic Profiling Reveals Neurochemical Alterations in the Brain of Rats Treated with Sorafenib.

    PubMed

    Du, Changman; Shao, Xue; Zhu, Ruiming; Li, Yan; Zhao, Qian; Fu, Dengqi; Gu, Hui; Kong, Jueying; Luo, Li; Long, Hailei; Deng, Pengchi; Wang, Huijuan; Hu, Chunyan; Zhao, Yinglan; Cen, Xiaobo

    2015-11-01

    Sorafenib, an active multi-kinase inhibitor, has been widely used as a chemotherapy drug to treat advanced clear-cell renal cell carcinoma patients. In spite of the relative safety, sorafenib has been shown to exert a negative impact on cognitive functioning in cancer patients, specifically on learning and memory; however, the underlying mechanism remains unclear. In this study, an NMR-based metabolomics approach was applied to investigate the neurochemical effects of sorafenib in rats. Male rats were once daily administrated with 120 mg/kg sorafenib by gavage for 3, 7, and 28 days, respectively. NMR-based metabolomics coupled with histopathology examinations for hippocampus, prefrontal cortex (PFC), and striatum were performed. The (1)H NMR spectra data were analyzed by using multivariate pattern recognition techniques to show the time-dependent biochemical variations induced by sorafenib. Excellent separation was obtained and distinguishing metabolites were observed between sorafenib-treated and control rats. A total of 36 differential metabolites in hippocampus of rats treated with sorafenib were identified, some of which were significantly changed. Furthermore, these modified metabolites mainly reflected the disturbances in neurotransmitters, energy metabolism, membrane, and amino acids. However, only a few metabolites in PFC and striatum were altered by sorafenib. Additionally, no apparent histological changes in these three brain regions were observed in sorafenib-treated rats. Together, our findings demonstrate the disturbed metabonomics pathways, especially, in hippocampus, which may underlie the sorafenib-induced cognitive deficits in patients. This work also shows the advantage of NMR-based metabolomics over traditional approach on the study of biochemical effects of drugs. PMID:26233726

  13. 1H-NMR Spectroscopy Revealed Mycobacterium tuberculosis Caused Abnormal Serum Metabolic Profile of Cattle

    PubMed Central

    Xiong, Xuekai; Hu, Xidan; Huang, Jiong; Xu, Zhiguang; Zhang, Xiansong; Hu, Changmin; Hu, Xueying; Guo, Aizhen; Wang, Yulan; Chen, Huanchun

    2013-01-01

    To re-evaluate virulence of Mycobacterium tuberculosis (M. tb) in cattle, we experimentally infected calves with M. tb andMycobacterium bovisvia intratracheal injection at a dose of 2.0×107 CFU and observed the animals for 33 weeks. The intradermal tuberculin test and IFN-γin vitro release assay showed that both M. tb and M. bovis induced similar responses. Immunohistochemical staining of pulmonary lymph nodes indicated that the antigen MPB83 of both M. tb and M. bovis were similarly distributed in the tissue samples. Histological examinations showed all of the infected groups exhibited neutrophil infiltration to similar extents. Although the infected cattle did not develop granulomatous inflammation, the metabolic profiles changed significantly, which were characterized by a change in energy production pathways and increased concentrations of N-acetyl glycoproteins. Glycolysis was induced in the infected cattle by decreased glucose and increased lactate content, and enhanced fatty acid β-oxidation was induced by decreased TG content, and decreased gluconeogenesis indicated by the decreased concentration of glucogenic and ketogenic amino acids promoted utilization of substances other than glucose as energy sources. In addition, an increase in acute phase reactive serum glycoproteins, together with neutrophil infiltration and increased of IL-1β production indicated an early inflammatory response before granuloma formation. In conclusion, this study indicated that both M. tb and M.bovis were virulent to cattle. Therefore, it is likely that cattle with M. tb infections would be critical to tuberculosis transmission from cattle to humans. Nuclear magnetic resonance was demonstrated to be an efficient method to systematically evaluate M. tb and M. bovi sinfection in cattle. PMID:24098654

  14. Metatranscriptomics reveals metabolic adaptation and induction of virulence factors by Haemophilus parasuis during lung infection.

    PubMed

    Bello-Ortí, Bernardo; Howell, Kate J; Tucker, Alexander W; Maskell, Duncan J; Aragon, Virginia

    2015-01-01

    Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs, and the causative agent of Glässer's disease. This disease is characterized by polyserositis and arthritis, produced by the severe inflammation caused by the systemic spread of the bacterium. After an initial colonization of the upper respiratory tract, H. parasuis enters the lung during the early stages of pig infection. In order to study gene expression at this location, we sequenced the ex vivo and in vivo H. parasuis Nagasaki transcriptome in the lung using a metatranscriptomic approach. Comparison of gene expression under these conditions with that found in conventional plate culture showed generally reduced expression of genes associated with anabolic and catabolic pathways, coupled with up-regulation of membrane-related genes involved in carbon acquisition, iron binding and pathogenesis. Some of the up-regulated membrane genes, including ABC transporters, virulence-associated autotransporters (vtaAs) and several hypothetical proteins, were only present in virulent H. parasuis strains, highlighting their significance as markers of disease potential. Finally, the analysis also revealed the presence of numerous antisense transcripts with possible roles in gene regulation. In summary, this data sheds some light on the scarcely studied in vivo transcriptome of H. parasuis, revealing nutritional virulence as an adaptive strategy for host survival, besides induction of classical virulence factors. PMID:26395877

  15. CD1d- and MR1-Restricted T Cells in Sepsis

    PubMed Central

    Szabo, Peter A.; Anantha, Ram V.; Shaler, Christopher R.; McCormick, John K.; Haeryfar, S.M. Mansour

    2015-01-01

    Dysregulated immune responses to infection, such as those encountered in sepsis, can be catastrophic. Sepsis is typically triggered by an overwhelming systemic response to an infectious agent(s) and is associated with high morbidity and mortality even under optimal critical care. Recent studies have implicated unconventional, innate-like T lymphocytes, including CD1d- and MR1-restricted T cells as effectors and/or regulators of inflammatory responses during sepsis. These cell types are typified by invariant natural killer T (iNKT) cells, variant NKT (vNKT) cells, and mucosa-associated invariant T (MAIT) cells. iNKT and vNKT cells are CD1d-restricted, lipid-reactive cells with remarkable immunoregulatory properties. MAIT cells participate in antimicrobial defense, and are restricted by major histocompatibility complex-related protein 1 (MR1), which displays microbe-derived vitamin B metabolites. Importantly, NKT and MAIT cells are rapid and potent producers of immunomodulatory cytokines. Therefore, they may be considered attractive targets during the early hyperinflammatory phase of sepsis when immediate interventions are urgently needed, and also in later phases when adjuvant immunotherapies could potentially reverse the dangerous state of immunosuppression. We will highlight recent findings that point to the significance or the therapeutic potentials of NKT and MAIT cells in sepsis and will also discuss what lies ahead in research in this area. PMID:26322041

  16. Photocatalytic properties of zinc sulfide nanocrystals biofabricated by metal-reducing bacterium Shewanella oneidensis MR-1.

    PubMed

    Xiao, Xiang; Ma, Xiao-Bo; Yuan, Hang; Liu, Peng-Cheng; Lei, Yu-Bin; Xu, Hui; Du, Dao-Lin; Sun, Jian-Fan; Feng, Yu-Jie

    2015-05-15

    Accumulation and utilization of heavy metals from wastewater by biological treatment system has aroused great interest. In the present study, a metal-reducing bacterium Shewanella oneidensis MR-1 was used to explore the biofabrication of ZnS nanocrystals from the artificial wastewater. The biogenic H2S produced via the reduction of thiosulfate precipitated the Zn(II) as sulfide extracellularly. Characterization by X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), and field emission scanning electron microscope (FESEM) confirmed the precipitates as ZnS nanocrystals. The biogenic ZnS nanocrystals appeared spherical in shape with an average diameter of 5 nm and mainly aggregated in the medium and cell surface of S. oneidensis MR-1. UV-vis DRS spectra showed ZnS nanoparticles appeared a strong absorption below 360 nm. Thus, the photocatalytic activity of ZnS was evaluated by the photodegradation of rhodamine B (RhB) under UV irradiation. The biogenic ZnS nanocrystals showed a high level of photodegradation efficiency to RhB coupled with a significant blue-shift of maximum adsorption peak. A detailed analysis indicated the photogenerated holes, rather than hydroxyl radicals, contributed to the photocatalytic decolorization of RhB. This approach of coupling biosynthesis of nanoparticles with heavy metal removal may offer a potential avenue for efficient bioremediation of heavy metal wastewater. PMID:25698574

  17. Bat breath reveals metabolic substrate use in free-ranging vampires.

    PubMed

    Voigt, Christian C; Grasse, Patricia; Rex, Katja; Hetz, Stefan K; Speakman, John R

    2008-01-01

    We analysed the stable carbon isotope ratio in exhaled CO(2) (delta(13)C(breath)) of free-ranging vampires to assess the type of metabolized substrate (endogenous or exogenous substrate) and its origin, i.e. whether the carbon atoms came from a C(4) food web (grass and cattle) or the C(3) food web in which they were captured (a rainforest remnant and its mammals). For an improved understanding of factors influencing the delta(13)C(breath) of vampires, we conducted feeding experiments with captive animals. The mean delta(13)C(breath) of starved bats was depleted in (13)C in relation to the diet by 4.6 per thousand (n = 10). Once fed with blood, delta(13)C(breath )levelled off within a short time approximately 2.2 per thousand above the stable carbon isotope signature of the diet. The median time required to exchange 50% of the carbon atoms in exhaled CO(2) with carbon atoms from the ingested blood was 18.6 min (mean 29.5 +/- 19.0 min, n = 5). The average delta(13)C of wing membrane and fur in free-ranging vampire bats suggested that bats almost exclusively foraged for cattle blood during the past weeks. The delta(13)C(breath) of the same bats averaged -19.1 per thousand. Given that all free-ranging vampires were starving and that the delta(13)C of cattle was more in enriched in (13)C by 5-6 per thousand than the delta(13)C(breath) of vampires, we conclude that the vampire bats of our study metabolised fat that was predominantly built from carbon atoms originating from cattle blood. Since delta(13)C of wing membrane and fur integrates over weeks and months respectively and delta(13)C(breath) over hours and days, we also conclude that vampire bats of the studied population consistently ignored rainforest mammals and chose cattle as their prey during and prior to our study. PMID:17701185

  18. Enhanced performance of hexavalent chromium reducing cathodes in the presence of Shewanella oneidensis MR-1 and lactate.

    PubMed

    Xafenias, Nikolaos; Zhang, Yue; Banks, Charles J

    2013-05-01

    Biocathodes for the reduction of the highly toxic hexavalent chromium (Cr(VI)) were investigated using Shewanella oneidensis MR-1 (MR-1) as a biocatalyst and performance was assessed in terms of current production and Cr(VI) reduction. Potentiostatically controlled experiments (-500 mV vs Ag/AgCl) showed that a mediatorless MR-1 biocathode started up under aerated conditions in the presence of lactate, received 5.5 and 1.7 times more electrons for Cr(VI) reduction over a 4 h operating period than controls without lactate and with lactate but without MR-1, respectively. Cr(VI) reduction was also enhanced, with a decrease in concentration over the 4 h operating period of 9 mg/L Cr(VI), compared to only 1 and 3 mg/L, respectively, in the controls. Riboflavin, an electron shuttle mediator naturally produced by MR-1, was also found to have a positive impact in potentiostatically controlled cathodes. Additionally, a microbial fuel cell (MFC) with MR-1 and lactate present in both anode and cathode produced a maximum current density of 32.5 mA/m(2) (1000 Ω external load) after receiving a 10 mg/L Cr(VI) addition in the cathode, and cathodic efficiency increased steadily over an 8 day operation period with successive Cr(VI) additions. In conclusion, effective and continuous Cr(VI) reduction with associated current production were achieved when MR-1 and lactate were both present in the biocathodes. PMID:23517384

  19. The Crystal Structure of Nitrosomonas europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into Sucrose Metabolism in Prokaryotes

    PubMed Central

    Wu, Rui; Asención Diez, Matías D.; Figueroa, Carlos M.; Machtey, Matías; Iglesias, Alberto A.; Ballicora, Miguel A.

    2015-01-01

    ABSTRACT In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the nonphotosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure, from the plant Arabidopsis thaliana, was in a closed conformation. Comparative structural analysis revealed a “hinge-latch” combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family was shown to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, and Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded. IMPORTANCE We obtained biochemical and structural evidence of sucrose metabolism in nonphotosynthetic bacteria. Until now, only sucrose synthases from photosynthetic organisms have been characterized. Here, we provide the crystal structure of the sucrose synthase from the chemolithoautotroph N. europaea. The structure supported that the enzyme functions with an open/close induced fit mechanism. The enzyme prefers as the substrate adenine-based nucleotides rather than uridine-based like the eukaryotic counterparts, implying a strong connection between sucrose and glycogen metabolism in these bacteria. Mutagenesis data showed that the catalytic mechanism must be conserved not only in sucrose synthases but also in all other retaining GT-B glycosyltransferases. PMID:26013491

  20. Metabolic analysis of Chlorobium chlorochromatii CaD3 reveals clues of the symbiosis in ‘Chlorochromatium aggregatum'.

    PubMed Central

    Cerqueda-García, Daniel; Martínez-Castilla, León P; Falcón, Luisa I; Delaye, Luis

    2014-01-01

    A symbiotic association occurs in ‘Chlorochromatium aggregatum', a phototrophic consortium integrated by two species of phylogenetically distant bacteria composed by the green-sulfur Chlorobium chlorochromatii CaD3 epibiont that surrounds a central β-proteobacterium. The non-motile chlorobia can perform nitrogen and carbon fixation, using sulfide as electron donors for anoxygenic photosynthesis. The consortium can move due to the flagella present in the central β-protobacterium. Although Chl. chlorochromatii CaD3 is never found as free-living bacteria in nature, previous transcriptomic and proteomic studies have revealed that there are differential transcription patterns between the symbiotic and free-living status of Chl. chlorocromatii CaD3 when grown in laboratory conditions. The differences occur mainly in genes encoding the enzymatic reactions involved in nitrogen and amino acid metabolism. We performed a metabolic reconstruction of Chl. chlorochromatii CaD3 and an in silico analysis of its amino acid metabolism using an elementary flux modes approach (EFM). Our study suggests that in symbiosis, Chl. chlorochromatii CaD3 is under limited nitrogen conditions where the GS/GOGAT (glutamine synthetase/glutamate synthetase) pathway is actively assimilating ammonia obtained via N2 fixation. In contrast, when free-living, Chl. chlorochromatii CaD3 is in a condition of nitrogen excess and ammonia is assimilated by the alanine dehydrogenase (AlaDH) pathway. We postulate that ‘Chlorochromatium aggregatum' originated from a parasitic interaction where the N2 fixation capacity of the chlorobia would be enhanced by injection of 2-oxoglutarate from the β-proteobacterium via the periplasm. This consortium would have the advantage of motility, which is fundamental to a phototrophic bacterium, and the syntrophy of nitrogen and carbon sources. PMID:24285361

  1. Metabolomics reveals the metabolic shifts following an intervention with rye bread in postmenopausal women- a randomized control trial

    PubMed Central

    2012-01-01

    Background Epidemiological studies have consistently shown that whole grain (WG) cereals can protect against the development of chronic diseases, but the underlying mechanism is not fully understood. Among WG products, WG rye is considered even more potent because of its unique discrepancy in postprandial insulin and glucose responses known as the rye factor. In this study, an NMR-based metabolomics approach was applied to study the metabolic effects of WG rye as a tool to determine the beneficial effects of WG rye on human health. Methods Thirty-three postmenopausal Finnish women with elevated serum total cholesterol (5.0-8.5 mmol/L) and BMI of 20–33 kg/m2 consumed a minimum of 20% of their daily energy intake as high fiber WG rye bread (RB) or refined wheat bread (WB) in a randomized, controlled, crossover design with two 8-wk intervention periods separated by an 8-wk washout period. At the end of each intervention period, fasting serum was collected for NMR-based metabolomics and the analysis of cholesterol fractions. Multilevel partial least squares discriminant analysis was used for paired comparisons of multivariate data. Results The metabolomics analysis of serum showed lower leucine and isoleucine and higher betaine and N,N-dimethylglycine levels after RB than WB intake. To further investigate the metabolic effects of RB, the serum cholesterol fractions were measured. Total- and LDL-cholesterol levels were higher after RB intake than after WB (p<0.05). Conclusions This study revealed favorable shifts in branched amino acid and single carbon metabolism and an unfavorable shift in serum cholesterol levels after RB intake in postmenopausal women, which should be considered for evaluating health beneficial effects of rye products. PMID:23088297

  2. Sulfur isotopes of organic matter preserved in 3.45-billion-year-old stromatolites reveal microbial metabolism

    PubMed Central

    Bontognali, Tomaso R. R.; Sessions, Alex L.; Allwood, Abigail C.; Fischer, Woodward W.; Grotzinger, John P.; Summons, Roger E.; Eiler, John M.

    2012-01-01

    The 3.45-billion-year-old Strelley Pool Formation of Western Australia preserves stromatolites that are considered among the oldest evidence for life on Earth. In places of exceptional preservation, these stromatolites contain laminae rich in organic carbon, interpreted as the fossil remains of ancient microbial mats. To better understand the biogeochemistry of these rocks, we performed microscale in situ sulfur isotope measurements of the preserved organic sulfur, including both Δ33S and . This approach allows us to tie physiological inference from isotope ratios directly to fossil biomass, providing a means to understand sulfur metabolism that is complimentary to, and independent from, inorganic proxies (e.g., pyrite). Δ33S values of the kerogen reveal mass-anomalous fractionations expected of the Archean sulfur cycle, whereas values show large fractionations at very small spatial scales, including values below -15‰. We interpret these isotopic patterns as recording the process of sulfurization of organic matter by H2S in heterogeneous mat pore-waters influenced by respiratory S metabolism. Positive Δ33S anomalies suggest that disproportionation of elemental sulfur would have been a prominent microbial process in these communities. PMID:22949693

  3. Sulfur isotopes of organic matter preserved in 3.45-billion-year-old stromatolites reveal microbial metabolism.

    PubMed

    Bontognali, Tomaso R R; Sessions, Alex L; Allwood, Abigail C; Fischer, Woodward W; Grotzinger, John P; Summons, Roger E; Eiler, John M

    2012-09-18

    The 3.45-billion-year-old Strelley Pool Formation of Western Australia preserves stromatolites that are considered among the oldest evidence for life on Earth. In places of exceptional preservation, these stromatolites contain laminae rich in organic carbon, interpreted as the fossil remains of ancient microbial mats. To better understand the biogeochemistry of these rocks, we performed microscale in situ sulfur isotope measurements of the preserved organic sulfur, including both Δ(33)S and . This approach allows us to tie physiological inference from isotope ratios directly to fossil biomass, providing a means to understand sulfur metabolism that is complimentary to, and independent from, inorganic proxies (e.g., pyrite). Δ(33)S values of the kerogen reveal mass-anomalous fractionations expected of the Archean sulfur cycle, whereas values show large fractionations at very small spatial scales, including values below -15‰. We interpret these isotopic patterns as recording the process of sulfurization of organic matter by H(2)S in heterogeneous mat pore-waters influenced by respiratory S metabolism. Positive Δ(33)S anomalies suggest that disproportionation of elemental sulfur would have been a prominent microbial process in these communities. PMID:22949693

  4. Transcriptome Analysis of Skin Photoaging in Chinese Females Reveals the Involvement of Skin Homeostasis and Metabolic Changes

    PubMed Central

    Yan, Li; Zhang, Feng; Yin, Ning-Bei; Lin, Hong-Bin; Huang, Chen-Yu; Wang, Lei; Yu, Jun; Wang, Duen-Mei; Zhao, Zhen-Min

    2013-01-01

    Background Photoaging is cumulative damage to skin, caused by chronic, repeated solar radiation exposure. Its molecular mechanisms are poorly understood at the level of global gene expression. Objective This study set out to uncover genes and functional modules involved in photoaging at the level of transcription, with the use of skin samples from Chinese women. Methods Using the Illumina microarray platform, we compared the genome-wide expression profiles of 21 pairs of sun-exposed pre-auricular and sun-protected post-auricular skin samples from northern Chinese women. Results With microarray analysis, 1,621 significantly regulated genes due to photoaging were identified from skin samples. These genes were subjected to functional enrichment analyses with both the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation databases. As revealed by the functional analyses, the up-regulated functional modules in sun-exposed pre-auricular skin were related to various cellular activities in regulation of the skin homeostasis (e.g., the KEGG pathways TGF-beta signaling pathway and ECM-receptor interaction), whereas the down-regulated functional modules were mostly metabolic-related. Additionally, five selected genes (HOXA5, LEPR, CLDN5, LAMC3, and CGA) identified as differentially-expressed were further confirmed by quantitative real-time PCR (Q-RT-PCR). Conclusion Our findings suggest that disruption of skin homeostasis and down-regulation of skin metabolism may play important roles in the process of photoaging. PMID:23637934

  5. Alterations in Hepatic Metabolism in fld Mice Reveal a Role for Lipin 1 in Regulating VLDL-Triacylglyceride Secretion

    PubMed Central

    Chen, Zhouji; Gropler, Matthew C.; Norris, Jin; Lawrence, John C.; Harris, Thurl E.; Finck, Brian N.

    2009-01-01

    Objective Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches. Methods and Results Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1—deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling. Conclusions Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance. PMID:18669885

  6. Transcriptomics and physiological analyses reveal co-ordinated alteration of metabolic pathways in Jatropha curcas drought tolerance.

    PubMed

    Sapeta, Helena; Lourenço, Tiago; Lorenz, Stefan; Grumaz, Christian; Kirstahler, Philipp; Barros, Pedro M; Costa, Joaquim Miguel; Sohn, Kai; Oliveira, M Margarida

    2016-02-01

    Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway. PMID:26602946

  7. RNA-seq reveals the critical role of CspA in regulating Brucella melitensis metabolism and virulence.

    PubMed

    Wang, Zhen; Liu, Wenxiao; Wu, Tonglei; Bie, Pengfei; Wu, Qingmin

    2016-04-01

    Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of Brucella to survive and multiply in the hostile environment of host macrophages is essential for its virulence. The cold shock protein CspA plays an important role in the virulence of B. melitensis. To analyze the genes regulated by CspA, the whole transcriptomes of B. melitensis NIΔcspA and its parental wild-type strain, B. melitensis NI, were sequenced and analyzed using the Solexa/Illumina sequencing platform. A total of 446 differentially expressed genes were identified, including 324 up-regulated and 122 down-regulated genes. Numerous genes identified are involved in amino acid, fatty acid, nitrogen, and energy metabolism. Interestingly, all genes involved in the type IV secretion system and LuxR-type regulatory protein VjbR were significantly down-regulated in NIΔcspA. In addition, an effector translocation assay confirmed that the function of T4SS in NIΔcspA is influenced by deletion of the cspA gene. These results revealed the differential phenomena associated with virulence and metabolism in NIΔcspA and NI, providing important information for understanding detailed CspA-regulated interaction networks and Brucella pathogenesis. PMID:26740105

  8. Metaproteomic Analysis of a Chemosynthetic Hydrothermal Vent Community Reveals Insights into Key-Metabolic Processes

    NASA Astrophysics Data System (ADS)

    Steen, I.; Stokke, R.; Lanzen, A.; Pedersen, R.; Øvreås, L.; Urich, T.

    2010-12-01

    internal sulfur cycle within the community. The community contained expressed enzymes of a variety of carbon metabolism pathways. Key enzymes of the reverse TCA cycle for fixation of CO2 and the Wood-Ljungdahl pathway for oxidation of acetyl-CoA and / or the fixation of CO2 were found. Key enzymes of aerobic and anaerobic methane-oxidation pathways were identified as well, namely particulate methane monooxygenase and methyl-Coenzyme M reductase. Various house-keeping gene-products, like cold- and heat shock proteins as well as ribosomal proteins and ATP synthases were identified. This approach has a future potential of broadening our understanding of environmental complexity and regulation in response to geochemical constraints. [1] Dupierris, V., Masselon, C., Court, M., Kieffer-Jaquinod, S., and Bruley, C. (2009) A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25, 1980-1981.

  9. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    SciTech Connect

    Khotin, Mikhail; Turoverova, Lidia; Aksenova, Vasilisa; Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7 Barlev, Nikolai; Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN ; Borutinskaite, Veronika Viktorija; Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius ; Vener, Alexander; Bajenova, Olga; Pinaev, George P.; Tentler, Dmitri

    2010-06-25

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  10. Photoactivation of Mutant Isocitrate Dehydrogenase 2 Reveals Rapid Cancer-Associated Metabolic and Epigenetic Changes

    PubMed Central

    2016-01-01

    Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation. PMID:26761588

  11. Photoactivation of Mutant Isocitrate Dehydrogenase 2 Reveals Rapid Cancer-Associated Metabolic and Epigenetic Changes.

    PubMed

    Walker, Olivia S; Elsässer, Simon J; Mahesh, Mohan; Bachman, Martin; Balasubramanian, Shankar; Chin, Jason W

    2016-01-27

    Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation. PMID:26761588

  12. Single-cell genomics reveal metabolic strategies for microbial growth and survival in an oligotrophic aquifer

    SciTech Connect

    Wilkins, Michael J.; Kennedy, David W.; Castelle, Cindy; Field, Erin; Stepanauskas, Ramunas; Fredrickson, Jim K.; Konopka, Allan

    2014-02-09

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site and have been shown to significantly change in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single cell genomics techniques to shed light on the physiological niche of these microorganisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3­-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide-range of both intra-and extra­-cellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors (TBDRs), which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. CRISPR-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic microorganisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area subsurface and biogeochemical shifts associated with Columbia River water intrusion.

  13. Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism.

    PubMed

    Spain, Anne M; Elshahed, Mostafa S; Najar, Fares Z; Krumholz, Lee R

    2015-01-01

    Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring's source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons. PMID:26417542

  14. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

    PubMed Central

    van der Meulen, Sjoerd B.; de Jong, Anne; Kok, Jan

    2016-01-01

    ABSTRACT RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  15. Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism

    PubMed Central

    Iwata, Terri N.; Ramírez, Julita A.; Tsang, Mark; Park, Heon; Margineantu, Daciana H.; Hockenbery, David M.

    2016-01-01

    Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism. PMID:27521345

  16. Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism

    PubMed Central

    Elshahed, Mostafa S.; Najar, Fares Z.; Krumholz, Lee R.

    2015-01-01

    Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring’s source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons. PMID:26417542

  17. Metagenomic approach reveals variation of microbes with arsenic and antimony metabolism genes from highly contaminated soil.

    PubMed

    Luo, Jinming; Bai, Yaohui; Liang, Jinsong; Qu, Jiuhui

    2014-01-01

    Microbes have great potential for arsenic (As) and antimony (Sb) bioremediation in heavily contaminated soil because they have the ability to biotransform As and Sb to species that have less toxicity or are more easily removed. In this study, we integrated a metagenomic method with physicochemical characterization to elucidate the composition of microbial community and functional genes (related to As and Sb) in a high As (range from 34.11 to 821.23 mg kg-1) and Sb (range from 226.67 to 3923.07 mg kg-1) contaminated mine field. Metagenomic analysis revealed that microbes from 18 phyla were present in the 5 samples of soil contaminated with high As and Sb. Moreover, redundancy analysis (RDA) of the relationship between the 18 phyla and the concentration of As and Sb demonstrated that 5 phyla of microbes, i.e. Actinobacteria, Firmicutes, Nitrospirae, Tenericutes and Gemmatimonadetes were positively correlated with As and Sb concentration. The distribution, diversity and abundance of functional genes (including arsC, arrA, aioA, arsB and ACR3) were much higher for the samples containing higher As and Sb concentrations. Based on correlation analysis, the results showed a positive relationship between arsC-like (R2 = 0.871) and aioA-like (R2 = 0.675) gene abundance and As concentration, and indicated that intracellular As(V) reduction and As(III) oxidation could be the dominant As detoxification mechanism enabling the microbes to survive in the environment. This study provides a direct and reliable reference on the diversity of microbial community and functional genes in an extremely high concentration As- and Sb-contaminated environment. PMID:25299175

  18. Metagenomic Approach Reveals Variation of Microbes with Arsenic and Antimony Metabolism Genes from Highly Contaminated Soil

    PubMed Central

    Luo, Jinming; Bai, Yaohui; Liang, Jinsong; Qu, Jiuhui

    2014-01-01

    Microbes have great potential for arsenic (As) and antimony (Sb) bioremediation in heavily contaminated soil because they have the ability to biotransform As and Sb to species that have less toxicity or are more easily removed. In this study, we integrated a metagenomic method with physicochemical characterization to elucidate the composition of microbial community and functional genes (related to As and Sb) in a high As (range from 34.11 to 821.23 mg kg−1) and Sb (range from 226.67 to 3923.07 mg kg−1) contaminated mine field. Metagenomic analysis revealed that microbes from 18 phyla were present in the 5 samples of soil contaminated with high As and Sb. Moreover, redundancy analysis (RDA) of the relationship between the 18 phyla and the concentration of As and Sb demonstrated that 5 phyla of microbes, i.e. Actinobacteria, Firmicutes, Nitrospirae, Tenericutes and Gemmatimonadetes were positively correlated with As and Sb concentration. The distribution, diversity and abundance of functional genes (including arsC, arrA, aioA, arsB and ACR3) were much higher for the samples containing higher As and Sb concentrations. Based on correlation analysis, the results showed a positive relationship between arsC-like (R2 = 0.871) and aioA-like (R2 = 0.675) gene abundance and As concentration, and indicated that intracellular As(V) reduction and As(III) oxidation could be the dominant As detoxification mechanism enabling the microbes to survive in the environment. This study provides a direct and reliable reference on the diversity of microbial community and functional genes in an extremely high concentration As- and Sb-contaminated environment. PMID:25299175

  19. Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation

    PubMed Central

    2012-01-01

    Background The lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor. Results After growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the β-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. Conclusions Our quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic

  20. Single-cell genomics reveals metabolic strategies for microbial growth and survival in an oligotrophic aquifer.

    PubMed

    Wilkins, Michael J; Kennedy, David W; Castelle, Cindy J; Field, Erin K; Stepanauskas, Ramunas; Fredrickson, James K; Konopka, Allan E

    2014-02-01

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site (a largely decommissioned nuclear production complex) in eastern Washington state, USA, and have been shown to change significantly in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single-cell genomics techniques to shed light on the physiological niche of these micro-organisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide range of both intra- and extracellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors, which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic micro-organisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area (in the south-eastern corner of the site) subsurface and biogeochemical shifts associated with Columbia River water intrusion. PMID:24324032

  1. The Role of 4-Hydroxyphenylpyruvate Dioxygenase in Enhancement of Solid-Phase Electron Transfer by Shewanella oneidensis MR-1

    SciTech Connect

    Turick, Charles E.; Beliaev, Alex S.; Zakrajsek, Brian A.; Reardon, Catherine L.; Lowy, Daniel A.; Poppy, Tara E.; Maloney, Andrea; Ekechukwu, Amy A.

    2009-05-01

    ABSTRACT - While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane associated c-type cytochromes and electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of the tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione ([2-(2- chloro- 4- methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA, which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates at which MR-1 reduces hydrous ferric oxide were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E°') of S. oneidensis MR-1. Based on our findings, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in S. oneidensis MR-1.

  2. Cu-doped TiO(2) nanoparticles enhance survival of Shewanella oneidensis MR-1 under ultraviolet light (UV) exposure.

    PubMed

    Wu, Bing; Zhuang, Wei-Qin; Sahu, Manoranjan; Biswas, Pratim; Tang, Yinjie J

    2011-10-01

    It has been shown that photocatalytic TiO(2) nanoparticles (NPs) can be used as an efficient anti-microbial agent under UV light due to generation of reactive oxygen species (ROS), while Shewanella oneidensis MR-1 is a metal-reducing bacterium highly susceptible to UV radiation. Interestingly, we found that the presence of Cu-doped TiO(2) NPs in the cultural medium dramatically increased the survival rates (based on colony-forming unit) of strain MR-1 by over 10,000-fold (incubation without shaking) and ~200 fold (incubation with shaking) after a 2-h exposure to UV light. Gene expression results (via qPCR measurement) indicated that the DNA repair gene recA in MR-1 was significantly induced by UV exposure (indicating cellular damage under UV stress), but the influence of NPs on recA expression was not statistically evident. Plausible explanations to NP attenuation of UV stresses are: 1. TiO(2) based NPs are capable of scattering and absorbing UV light and thus create a shading effect to protect MR-1 from UV radiation; 2. more importantly, Cu-doped TiO(2) NPs can co-agglomerate with MR-1 to form large flocs that improves cells' survival against the environmental stresses. This study improves our understanding of NP ecological impacts under natural solar radiation and provides useful insights to application of photocatalytic-NPs for bacterial disinfection. PMID:21855961

  3. Growth Inhibition and Stimulation of Shewanella oneidensis MR-1 by Surfactants and Calcium Polysulfide

    SciTech Connect

    Bailey, Kathryn L.; Tilton, Fred A.; Jansik, Danielle P.; Ergas, Sarina J.; Marshall, Matthew J.; Miracle, Ann L.; Wellman, Dawn M.

    2012-06-14

    Foam delivery technology (FDT) uses surfactant based foam to immobilize subsurface contaminants in situ. Where traditional approaches are impractical, FDT has the potential to overcome many of the technical challenges facing the remediation of contaminated deep vadose zone environments. However, little is known about the effects these reactive chemicals may have on microorganisms inhabiting the contaminated subsurface. In addition, there are currently no standard assays to assess microbial responses to subsurface remedial treatments while these agents are under development. The objective of this study was to develop a rapid laboratory assay to assess the potential growth inhibition and/or stimulation of microorganisms following exposure to candidate FDT components. Calcium polysulfide (CPS) and several surfactants (i.e. sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), cocamidopropyl betaine (CAPB) and NINOL40-CO) have diverse chemistries and are candidate components of FDT. Shewanella oneidensis MR-1 cultures were exposed to a range of concentrations of these chemicals to determine the minimum bactericidal concentration (MBC) and the growth and viability potential of these components. Concentrations of SDS higher than 700 {micro}M were toxic to S. oneidensis MR-1 growth over the course of four days of exposure. The relative acute toxicity order for these compounds was SDS>>CPS>>NINOL40-CO>SLES-CAPB. Dose dependent growth decreases (20 to 100 mM) were observed in the CAPB and SLES treated cultures and both CPS and NINOL 40-CO were toxic at all concentrations tested (1.45 to 7.25 mM CPS). Both SLES (20 to 100 mM) and SDS at lower concentrations (20 to 500 {micro}M) were stimulatory to S. oneidensis MR-1 indicating a capacity to be used as a carbon source. These studies also identified potentially key component characteristics, such as precipitate formation and oxygen availability, which may prove valuable in assessing the response of subsurface

  4. Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella Oneidensis MR-1

    PubMed Central

    Shi, Liang; Rosso, Kevin M.; Clarke, Tomas A.; Richardson, David J.; Zachara, John M.; Fredrickson, James K.

    2012-01-01

    In the absence of O2 and other electron acceptors, the Gram-negative bacterium Shewanella oneidensis MR-1 can use ferric [Fe(III)] (oxy)(hydr)oxide minerals as the terminal electron acceptors for anaerobic respiration. At circumneutral pH and in the absence of strong complexing ligands, Fe(III) oxides are relatively insoluble and thus are external to the bacterial cells. S. oneidensis MR-1 and related strains of metal-reducing Shewanella have evolved machinery (i.e., metal-reducing or Mtr pathway) for transferring electrons from the inner-membrane, through the periplasm and across the outer-membrane to the surface of extracellular Fe(III) oxides. The protein components identified to date for the Mtr pathway include CymA, MtrA, MtrB, MtrC, and OmcA. CymA is an inner-membrane tetraheme c-type cytochrome (c-Cyt) that belongs to the NapC/NrfH family of quinol dehydrogenases. It is proposed that CymA oxidizes the quinol in the inner-membrane and transfers the released electrons to MtrA either directly or indirectly through other periplasmic proteins. A decaheme c-Cyt, MtrA is thought to be embedded in the trans outer-membrane and porin-like protein MtrB. Together, MtrAB deliver the electrons through the outer-membrane to the MtrC and OmcA on the outmost bacterial surface. MtrC and OmcA are the outer-membrane decaheme c-Cyts that are translocated across the outer-membrane by the bacterial type II secretion system. Functioning as terminal reductases, MtrC and OmcA can bind the surface of Fe(III) oxides and transfer electrons directly to these minerals via their solvent-exposed hemes. To increase their reaction rates, MtrC and OmcA can use the flavins secreted by S. oneidensis MR-1 cells as diffusible co-factors for reduction of Fe(III) oxides. Because of their extracellular location and broad redox potentials, MtrC and OmcA can also serve as the terminal reductases for soluble forms of Fe(III). In addition to Fe(III) oxides, Mtr pathway is also involved in reduction of

  5. Genome-scale study reveals reduced metabolic adaptability in patients with non-alcoholic fatty liver disease.

    PubMed

    Hyötyläinen, Tuulia; Jerby, Livnat; Petäjä, Elina M; Mattila, Ismo; Jäntti, Sirkku; Auvinen, Petri; Gastaldelli, Amalia; Yki-Järvinen, Hannele; Ruppin, Eytan; Orešič, Matej

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is a major risk factor leading to chronic liver disease and type 2 diabetes. Here we chart liver metabolic activity and functionality in NAFLD by integrating global transcriptomic data, from human liver biopsies, and metabolic flux data, measured across the human splanchnic vascular bed, within a genome-scale model of human metabolism. We show that an increased amount of liver fat induces mitochondrial metabolism, lipolysis, glyceroneogenesis and a switch from lactate to glycerol as substrate for gluconeogenesis, indicating an intricate balance of exacerbated opposite metabolic processes in glycemic regulation. These changes were associated with reduced metabolic adaptability on a network level in the sense that liver fat accumulation puts increasing demands on the liver to adaptively regulate metabolic responses to maintain basic liver functions. We propose that failure to meet excessive metabolic challenges coupled with reduced metabolic adaptability may lead to a vicious pathogenic cycle leading to the co-morbidities of NAFLD. PMID:26839171

  6. Genome-scale study reveals reduced metabolic adaptability in patients with non-alcoholic fatty liver disease

    PubMed Central

    Hyötyläinen, Tuulia; Jerby, Livnat; Petäjä, Elina M.; Mattila, Ismo; Jäntti, Sirkku; Auvinen, Petri; Gastaldelli, Amalia; Yki-Järvinen, Hannele; Ruppin, Eytan; Orešič, Matej

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is a major risk factor leading to chronic liver disease and type 2 diabetes. Here we chart liver metabolic activity and functionality in NAFLD by integrating global transcriptomic data, from human liver biopsies, and metabolic flux data, measured across the human splanchnic vascular bed, within a genome-scale model of human metabolism. We show that an increased amount of liver fat induces mitochondrial metabolism, lipolysis, glyceroneogenesis and a switch from lactate to glycerol as substrate for gluconeogenesis, indicating an intricate balance of exacerbated opposite metabolic processes in glycemic regulation. These changes were associated with reduced metabolic adaptability on a network level in the sense that liver fat accumulation puts increasing demands on the liver to adaptively regulate metabolic responses to maintain basic liver functions. We propose that failure to meet excessive metabolic challenges coupled with reduced metabolic adaptability may lead to a vicious pathogenic cycle leading to the co-morbidities of NAFLD. PMID:26839171

  7. Mutants Shewanella Oneidensis MR-1 Effective in Generating Bioelectricity in Microbial Fuel Cells

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Shakulov, R. S.; Sidoruk, K. V.; Smirnov, I. A.; Ilyin, V. K.; Soldatov, P. E.; Tyurin-Kuz'min, A. Y.; Smolenskaya, T. S.; Debabov, V. G.

    2013-02-01

    The capability of Shewanella oneidensis MR-1 mutants FRS1 and FRB1, characterized by a high reducing activity, to generate electricity was measured in microbial fuel cells (MFC) of different types. The reducing activity of these mutants was previously identified with respect to the rate of discoloration (reduction) of methylene blueё, a dye that served as an electron transfer mediator. The present study demonstrated that the rate of MB discoloration was in direct correlation with the level of voltage in MFC of the above strains. In case when the mutants were used, the parameter in MFC proved to be 30-40% greater than in the case of the parent strain. In the course of this investigation MFC characteristics were identified, optimal electrode materials and ion-selective membranes selected, and MFC laboratory mockups manufactured.

  8. A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

    PubMed Central

    Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

    2015-01-01

    Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

  9. A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum.

    PubMed

    Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

    2015-01-01

    Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

  10. Comparative Genomics Revealed Genetic Diversity and Species/Strain-Level Differences in Carbohydrate Metabolism of Three Probiotic Bifidobacterial Species

    PubMed Central

    Odamaki, Toshitaka; Horigome, Ayako; Sugahara, Hirosuke; Hashikura, Nanami; Minami, Junichi; Xiao, Jin-zhong; Abe, Fumiaki

    2015-01-01

    Strains of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis are widely used as probiotics in the food industry. Although numerous studies have revealed the properties and functionality of these strains, it is uncertain whether these characteristics are species common or strain specific. To address this issue, we performed a comparative genomic analysis of 49 strains belonging to these three bifidobacterial species to describe their genetic diversity and to evaluate species-level differences. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve. Further analysis focused on carbohydrate metabolism revealed the existence of certain strain-dependent genes, such as those encoding enzymes for host glycan utilisation or certain membrane transporters, and many genes commonly distributed at the species level, as was previously reported in studies with limited strains. As B. longum and B. breve are human-residential bifidobacteria (HRB), whereas B. animalis is a non-HRB species, several of the differences in these species' gene distributions might be the result of their adaptations to the nutrient environment. This information may aid both in selecting probiotic candidates and in understanding their potential function as probiotics. PMID:26236711

  11. Global Transcriptome Analysis of the Cold Shock Response ofShewanella oneidensis MR-1 and Mutational Analysis of Its Classical ColdShock Proteins.

    SciTech Connect

    Gao, H.; Thompson, D.K.; Zhou, J.-Z.

    2006-04-01

    This study presents a global transcriptional analysis of thecold shock response of Shewanella oneidensis MR-1 after a temperaturedownshift from 30oC to 8 or 15oC based on time series microarrayexperiments. More than 700 genes were found to be significantly affected(P<0.05) upon cold shock challenge, especially at 8oC. The temporalgene expression patterns of the classical cold shock genes varied, andonly some of them, most notably So1648 and So2787, were differentiallyregulated in response to a temperature downshift. The global response ofS. oneidensis to cold shock was also characterized by the up-regulationof genes encoding membraneproteins, DNA metabolism and translationapparatus components, metabolic proteins, regulatory proteins, andhypothetical proteins. Most of the metabolic proteins affected areinvolved in catalytic processes that generate NADH or NADPH. Mutationalanalyses confirmed that the small cold shock proteins, So1648 and So2787,are involved in the cold shock response of S. oneidensis. The analysesalso indicated that So1648 may function only at very lowtemperatures.

  12. A Ferrous Iron Exporter Mediates Iron Resistance in Shewanella oneidensis MR-1

    PubMed Central

    Bennett, Brittany D.; Brutinel, Evan D.

    2015-01-01

    Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium frequently found in aquatic sediments. In the absence of oxygen, S. oneidensis can respire extracellular, insoluble oxidized metals, such as iron (hydr)oxides, making it intimately involved in environmental metal and nutrient cycling. The reduction of ferric iron (Fe3+) results in the production of ferrous iron (Fe2+) ions, which remain soluble under certain conditions and are toxic to cells at higher concentrations. We have identified an inner membrane protein in S. oneidensis, encoded by the gene SO_4475 and here called FeoE, which is important for survival during anaerobic iron respiration. FeoE, a member of the cation diffusion facilitator (CDF) protein family, functions to export excess Fe2+ from the MR-1 cytoplasm. Mutants lacking feoE exhibit an increased sensitivity to Fe2+. The export function of FeoE is specific for Fe2+, as an feoE mutant is equally sensitive to other metal ions known to be substrates of other CDF proteins (Cd2+, Co2+, Cu2+, Mn2+, Ni2+, or Zn2+). The substrate specificity of FeoE differs from that of FieF, the Escherichia coli homolog of FeoE, which has been reported to be a Cd2+/Zn2+ or Fe2+/Zn2+ exporter. A complemented feoE mutant has an increased growth rate in the presence of excess Fe2+ compared to that of the ΔfeoE mutant complemented with fieF. It is possible that FeoE has evolved to become an efficient and specific Fe2+ exporter in response to the high levels of iron often present in the types of environmental niches in which Shewanella species can be found. PMID:26341213

  13. A Ferrous Iron Exporter Mediates Iron Resistance in Shewanella oneidensis MR-1.

    PubMed

    Bennett, Brittany D; Brutinel, Evan D; Gralnick, Jeffrey A

    2015-11-01

    Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium frequently found in aquatic sediments. In the absence of oxygen, S. oneidensis can respire extracellular, insoluble oxidized metals, such as iron (hydr)oxides, making it intimately involved in environmental metal and nutrient cycling. The reduction of ferric iron (Fe(3+)) results in the production of ferrous iron (Fe(2+)) ions, which remain soluble under certain conditions and are toxic to cells at higher concentrations. We have identified an inner membrane protein in S. oneidensis, encoded by the gene SO_4475 and here called FeoE, which is important for survival during anaerobic iron respiration. FeoE, a member of the cation diffusion facilitator (CDF) protein family, functions to export excess Fe(2+) from the MR-1 cytoplasm. Mutants lacking feoE exhibit an increased sensitivity to Fe(2+). The export function of FeoE is specific for Fe(2+), as an feoE mutant is equally sensitive to other metal ions known to be substrates of other CDF proteins (Cd(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), or Zn(2+)). The substrate specificity of FeoE differs from that of FieF, the Escherichia coli homolog of FeoE, which has been reported to be a Cd(2+)/Zn(2+) or Fe(2+)/Zn(2+) exporter. A complemented feoE mutant has an increased growth rate in the presence of excess Fe(2+) compared to that of the ΔfeoE mutant complemented with fieF. It is possible that FeoE has evolved to become an efficient and specific Fe(2+) exporter in response to the high levels of iron often present in the types of environmental niches in which Shewanella species can be found. PMID:26341213

  14. Metabolic Profiling with Gas Chromatography-Mass Spectrometry and Capillary Electrophoresis-Mass Spectrometry Reveals the Carbon-Nitrogen Status of Tobacco Leaves Across Different Planting Areas.

    PubMed

    Zhao, Jieyu; Zhao, Yanni; Hu, Chunxiu; Zhao, Chunxia; Zhang, Junjie; Li, Lili; Zeng, Jun; Peng, Xiaojun; Lu, Xin; Xu, Guowang

    2016-02-01

    The interaction between carbon (C) and nitrogen (N) metabolism can reflect plant growth status and environmental factors. Little is known regarding the connections between C-N metabolism and growing regions under field conditions. To comprehensively investigate the relationship in mature tobacco leaves, we established metabolomics approaches based on gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis-time-of-flight-mass spectrometry (CE-TOF-MS). Approximately 240 polar metabolites were determined. Multivariate statistical analysis revealed that the growing region greatly influenced the metabolic profiles of tobacco leaves. A metabolic correlation network and related pathway maps were used to reveal the global overview of the alteration of C-N metabolism across three typical regions. In Yunnan, sugars and tricarboxylic acid (TCA) cycle intermediates were closely correlated with amino acid pools. Henan tobacco leaves showed positive correlation between the pentose phosphate pathway (PPP) intermediates and C-rich secondary metabolism. In Guizhou, the proline and asparagine had significant links with TCA cycle intermediates and urea cycle, and antioxidant accumulation was observed in response to drought. These results demonstrate that combined analytical approaches have great potential to detect polar metabolites and provide information on C-N metabolism related to planting regional characteristics. PMID:26784525

  15. Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress.

    PubMed

    Shen, Tie; Rui, Bin; Zhou, Hong; Zhang, Ximing; Yi, Yin; Wen, Han; Zheng, Haoran; Wu, Jihui; Shi, Yunyu

    2013-01-27

    The ability of a microorganism to adapt to changes in the environment, such as in nutrient or oxygen availability, is essential for its competitive fitness and survival. The cellular objective and the strategy of the metabolic response to an extreme environment are therefore of tremendous interest and, thus, have been increasingly explored. However, the cellular objective of the complex regulatory structure of the metabolic changes has not yet been fully elucidated and more details regarding the quantitative behaviour of the metabolic flux redistribution are required to understand the systems-wide biological significance of this response. In this study, the intracellular metabolic flux ratios involved in the central carbon metabolism were determined by fractional (13)C-labeling and metabolic flux ratio analysis (MetaFoR) of the wild-type E. coli strain JM101 at an oxidative environment in a chemostat. We observed a significant increase in the flux through phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (MEZ) and serine hydroxymethyltransferase (SHMT). We applied an ε-constraint based multi-objective optimization to investigate the trade-off relationships between the biomass yield and the generation of reductive power using the in silico iJR904 genome-scale model of E. coli K-12. The theoretical metabolic redistribution supports that the trans-hydrogenase pathway should not play a direct role in the defence mounted by E. coli against oxidative stress. The agreement between the measured ratio and the theoretical redistribution established the significance of NADPH synthesis as the goal of the metabolic reprogramming that occurs in response to oxidative stress. Our work presents a framework that combines metabolic flux ratio analysis and multi-objective optimization to investigate the metabolic trade-offs that occur under varied environmental conditions. Our results led to the proposal that the metabolic response of E

  16. Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

    PubMed Central

    2014-01-01

    Background Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. Results Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis. Conclusions This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance

  17. Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics.

    PubMed

    Boone, Cory H T; Grove, Ryan A; Adamcova, Dana; Braga, Camila P; Adamec, Jiri

    2016-07-01

    Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death. PMID:27193513

  18. Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease

    PubMed Central

    Zeng, Lin

    2013-01-01

    Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA. PMID:23222725

  19. Benzaldehyde is a precursor of phenylpropylamino alkaloids as revealed by targeted metabolic profiling and comparative biochemical analyses in Ephedra spp.

    PubMed

    Krizevski, Raz; Bar, Einat; Shalit, O R; Levy, Asaf; Hagel, Jillian M; Kilpatrick, Korey; Marsolais, Frédéric; Facchini, Peter J; Ben-Shabat, Shimon; Sitrit, Yaron; Lewinsohn, Efraim

    2012-09-01

    Ephedrine and pseudoephedrine are phenylpropylamino alkaloids widely used in modern medicine. Some Ephedra species such as E. sinica Stapf (Ephedraceae), a widely used Chinese medicinal plant (Chinese name: Ma Huang), accumulate ephedrine alkaloids as active constituents. Other Ephedra species, such as E. foeminea Forssk. (syn. E. campylopoda C.A. Mey) lack ephedrine alkaloids and their postulated metabolic precursors 1-phenylpropane-1,2-dione and (S)-cathinone. Solid-phase microextraction analysis of freshly picked young E. sinica and E. foeminea stems revealed the presence of increased benzaldehyde levels in E. foeminea, whereas 1-phenylpropane-1,2-dione was detected only in E. sinica. Soluble protein preparations from E. sinica and E. foeminea stems catalyzed the conversion of benzaldehyde and pyruvate to (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)-2-hydroxypropiophenone (S)-2-hydroxypropiophenone and 1-phenylpropane-1,2-dione. The activity, termed benzaldehyde carboxyligase (BCL) required the presence of magnesium and thiamine pyrophosphate and was 40 times higher in E. sinica as compared to E. foeminea. The distribution patterns of BCL activity in E. sinica tissues correlates well with the distribution pattern of the ephedrine alkaloids. (S)-Cathinone reductase enzymatic activities generating (1R,2S)-norephedrine and (1S,1R)-norephedrine were significantly higher in E. sinica relative to the levels displayed by E. foeminea. Surprisingly, (1R,2S)-norephedrine N-methyltransferase activity which is a downstream enzyme in ephedrine biosynthesis was significantly higher in E. foeminea than in E. sinica. Our studies further support that benzaldehyde is the metabolic precursor to phenylpropylamino alkaloids in E. sinica. PMID:22727117

  20. Multi-omic profiling -of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.

    PubMed

    Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2015-11-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)(+) , adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)(+) and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. PMID:25995028

  1. Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c(3), in Shewanella oneidensis MR-1

    NASA Technical Reports Server (NTRS)

    Ozawa, K.; Tsapin, A. I.; Nealson, K. H.; Cusanovich, M. A.; Akutsu, H.

    2000-01-01

    Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c(3) gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.

  2. Urinary Metabolic Phenotyping Reveals Differences in the Metabolic Status of Healthy and Inflammatory Bowel Disease (IBD) Children in Relation to Growth and Disease Activity

    PubMed Central

    Martin, Francois-Pierre; Ezri, Jessica; Cominetti, Ornella; Da Silva, Laeticia; Kussmann, Martin; Godin, Jean-Philippe; Nydegger, Andreas

    2016-01-01

    Background: Growth failure and delayed puberty are well known features of children and adolescents with inflammatory bowel disease (IBD), in addition to the chronic course of the disease. Urinary metabonomics was applied in order to better understand metabolic changes between healthy and IBD children. Methods: 21 Pediatric patients with IBD (mean age 14.8 years, 8 males) were enrolled from the Pediatric Gastroenterology Outpatient Clinic over two years. Clinical and biological data were collected at baseline, 6, and 12 months. 27 healthy children (mean age 12.9 years, 16 males) were assessed at baseline. Urine samples were collected at each visit and subjected to 1H Nuclear Magnetic Resonance (NMR) spectroscopy. Results: Using 1H NMR metabonomics, we determined that urine metabolic profiles of IBD children differ significantly from healthy controls. Metabolic differences include central energy metabolism, amino acid, and gut microbial metabolic pathways. The analysis described that combined urinary urea and phenylacetylglutamine—two readouts of nitrogen metabolism—may be relevant to monitor metabolic status in the course of disease. Conclusion: Non-invasive sampling of urine followed by metabonomic profiling can elucidate and monitor the metabolic status of children in relation to disease status. Further developments of omic-approaches in pediatric research might deliver novel nutritional and metabolic hypotheses. PMID:27529220

  3. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

    SciTech Connect

    Shi, CY; Yang, H; Wei, CL; Yu, O; Zhang, ZZ; Sun, J; Wan, XC

    2011-01-01

    Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real

  4. SO2907, A Putative TonB-dependent Receptor, Is Involved in Dissimilatory Iron Reduction by Shewanella oneidensis Strain MR-1

    SciTech Connect

    Qian, Yufeng; Shi, Liang; Tien, Ming

    2011-09-30

    Shewanella oneidensis strain MR-1 utilizes soluble and insoluble ferric ions as terminal electron acceptors during anaerobic respiration. The components of respiratory metabolism are localized in the membrane fractions which include the outer membrane and cytoplasmic membrane. Many of the biological components that interact with the various iron forms are proposed to be localized in these membrane fractions. To identify the iron-binding proteins acting either as an iron transporter or as a terminal iron reductase, we used metal-catalyzed oxidation reactions. This system catalyzed the oxidation of amino acids in close proximity to the iron binding site. The carbonyl groups formed from this oxidation can then be labeled with fluoresceinamine (FLNH2). The peptide harboring the FLNH2 can then be proteolytically digested, purified by HPLC and then identified by MALDI-TOF tandem MS. A predominant peptide was identified to be part of SO2907 that encodes a putative TonB-dependent receptor. Compared to wild type (wt), the so2097 gene deletion (ΔSO2907) mutant has impaired ability to reduce soluble Fe(III), but retains the same ability to respire oxygen or fumarate as the wt. The ΔSO2907 mutant was also impacted in reduction of insoluble iron. Iron binding assays using isothermal titration calorimetry and fluorescence tryptophan quenching demonstrated that a truncated form of heterologous-expressed SO2907 that contains the Fe(III) binding site, is capable of binding soluble Fe(III) forms with Kd of approximate 50 μM. To the best of our knowledge, this is the first report of the physiological role of SO2907 in Fe(III) reduction by MR-1.

  5. Depositional style of mass-flow lobes in Ulleung Basin (East Sea, Korea) interpreted from MR1 sonar image, chirp profiles, and piston cores

    NASA Astrophysics Data System (ADS)

    Lee, S. H.; Jung, W.; Bahk, J.; Gardner, J. M.

    2011-12-01

    A detailed analysis of MR1 sonar and chirp (2-7 kHz) seismic data with 5 (2.9-9.4 m long) piston cores and 17 AMS 14C ages reveals spatial and temporal variations in depositional style of mass-flow lobes in the Ulleung Basin. On MR1 sonar image, eight mass-flow lobes are identified in the western basin plain (>2100 m). They are covered by ca. 2-m thick Holocene pelagic sediments, and show the northward flow direction. Lobes 1-4 have large dimensions (>27 km long and 15-25 km wide) and occupy in the lower stratigraphic position. On the other hand, lobes 5-8, deposited in the more proximal area, have small dimensions (8.8-31.5 km long and 1.2-12 km wide) and occur in the upper stratigraphic position. Lobes 1-4 deposited retrogressively. Lobes 1 and 2 are characterized by relatively strong back-scattering intensity with smooth surfaces on MR1 image, and show flat, sharp bottom echo and several distinct to diffuse internal reflectors in chirp profiles. Sediments near their edges consist of fine-grained turbidites (laminated sand/mud and homogeneous mud) with minor massive clay-rich sand. However, they change to mud-matrix disorganized gravel and massive sand with the overlying fine-grained turbidites toward the proximal part. Lobes 1 and 2 deposited between ca. 20 and 18 cal. ka B.P. Lobes 3 and 4, overlying lobe 2, show weak to medium back-scattering intensity on MR1 image with slightly irregular to hummocky surfaces corresponding to small-scale (<100 m wide and <5 m high) hyperbolic bottom echoes in chirp profiles. Upper surfaces of the lobes are nearly flat to convex-up in cross section. Lobes 5-8 deposited on the upper surfaces of lobes 3 and 4. Their widths abruptly decrease to the proximal part, forming a bottle-neck morphology. They generally show weak to medium back-scattering intensity with relatively strong back-scattering intensity along the margins on MR1 image. They exhibit convex-up upper surfaces with distinct lateral margins in chirp profiles as their

  6. Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

    PubMed Central

    2013-01-01

    Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that

  7. Ferrous phosphate surface precipitates resulting from the reduction of intragrain 6-line ferrihydrite by Shewanella oneidensis MR-1

    SciTech Connect

    Peretyazhko, Tetyana; Zachara, John M.; Kennedy, David W.; Fredrickson, Jim K.; Arey, Bruce W.; McKinley, James P.; Wang, Chong M.; Dohnalkova, Alice; Xia, Yuanxian

    2010-07-01

    The reductive biotransformation of 6-line ferrihydrite located within porous silica (intragrain ferrihydrite) by Shewanella oneidensis MR-1 was investigated and compared to the behavior of 6-line ferrihydrite in suspension (free ferrihydrite). The effect of buffer type (PIPES and NaHCO3), phosphate (P), and an electron shuttle (AQDS) on the extent of reduction and formation of Fe(II) secondary phases was investigated under anoxic conditions. Electron microscopy and micro X-ray diffraction were applied to evaluate the morphology and mineralogy of the biogenic precipitates and to study the distribution of microorganisms on the surface of porous silica after bioreduction. Kinetic reduction experiments with free and intragrain ferrihydrite revealed contrasting behaviour with respect to the buffer and presence of P. The overall amount of intragrain ferrihydrite reduction was less than that of free ferrihydrite [at 5 mmol L-1 Fe(III)T]. Reductive mineralization was not observed in the intragrain ferrihydrite incubations without P, and all biogenic Fe(II) concentrated in the aqueous phase. Irrespective of buffer and AQDS addition, rosettes of Fe(II) phosphate of approximate 20-30 μm size were observed on porous silica when P was present. The rosettes grew not only on the silica surface but also within it, forming a coherent spherical structure. These precipitates were well colonized by microorganisms and contained extracellular materials at the end of incubation. Microbial extracellular polymeric substances may have adsorbed Fe(II) promoting Fe(II) phosphate nucleation with subsequent crystal growth proceeding in different directions from a common center.

  8. Comparative Genomics and Metabolic Analysis Reveals Peculiar Characteristics of Rhodococcus opacus Strain M213 Particularly for Naphthalene Degradation

    PubMed Central

    Blom, Jochen; Indest, Karl J.; Jung, Carina M.; Stothard, Paul; Bera, Gopal; Green, Stefan J.; Ogram, Andrew

    2016-01-01

    The genome of Rhodococcus opacus strain M213, isolated from a fuel-oil contaminated soil, was sequenced and annotated which revealed a genome size of 9,194,165 bp encoding 8680 putative genes and a G+C content of 66.72%. Among the protein coding genes, 71.77% were annotated as clusters of orthologous groups of proteins (COGs); 55% of the COGs were present as paralog clusters. Pulsed field gel electrophoresis (PFGE) analysis of M213 revealed the presence of three different sized replicons- a circular chromosome and two megaplasmids (pNUO1 and pNUO2) estimated to be of 750Kb 350Kb in size, respectively. Conversely, using an alternative approach of optical mapping, the plasmid replicons appeared as a circular ~1.2 Mb megaplasmid and a linear, ~0.7 Mb megaplasmid. Genome-wide comparative analysis of M213 with a cohort of sequenced Rhodococcus species revealed low syntenic affiliation with other R. opacus species including strains B4 and PD630. Conversely, a closer affiliation of M213, at the functional (COG) level, was observed with the catabolically versatile R. jostii strain RHA1 and other Rhodococcii such as R. wratislaviensis strain IFP 2016, R. imtechensis strain RKJ300, Rhodococcus sp. strain JVH1, and Rhodococcus sp. strain DK17, respectively. An in-depth, genome-wide comparison between these functional relatives revealed 971 unique genes in M213 representing 11% of its total genome; many associating with catabolic functions. Of major interest was the identification of as many as 154 genomic islands (GEIs), many with duplicated catabolic genes, in particular for PAHs; a trait that was confirmed by PCR-based identification of naphthalene dioxygenase (NDO) as a representative gene, across PFGE-resolved replicons of strain M213. Interestingly, several plasmid/GEI-encoded genes, that likely participate in degrading naphthalene (NAP) via a peculiar pathway, were also identified in strain M213 using a combination of bioinformatics, metabolic analysis and gene

  9. Comparative Genomics and Metabolic Analysis Reveals Peculiar Characteristics of Rhodococcus opacus Strain M213 Particularly for Naphthalene Degradation.

    PubMed

    Pathak, Ashish; Chauhan, Ashvini; Blom, Jochen; Indest, Karl J; Jung, Carina M; Stothard, Paul; Bera, Gopal; Green, Stefan J; Ogram, Andrew

    2016-01-01

    The genome of Rhodococcus opacus strain M213, isolated from a fuel-oil contaminated soil, was sequenced and annotated which revealed a genome size of 9,194,165 bp encoding 8680 putative genes and a G+C content of 66.72%. Among the protein coding genes, 71.77% were annotated as clusters of orthologous groups of proteins (COGs); 55% of the COGs were present as paralog clusters. Pulsed field gel electrophoresis (PFGE) analysis of M213 revealed the presence of three different sized replicons- a circular chromosome and two megaplasmids (pNUO1 and pNUO2) estimated to be of 750Kb 350Kb in size, respectively. Conversely, using an alternative approach of optical mapping, the plasmid replicons appeared as a circular ~1.2 Mb megaplasmid and a linear, ~0.7 Mb megaplasmid. Genome-wide comparative analysis of M213 with a cohort of sequenced Rhodococcus species revealed low syntenic affiliation with other R. opacus species including strains B4 and PD630. Conversely, a closer affiliation of M213, at the functional (COG) level, was observed with the catabolically versatile R. jostii strain RHA1 and other Rhodococcii such as R. wratislaviensis strain IFP 2016, R. imtechensis strain RKJ300, Rhodococcus sp. strain JVH1, and Rhodococcus sp. strain DK17, respectively. An in-depth, genome-wide comparison between these functional relatives revealed 971 unique genes in M213 representing 11% of its total genome; many associating with catabolic functions. Of major interest was the identification of as many as 154 genomic islands (GEIs), many with duplicated catabolic genes, in particular for PAHs; a trait that was confirmed by PCR-based identification of naphthalene dioxygenase (NDO) as a representative gene, across PFGE-resolved replicons of strain M213. Interestingly, several plasmid/GEI-encoded genes, that likely participate in degrading naphthalene (NAP) via a peculiar pathway, were also identified in strain M213 using a combination of bioinformatics, metabolic analysis and gene

  10. Comparative Genomic and Transcriptomic Analyses Reveal Habitat Differentiation and Different Transcriptional Responses during Pectin Metabolism in Alishewanella Species

    PubMed Central

    Jung, Jaejoon

    2013-01-01

    Alishewanella species are expected to have high adaptability to diverse environments because they are isolated from different natural habitats. To investigate how the evolutionary history of Alishewanella species is reflected in their genomes, we performed comparative genomic and transcriptomic analyses of A. jeotgali, A. aestuarii, and A. agri, which were isolated from fermented seafood, tidal flat sediment, and soil, respectively. Genomic islands with variable GC contents indicated that invasion of prophage and transposition events occurred in A. jeotgali and A. agri but not in A. aestuarii. Habitat differentiation of A. agri from a marine environment to a terrestrial environment was proposed because the species-specific genes of A. agri were similar to those of soil bacteria, whereas those of A. jeotgali and A. aestuarii were more closely related to marine bacteria. Comparative transcriptomic analysis with pectin as a sole carbon source revealed different transcriptional responses in Alishewanella species, especially in oxidative stress-, methylglyoxal detoxification-, membrane maintenance-, and protease/chaperone activity-related genes. Transcriptomic and experimental data demonstrated that A. agri had a higher pectin degradation rate and more resistance to oxidative stress under pectin-amended conditions than the other 2 Alishewanella species. However, expression patterns of genes in the pectin metabolic pathway and of glyoxylate bypass genes were similar among all 3 Alishewanella species. Our comparative genomic and transcriptomic data revealed that Alishewanella species have evolved through horizontal gene transfer and habitat differentiation and that pectin degradation pathways in Alishewanella species are highly conserved, although stress responses of each Alishewanella species differed under pectin culture conditions. PMID:23934491

  11. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.

    PubMed

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  12. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  13. Integrated metabolomic and transcriptome analyses reveal finishing forage affects metabolic pathways related to beef quality and animal welfare.

    PubMed

    Carrillo, José A; He, Yanghua; Li, Yaokun; Liu, Jianan; Erdman, Richard A; Sonstegard, Tad S; Song, Jiuzhou

    2016-01-01

    Beef represents a major dietary component and source of protein in many countries. With an increasing demand for beef, the industry is currently undergoing changes towards naturally produced beef. However, the true differences between the feeding systems, especially the biochemical and nutritional aspects, are still unclear. Using transcriptome and metabolome profiles, we identified biological pathways related to the differences between grass- and grain-fed Angus steers. In the latissimus dorsi muscle, we have recognized 241 differentially expressed genes (FDR < 0.1). The metabolome examinations of muscle and blood revealed 163 and 179 altered compounds in each tissue (P < 0.05), respectively. Accordingly, alterations in glucose metabolism, divergences in free fatty acids and carnitine conjugated lipid levels, and altered β-oxidation have been observed. The anti-inflammatory n3 polyunsaturated fatty acids are enriched in grass finished beef, while higher levels of n6 PUFAs in grain finished animals may promote inflammation and oxidative stress. Furthermore, grass-fed animals produce tender beef with lower total fat and a higher omega3/omega6 ratio than grain-fed ones, which could potentially benefit consumer health. Most importantly, blood cortisol levels strongly indicate that grass-fed animals may experience less stress than the grain-fed individuals. These results will provide deeper insights into the merits and mechanisms of muscle development. PMID:27185157

  14. Pathway analysis using (13) C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila.

    PubMed

    Häuslein, Ina; Manske, Christian; Goebel, Werner; Eisenreich, Wolfgang; Hilbi, Hubert

    2016-04-01

    Amino acids represent the prime carbon and energy source for Legionella pneumophila, a facultative intracellular pathogen, which can cause a life-threatening pneumonia termed Legionnaires' disease. Genome, transcriptome and proteome studies indicate that L. pneumophila also utilizes carbon substrates other than amino acids. We show here that glycerol promotes intracellular replication of L. pneumophila in amoeba or macrophages (but not extracellular growth) dependent on glycerol-3-phosphate dehydrogenase, GlpD. An L. pneumophila mutant strain lacking glpD was outcompeted by wild-type bacteria upon co-infection of amoeba, indicating an important role of glycerol during infection. Isotopologue profiling studies using (13) C-labelled substrates were performed in a novel minimal defined medium, MDM, comprising essential amino acids, proline and phenylalanine. In MDM, L. pneumophila utilized (13) C-labelled glycerol or glucose predominantly for gluconeogenesis and the pentose phosphate pathway, while the amino acid serine was used for energy generation via the citrate cycle. Similar results were obtained for L. pneumophila growing intracellularly in amoeba fed with (13) C-labelled glycerol, glucose or serine. Collectively, these results reveal a bipartite metabolism of L. pneumophila, where glycerol and carbohydrates like glucose are mainly fed into anabolic processes, while serine serves as major energy supply. PMID:26691313

  15. Transcriptome Characterization of Gnetum parvifolium Reveals Candidate Genes Involved in Important Secondary Metabolic Pathways of Flavonoids and Stilbenoids

    PubMed Central

    Deng, Nan; Chang, Ermei; Li, Minghe; Ji, Jing; Yao, Xiamei; Bartish, Igor V.; Liu, Jianfeng; Ma, Jing; Chen, Lanzhen; Jiang, Zeping; Shi, Shengqing

    2016-01-01

    Gnetum is a small, unique group of Gnetophyta with a controversial phylogenetic position. Gnetum parvifolium is an important Chinese traditional medicinal plant, which is rich in bioactive compounds such as flavonoids and stilbenoids. These compounds provide significant medicinal effects, mostly as antioxidant, anticancer, and antibacterial agents. However, the mechanisms involved in the biosynthesis and regulation of these compounds in G. parvifolium are still unknown. In this study, we found that flavonoids and stilbene compounds accumulated at different levels in various tissues of G. parvifolium. We further obtained and analyzed massive sequence information from pooled samples of G. parvifolium by transcriptome sequencing, which generated 94,816 unigenes with an average length of 724 bp. Functional annotation of all these unigenes revealed that many of them were associated with several important secondary metabolism pathways including flavonoids and stilbenoids. In particular, several candidate unigenes (PAL-, C4H-, 4CL-, and STS-like genes) involved in stilbenoids biosynthesis were highly expressed in leaves and mature fruits. Furthermore, high temperature and UV-C strongly induced the expression of these genes and enhanced stilbene production (i.e., resveratrol and piceatannol) in leaves of young seedlings. Our present transcriptomic and biochemical data on secondary metabolites in G. parvifolium should encourage further investigation on evolution, ecology, functional genomics, and breeding of this plant with strong pharmaceutical potential. PMID:26973657

  16. In vivo HMRS and lipidomic profiling reveals comprehensive changes of hippocampal metabolism during aging in mice.

    PubMed

    Lin, Lejun; Cao, Bofeng; Xu, Zhiying; Sui, Yanbin; Chen, Jiao; Luan, Qiang; Yang, Ruifang; Li, Shanchun; Li, Ke Feng

    2016-01-29

    Aging is characterized by various cellular changes in the brain. Hippocampus is important for systemic aging and lifespan control. There is still a lack of comprehensive overview of metabolic changes in hippocampus during aging. In this study, we first created an accelerated brain aging mice model through the chronic administration of d-galactose. We then performed a multiplatform metabolomic profiling of mice hippocampus using the combination of in vivo 9.4 T HMRS and in vitro LC-MS/MS based lipidomics. We found N-acetylaspartic acid (NAA), gama-aminobutyric acid (GABA), glutamate/glutamine, taurine, choline, sphingolipids (SMs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs) and phosphatidylserines (PSs), all of them decreasing with the aging process in mice hippocampus. The changes of sphingolipids and phospholipids were not limited to one single class or molecular species. In contrast, we found the significant accumulation of lactate, myoinositol and phosphatidylcholines (PCs) along with aging in hippocampus. SM (d18:1/20:2), PE (36:2), PG (34:1), PI (36:4), PS (18:0/20:4) and PC (36:0) have the most significant changes along with aging. Network analysis revealed the striking loss of biochemical connectivity and interactions between hippocampal metabolites with aging. The correlation pattern between metabolites in hippocampus could function as biomarkers for aging or diagnosis of aging-related diseases. PMID:26707637

  17. Metagenomic Analysis of the Pygmy Loris Fecal Microbiome Reveals Unique Functional Capacity Related to Metabolism of Aromatic Compounds

    PubMed Central

    Xu, Bo; Xu, Weijiang; Yang, Fuya; Li, Junjun; Yang, Yunjuan; Tang, Xianghua; Mu, Yuelin; Zhou, Junpei; Huang, Zunxi

    2013-01-01

    The animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. An analysis of 78,619 pyrosequencing reads generated from pygmy loris fecal DNA extracts was performed to help better understand the microbial diversity and functional capacity of the pygmy loris gut microbiome. The taxonomic analysis of the metagenomic reads indicated that pygmy loris fecal microbiomes were dominated by Bacteroidetes and Proteobacteria phyla. The hierarchical clustering of several gastrointestinal metagenomes demonstrated the similarities of the microbial community structures of pygmy loris and mouse gut systems despite their differences in functional capacity. The comparative analysis of function classification revealed that the metagenome of the pygmy loris was characterized by an overrepresentation of those sequences involved in aromatic compound metabolism compared with humans and other animals. The key enzymes related to the benzoate degradation pathway were identified based on the Kyoto Encyclopedia of Genes and Genomes pathway assignment. These results would contribute to the limited body of primate metagenome studies and provide a framework for comparative metagenomic analysis between human and non-human primates, as well as a comparative understanding of the evolution of humans and their microbiome. However, future studies on the metagenome sequencing of pygmy loris and other prosimians regarding the effects of age, genetics, and environment on the composition and activity of the metagenomes are required. PMID:23457582

  18. Integrated metabolomic and transcriptome analyses reveal finishing forage affects metabolic pathways related to beef quality and animal welfare

    PubMed Central

    Carrillo, José A.; He, Yanghua; Li, Yaokun; Liu, Jianan; Erdman, Richard A.; Sonstegard, Tad S.; Song, Jiuzhou

    2016-01-01

    Beef represents a major dietary component and source of protein in many countries. With an increasing demand for beef, the industry is currently undergoing changes towards naturally produced beef. However, the true differences between the feeding systems, especially the biochemical and nutritional aspects, are still unclear. Using transcriptome and metabolome profiles, we identified biological pathways related to the differences between grass- and grain-fed Angus steers. In the latissimus dorsi muscle, we have recognized 241 differentially expressed genes (FDR < 0.1). The metabolome examinations of muscle and blood revealed 163 and 179 altered compounds in each tissue (P < 0.05), respectively. Accordingly, alterations in glucose metabolism, divergences in free fatty acids and carnitine conjugated lipid levels, and altered β-oxidation have been observed. The anti-inflammatory n3 polyunsaturated fatty acids are enriched in grass finished beef, while higher levels of n6 PUFAs in grain finished animals may promote inflammation and oxidative stress. Furthermore, grass-fed animals produce tender beef with lower total fat and a higher omega3/omega6 ratio than grain-fed ones, which could potentially benefit consumer health. Most importantly, blood cortisol levels strongly indicate that grass-fed animals may experience less stress than the grain-fed individuals. These results will provide deeper insights into the merits and mechanisms of muscle development. PMID:27185157

  19. In-Depth Characterization and Validation of Human Urine Metabolomes Reveal Novel Metabolic Signatures of Lower Urinary Tract Symptoms

    NASA Astrophysics Data System (ADS)

    Hao, Ling; Greer, Tyler; Page, David; Shi, Yatao; Vezina, Chad M.; Macoska, Jill A.; Marker, Paul C.; Bjorling, Dale E.; Bushman, Wade; Ricke, William A.; Li, Lingjun

    2016-08-01

    Lower urinary tract symptoms (LUTS) are a range of irritative or obstructive symptoms that commonly afflict aging population. The diagnosis is mostly based on patient-reported symptoms, and current medication often fails to completely eliminate these symptoms. There is a pressing need for objective non-invasive approaches to measure symptoms and understand disease mechanisms. We developed an in-depth workflow combining urine metabolomics analysis and machine learning bioinformatics to characterize metabolic alterations and support objective diagnosis of LUTS. Machine learning feature selection and statistical tests were combined to identify candidate biomarkers, which were statistically validated with leave-one-patient-out cross-validation and absolutely quantified by selected reaction monitoring assay. Receiver operating characteristic analysis showed highly-accurate prediction power of candidate biomarkers to stratify patients into disease or non-diseased categories. The key metabolites and pathways may be possibly correlated with smooth muscle tone changes, increased collagen content, and inflammation, which have been identified as potential contributors to urinary dysfunction in humans and rodents. Periurethral tissue staining revealed a significant increase in collagen content and tissue stiffness in men with LUTS. Together, our study provides the first characterization and validation of LUTS urinary metabolites and pathways to support the future development of a urine-based diagnostic test for LUTS.

  20. Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis

    PubMed Central

    Matz-Soja, Madlen; Rennert, Christiane; Schönefeld, Kristin; Aleithe, Susanne; Boettger, Jan; Schmidt-Heck, Wolfgang; Weiss, Thomas S; Hovhannisyan, Amalya; Zellmer, Sebastian; Klöting, Nora; Schulz, Angela; Kratzsch, Jürgen; Guthke, Reinhardt; Gebhardt, Rolf

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond. DOI: http://dx.doi.org/10.7554/eLife.13308.001 PMID:27185526

  1. Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis.

    PubMed

    Matz-Soja, Madlen; Rennert, Christiane; Schönefeld, Kristin; Aleithe, Susanne; Boettger, Jan; Schmidt-Heck, Wolfgang; Weiss, Thomas S; Hovhannisyan, Amalya; Zellmer, Sebastian; Klöting, Nora; Schulz, Angela; Kratzsch, Jürgen; Guthke, Reinhardt; Gebhardt, Rolf

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond. PMID:27185526

  2. Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening

    PubMed Central

    Mou, Wangshu; Li, Dongdong; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

    2015-01-01

    Abscisic acid (ABA) has been proven to be involved in the regulation of climacteric fruit ripening, but a comprehensive investigation of its influence on ripening related processes is still lacking. By applying the next generation sequencing technology, we conducted a comparative analysis of the effects of exogenous ABA and NDGA (Nordihydroguaiaretic acid, an inhibitor of ABA biosynthesis) on tomato fruit ripening. The high throughput sequencing results showed that out of the 25728 genes expressed across all three samples, 10388 were identified as significantly differently expressed genes. Exogenous ABA was found to enhance the transcription of genes involved in pigments metabolism, including carotenoids biosynthesis and chlorophyll degradation, whereas NDGA treatment inhibited these processes. The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant system. Intriguingly, we also found that an inhibition of endogenous ABA significantly enhanced the transcriptional abundance of genes involved in photosynthesis. Our results highlighted the significance of ABA in regulating tomato ripening, which provided insight into the regulatory mechanism of fruit maturation and senescence process. PMID:26053166

  3. Microbial metagenomes from three aquifers in the Fennoscandian shield terrestrial deep biosphere reveal metabolic partitioning among populations.

    PubMed

    Wu, Xiaofen; Holmfeldt, Karin; Hubalek, Valerie; Lundin, Daniel; Åström, Mats; Bertilsson, Stefan; Dopson, Mark

    2016-05-01

    Microorganisms in the terrestrial deep biosphere host up to 20% of the earth's biomass and are suggested to be sustained by the gases hydrogen and carbon dioxide. A metagenome analysis of three deep subsurface water types of contrasting age (from <20 to several thousand years) and depth (171 to 448 m) revealed phylogenetically distinct microbial community subsets that either passed or were retained by a 0.22 μm filter. Such cells of <0.22 μm would have been overlooked in previous studies relying on membrane capture. Metagenomes from the three water types were used for reconstruction of 69 distinct microbial genomes, each with >86% coverage. The populations were dominated by Proteobacteria, Candidate divisions, unclassified archaea and unclassified bacteria. The estimated genome sizes of the <0.22 μm populations were generally smaller than their phylogenetically closest relatives, suggesting that small dimensions along with a reduced genome size may be adaptations to oligotrophy. Shallow 'modern marine' water showed community members with a predominantly heterotrophic lifestyle. In contrast, the deeper, 'old saline' water adhered more closely to the current paradigm of a hydrogen-driven deep biosphere. The data were finally used to create a combined metabolic model of the deep terrestrial biosphere microbial community. PMID:26484735

  4. In-Depth Characterization and Validation of Human Urine Metabolomes Reveal Novel Metabolic Signatures of Lower Urinary Tract Symptoms

    PubMed Central

    Hao, Ling; Greer, Tyler; Page, David; Shi, Yatao; Vezina, Chad M.; Macoska, Jill A.; Marker, Paul C.; Bjorling, Dale E.; Bushman, Wade; Ricke, William A.; Li, Lingjun

    2016-01-01

    Lower urinary tract symptoms (LUTS) are a range of irritative or obstructive symptoms that commonly afflict aging population. The diagnosis is mostly based on patient-reported symptoms, and current medication often fails to completely eliminate these symptoms. There is a pressing need for objective non-invasive approaches to measure symptoms and understand disease mechanisms. We developed an in-depth workflow combining urine metabolomics analysis and machine learning bioinformatics to characterize metabolic alterations and support objective diagnosis of LUTS. Machine learning feature selection and statistical tests were combined to identify candidate biomarkers, which were statistically validated with leave-one-patient-out cross-validation and absolutely quantified by selected reaction monitoring assay. Receiver operating characteristic analysis showed highly-accurate prediction power of candidate biomarkers to stratify patients into disease or non-diseased categories. The key metabolites and pathways may be possibly correlated with smooth muscle tone changes, increased collagen content, and inflammation, which have been identified as potential contributors to urinary dysfunction in humans and rodents. Periurethral tissue staining revealed a significant increase in collagen content and tissue stiffness in men with LUTS. Together, our study provides the first characterization and validation of LUTS urinary metabolites and pathways to support the future development of a urine-based diagnostic test for LUTS. PMID:27502322

  5. Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella oneidensis MR-1

    SciTech Connect

    Shi, Liang; Rosso, Kevin M.; Clarke, Thomas A.; Richardson, David J.; Zachara, John M.; Fredrickson, Jim K.

    2012-02-15

    In the absence of O2 and other electron acceptors, the Gram-negative bacterium Shewanella oneidensis MR-1 can use ferric [Fe(III)] (oxy)(hydr)oxide minerals as the terminal electron acceptors for anaerobic respiration. At circumneutral pH and in the absence of strong complexing ligands, Fe(III) oxides are relatively insoluble and thus are external to the bacterial cells. S. oneidensis MR-1 and related strains of metal-reducing Shewanella have evolved the machinery (i.e., metal-reducing or Mtr pathway) for transferring electrons from the inner-membrane, through the periplasm and across the outer-membrane to the surface of extracellular Fe(III) oxides. The protein components identified to date for the Mtr pathway include CymA, MtrA, MtrB, MtrC and OmcA. CymA is an inner-membrane tetraheme c-type cytochrome (c-Cyt) that belongs to the NapC/NrfH family of quinol dehydrogenases. It is proposed that CymA oxidizes the quinol in the inner-membrane and transfers the released electrons to redox proteins in the periplasm. Although the periplasmic proteins receiving electrons from CymA during Fe(III) oxidation have not been identified, they are believed to relay the electrons in the periplasm to MtrA. A decaheme c-Cyt, MtrA is thought to be embedded in the trans outer-membrane and porin-like protein MtrB. Together, MtrAB deliver the electrons through the outer-membrane to the MtrC and OmcA on the outmost bacterial surface. MtrC and OmcA are the outer-membrane decaheme c-Cyts that are translocated across the outer-membrane by the bacterial type II secretion system. Functioning as terminal reductases, MtrC and OmcA can bind the surface of Fe(III) oxides and transfer electrons directly to these minerals via their solvent-exposed hemes. To increase their reaction rates, MtrC and OmcA can use the flavins secreted by S. oneidensis MR-1 cells as diffusible co-factors for reduction of Fe(III) oxides. Because of their extracellular location and broad redox potentials, MtrC and OmcA can

  6. Deciphering the Electron Transport Pathway for Graphene Oxide Reduction by Shewanella oneidensis MR-1 ▿†‡

    PubMed Central

    Jiao, Yongqin; Qian, Fang; Li, Yat; Wang, Gongming; Saltikov, Chad W.; Gralnick, Jeffrey A.

    2011-01-01

    We determined that graphene oxide reduction by Shewanella oneidensis MR-1 requires the Mtr respiratory pathway by analyzing a range of mutants lacking these proteins. Electron shuttling compounds increased the graphene oxide reduction rate 3- to 5-fold. These results may help facilitate the use of bacteria for large-scale graphene production. PMID:21602337

  7. THE ROLE OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE IN ENHANCEMENT OF SOLID-PHASE ELECTRON TRANSFER BY SHEWANELLA ONEIDENSIS MR-1

    SciTech Connect

    Turick, C; Amy Ekechukwu, A

    2007-06-01

    While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane-associated c-type cytochromes and redox active electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. In this study, we determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione (2-(2-chloro-4-methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates, with which MR-1 reduces hydrous ferric oxide, were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E{sup o}{prime}) of S. oneidensis MR-1. Based on this work, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in Shewanella oneidensis.

  8. The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Turick, Charles E; Beliaev, Alex S; Zakrajsek, Brian A; Reardon, Catherine L; Lowy, Daniel A; Poppy, Tara E; Maloney, Andrea; Ekechukwu, Amy A

    2009-05-01

    We hypothesized that Shewanella oneidensis MR-1, a model dissimilatory metal-reducing bacterium, could utilize environmentally relevant concentrations of tyrosine to produce pyomelanin for enhanced Fe(III) oxide reduction. Because homogentisate is an intermediate of the tyrosine degradation pathway, and a precursor of a redox-cycling metabolite, pyomelanin, we evaluated the process of homogentisate production by S. oneidensis MR-1, in order to identify the key steps involved in pyomelanin production. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. We used genetic analysis and physiological characterization of MR-1 strains either deficient in or displaying substantially increased pyomelanin production. The relative significance imparted by pyomelanin on solid-phase electron transfer was also addressed using electrochemical techniques, which allowed us to extend the genetic and physiological findings to biogeochemical cycling of metals. Based on our findings, environmental production of pyomelanin from available organic precursors could contribute to the survival of S. oneidensis MR-1 when dissolved oxygen concentrations become low, by providing an increased capacity for solid-phase metal reduction. This study demonstrates the role of organic precursors and their concentrations in pyomelanin production, solid phase metal reduction and biogeochemical cycling of iron. PMID:19573203

  9. Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships between Gene Centrality and Essentiality

    PubMed Central

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alexander S.

    2015-01-01

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as “topologically important.” Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as “functionally important” genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles. PMID:25826650

  10. Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

    SciTech Connect

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alex S.

    2015-03-27

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as ‘topologically important.’ Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as ‘functionally important’ genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.

  11. Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

    DOE PAGESBeta

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alex S.

    2015-03-27

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as ‘topologically important.’ Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termedmore » as ‘functionally important’ genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.« less

  12. Biofilm shows spatially stratified metabolic responses to contaminant exposure

    PubMed Central

    Cao, Bin; Majors, Paul D.; Ahmed, Bulbul; Renslow, Ryan S.; Silvia, Crystal P.; Shi, Liang; Kjelleberg, Staffan; Fredrickson, Jim K.; Beyenal, Haluk

    2012-01-01

    Summary Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic responses of metabolically active Shewanella oneidensis MR-1 biofilms to U(VI) (uranyl, UO22+) and Cr(VI) (chromate, CrO42−) using noninvasive nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) approaches to obtain insights into adaptation in biofilms during biofilm-contaminant interactions. While overall biomass distribution was not significantly altered upon exposure to U(VI) or Cr(VI), MRI and spatial mapping of the diffusion revealed localized changes in the water diffusion coefficients in the biofilms, suggesting significant contaminant-induced changes in structural or hydrodynamic properties during bioremediation. Finally, we quantitatively demonstrated that the metabolic responses of biofilms to contaminant exposure are spatially stratified, implying that adaptation in biofilms is custom-developed based on local microenvironments. PMID:22925136

  13. High-Throughput Tissue Bioenergetics Analysis Reveals Identical Metabolic Allometric Scaling for Teleost Hearts and Whole Organisms

    PubMed Central

    Jayasundara, Nishad; Kozal, Jordan S.; Arnold, Mariah C.; Chan, Sherine S. L.; Di Giulio, Richard T.

    2015-01-01

    Organismal metabolic rate, a fundamental metric in biology, demonstrates an allometric scaling relationship with body size. Fractal-like vascular distribution networks of biological systems are proposed to underlie metabolic rate allometric scaling laws from individual organisms to cells, mitochondria, and enzymes. Tissue-specific metabolic scaling is notably absent from this paradigm. In the current study, metabolic scaling relationships of hearts and brains with body size were examined by improving on a high-throughput whole-organ oxygen consumption rate (OCR) analysis method in five biomedically and environmentally relevant teleost model species. Tissue-specific metabolic scaling was compared with organismal routine metabolism (RMO2), which was measured using whole organismal respirometry. Basal heart OCR and organismal RMO2 scaled identically with body mass in a species-specific fashion across all five species tested. However, organismal maximum metabolic rates (MMO2) and pharmacologically-induced maximum cardiac metabolic rates in zebrafish Danio rerio did not show a similar relationship with body mass. Brain metabolic rates did not scale with body size. The identical allometric scaling of heart and organismal metabolic rates with body size suggests that hearts, the power generator of an organism’s vascular distribution network, might be crucial in determining teleost metabolic rate scaling under routine conditions. Furthermore, these findings indicate the possibility of measuring heart OCR utilizing the high-throughput approach presented here as a proxy for organismal metabolic rate—a useful metric in characterizing organismal fitness. In addition to heart and brain OCR, the current approach was also used to measure whole liver OCR, partition cardiac mitochondrial bioenergetic parameters using pharmacological agents, and estimate heart and brain glycolytic rates. This high-throughput whole-organ bioenergetic analysis method has important applications in

  14. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

    PubMed Central

    Zulak, Katherine G; Weljie, Aalim M; Vogel, Hans J; Facchini, Peter J

    2008-01-01

    Background Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor. Results Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary

  15. Validation of Shewanella oneidensis MR-1 Small Proteins by AMT Tag-based Proteome Analysis

    SciTech Connect

    Romine, Margaret F.; Elias, Dwayne A.; Monroe, Matthew E.; Auberry, Kenneth J.; Fang, Ruihua; Fredrickson, Jim K.; Anderson, Gordon A.; Smith, Richard D.; Lipton, Mary S.

    2004-09-01

    Using stringent criteria for protein identification by accurate mass and time (AMT) tag mass spectrometric methodology, we detected 36 proteins <101 amino acids in length, including 10 that were annotated as hypothetical proteins, in 172 global tryptic digests of Shewanella oneidensis MR-1 proteins analyzed. Peptides that map to the conserved, but functionally uncharacterized proteins SO4134 and SO2787, were the most frequently detected small proteins in these samples, while hypotheticals SO2669 and SO2063, conserved hypotheticals SO0335 and SO2176, and the SlyX protein (SO1063) were observed at frequencies similar to small expected abundant ribosomal proteins and translation initiation factor IF-1 and consequently, likely to encode important cellular functions. In addition, 30 proteins including three of the small proteins that map to genes predicted to encode frameshifts, point mutations, or recoding signals were detected. Of these 30 genes, peptides that map to positions beyond internal stop codons were detected in 13 genes (SO0101, SO0419, SO0590, SO0738, SO1113, SO1211, SO3079, SO3130, SO3240, SO4231, SO4328, SO4422, and SO4657). While expression of the full-length formate dehydrogenase encoded by SO0101 can be explained by incorporation of selenocysteine at the internal stop codon, the mechanism of translating downstream sequences in the remaining genes remains unknown.

  16. Multi-heme Cytochromes in Shewanella oneidensis MR-1: Structures, functions and opportunities

    SciTech Connect

    Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen; Butt, Julea N.

    2014-11-05

    Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies.

  17. Reduction of jarosite by Shewanella oneidensis MR-1 and secondary mineralization

    NASA Astrophysics Data System (ADS)

    Bingjie, Ouyang; Xiancai, Lu; Huan, Liu; Juan, Li; Tingting, Zhu; Xiangyu, Zhu; Jianjun, Lu; Rucheng, Wang

    2014-01-01

    Jarosite is a common mineral in a variety of environments formed by the oxidation of iron sulfide normally accompanying with the generation of acid mine drainage (AMD) in mining areas or acid rock drainages (ARD) in many localities. Decomposition of jarosite by dissimilatory iron reducing bacteria (DIRB) influences the mobility of many heavy metals generally accommodated in natural jarosite. This study examined the anaerobic reduction of synthesized jarosite by Shewanella oneidensis strain MR-1, a typical facultative bacteria. The release of ferrous and ferric ion, as well as sulfate and potassium, in the inoculated experimental group lasting 80 days is much higher than that in abiotic control groups. The detection of bicarbonate and acetate in experimental solution further confirms the mechanism of microbial reduction of jarosite, in which lactate acts as the electron donor. The produced ferrous iron stimulates the subsequent secondary mineralization, leading to precipitation and transformation of various iron-containing minerals. Green rust and goethite are the intermediate minerals of the microbial reduction process under anoxic conditions, and the end products include magnetite and siderite. In aerobic environments, goethite, magnetite and siderite were also detected, but the contents were relatively lower. While in abiotic experiments, only goethite has been detected as a product. Thus, the microbial reduction and subsequent mineral transformation can remarkably influence the geochemical cycling of iron and sulfur in supergene environments, as well as the mobility of heavy metals commonly accommodated in jarosite.

  18. Mechanism and Consequences of Anaerobic Respiration of Cobalt by Shewanella oneidensis Strain MR-1

    PubMed Central

    Hau, Heidi H.; Gilbert, Alan; Coursolle, Dan; Gralnick, Jeffrey A.

    2008-01-01

    Bacteria from the genus Shewanella are the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability of Shewanella oneidensis to catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA]− reduction by S. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA]2−, the product of [Co(III)-EDTA]− respiration, inhibited the growth of S. oneidensis strain MR-1 and that this toxicity was partially abolished by the addition of MgSO4. We demonstrate that [Co(III)-EDTA]− reduction by S. oneidensis requires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (the c-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developing S. oneidensis strains as a tool for bioremediation. PMID:18836009

  19. Localization of cytochromes to the outer membrane of anaerobically grown Shewanella putrefaciens MR-1.

    PubMed Central

    Myers, C R; Myers, J M

    1992-01-01

    In gram-negative bacteria, numerous cell functions, including respiration-linked electron transport, have been ascribed to the cytoplasmic membrane. Gram-negative bacteria which use solid substrates (e.g., oxidized manganese or iron) as terminal electron acceptors for anaerobic respiration are presented with a unique problem: they must somehow establish an electron transport link across the outer membrane between large particulate metal oxides and the electron transport chain in the cytoplasmic membrane. When the metal-reducing bacterium Shewanella putrefaciens MR-1 is grown under anaerobic conditions and membrane fractions are purified from cells lysed by an EDTA-lysozyme-polyoxyethylene cetyl ether (Brij 58) protocol, approximately 80% of its membrane-bound cytochromes are localized in its outer membrane. These outer membrane cytochromes could not be dislodged by treatment with chaotropic agents or by increased concentrations of the nonionic detergent Brij 58, suggesting that they are integral membrane proteins. Cytochrome distribution in cells lysed by a French press protocol confirm the localization of cytochromes to the outer membrane of anaerobically grown cells. This novel cytochrome distribution could play a key role in the anaerobic respiratory capabilities of this bacterium, especially in its ability to mediate manganese and iron reduction. Images PMID:1592800

  20. Structure of biogenic uraninite produced by Shewanella oneidensis strain MR-1

    SciTech Connect

    Schofield, Eleanor J.; Veeramani, Harish; Sharp, Jonathan; Suvorova, Elena; Bernier-Latmani, Rizlan; Mehta, Apurva; STAHLMAN, JONATHAN O.; Webb, Samuel M.; Clark, David L.; Conradson, Steven D.; Ilton, Eugene S.; Bargar, John R.

    2008-11-01

    The stability of biogenic uraninite with respect to oxidation is seminal to the success of in-situ bioreduction strategies for remediation of subsurface U(VI) contamination. The properties and hence stability of uraninite are dependent on its size, structure and composition. In this study, the local-, intermediate-, and long-range molecular-scale structure of nanoscale uraninite produced by Shewanella oneidensis strain MR-1 was investigated using EXAFS, SR-based powder diffraction and TEM. The uraninite products were found to be structurally homologous with stoichiometric UO2 under all conditions considered. Significantly, there was no evidence for lattice strain of the biogenic uraninite nanoparticles. The fresh nanoparticles were found to exhibit a well-ordered interior core of diameter ca 1 nm and an outer region of thickness ca ~ 1 nm in which the structure is locally distorted. The lack of nanoparticle strain and structural homology with stoichiometric UO2 suggests that established thermodynamic parameters for the latter material are an appropriate starting point to model the behavior of nano-biogenic uraninite. The detailed structural analysis in this study provides an essential foundation for subsequent investigations of more environmentally relevant samples.

  1. Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution1[OPEN

    PubMed Central

    Päpke, Carola; Diab, Houssein; Fernie, Alisdair R.

    2016-01-01

    Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [13C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [13C]glutamate and [15N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the 13C/15N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia. PMID:26553649

  2. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens

    PubMed Central

    Meermeier, Erin W.; Laugel, Bruno F.; Sewell, Andrew K.; Corbett, Alexandra J.; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J.; Franks, Tamera; Gold, Marielle C.; Lewinsohn, David M.

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2+ MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2+ clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  3. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens.

    PubMed

    Meermeier, Erin W; Laugel, Bruno F; Sewell, Andrew K; Corbett, Alexandra J; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J; Franks, Tamera; Gold, Marielle C; Lewinsohn, David M

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  4. Gene Expression Analysis of Rice Seedling under Potassium Deprivation Reveals Major Changes in Metabolism and Signaling Components

    PubMed Central

    Shankar, Alka; Singh, Amarjeet; Kanwar, Poonam; Srivastava, Ashish Kumar; Pandey, Amita; Suprasanna, Penna; Kapoor, Sanjay; Pandey, Girdhar K.

    2013-01-01

    Plant nutrition is one of the important areas for improving the yield and quality in crops as well as non-crop plants. Potassium is an essential plant nutrient and is required in abundance for their proper growth and development. Potassium deficiency directly affects the plant growth and hence crop yield and production. Recently, potassium-dependent transcriptomic analysis has been performed in the model plant Arabidopsis, however in cereals and crop plants; such a transcriptome analysis has not been undertaken till date. In rice, the molecular mechanism for the regulation of potassium starvation responses has not been investigated in detail. Here, we present a combined physiological and whole genome transcriptomic study of rice seedlings exposed to a brief period of potassium deficiency then replenished with potassium. Our results reveal that the expressions of a diverse set of genes annotated with many distinct functions were altered under potassium deprivation. Our findings highlight altered expression patterns of potassium-responsive genes majorly involved in metabolic processes, stress responses, signaling pathways, transcriptional regulation, and transport of multiple molecules including K+. Interestingly, several genes responsive to low-potassium conditions show a reversal in expression upon resupply of potassium. The results of this study indicate that potassium deprivation leads to activation of multiple genes and gene networks, which may be acting in concert to sense the external potassium and mediate uptake, distribution and ultimately adaptation to low potassium conditions. The interplay of both upregulated and downregulated genes globally in response to potassium deprivation determines how plants cope with the stress of nutrient deficiency at different physiological as well as developmental stages of plants. PMID:23922980

  5. Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism

    PubMed Central

    Valencia, C. Alexander; Wang, Xinjian; Wang, Jin; Peters, Anna; Simmons, Julia R.; Moran, Molly C.; Mathur, Abhinav; Husami, Ammar; Qian, Yaping; Sheridan, Rachel; Bove, Kevin E.; Witte, David; Huang, Taosheng; Miethke, Alexander G.

    2016-01-01

    Background & Aims The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological factors in patients with idiopathic ALF and impaired energy metabolism. Methods Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis. Results A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9, POLG, POLG2, DGUOK, and RRM2B; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9, which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy. Conclusion Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF. PMID:27483465

  6. The metabolomic profile of gamma-irradiated human hepatoma and muscle cells reveals metabolic changes consistent with the Warburg effect

    PubMed Central

    Wang, Min; Keogh, Adrian; Treves, Susan; Idle, Jeffrey R.

    2016-01-01

    The two human cell lines HepG2 from hepatoma and HMCL-7304 from striated muscle were γ-irradiated with doses between 0 and 4 Gy. Abundant γH2AX foci were observed at 4 Gy after 4 h of culture post-irradiation. Sham-irradiated cells showed no γH2AX foci and therefore no signs of radiation-induced double-strand DNA breaks. Flow cytometry indicated that 41.5% of HepG2 cells were in G2/M and this rose statistically significantly with increasing radiation dose reaching a plateau at ∼47%. Cell lysates from both cell lines were subjected to metabolomic analysis using Gas Chromatography-Mass Spectrometry (GCMS). A total of 46 metabolites could be identified by GCMS in HepG2 cell lysates and 29 in HMCL-7304 lysates, most of which occurred in HepG2 cells. Principal Components Analysis (PCA) showed a clear separation of sham, 1, 2 and 4 Gy doses. Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA) revealed elevations in intracellular lactate, alanine, glucose, glucose 6-phosphate, fructose and 5-oxoproline, which were found by univariate statistics to be highly statistically significantly elevated at both 2 and 4 Gy compared with sham irradiated cells. These findings suggested upregulation of cytosolic aerobic glycolysis (the Warburg effect), with potential shunting of glucose through aldose reductase in the polyol pathway, and consumption of reduced Glutathione (GSH) due to γ-irradiation. In HMCL-7304 myotubes, a putative Warburg effect was also observed only at 2 Gy, albeit a lesser magnitude than in HepG2 cells. It is anticipated that these novel metabolic perturbations following γ-irradiation of cultured cells will lead to a fuller understanding of the mechanisms of tissue damage following ionizing radiation exposure. PMID:26823999

  7. Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose

    PubMed Central

    Schabort, Du Toit W. P.; Letebele, Precious K.; Steyn, Laurinda; Kilian, Stephanus G.; du Preez, James C.

    2016-01-01

    We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus. PMID:27315089

  8. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

    SciTech Connect

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2014-12-15

    We report that integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. We found that the result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Lastly, taken together, the

  9. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides1[OPEN

    PubMed Central

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2015-01-01

    Integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and 13C labeling

  10. Integrative Analysis of Circadian Transcriptome and Metabolic Network Reveals the Role of De Novo Purine Synthesis in Circadian Control of Cell Cycle