scholarly journals Carbon Flux Analysis by13C Nuclear Magnetic Resonance To Determine the Effect of CO2on Anaerobic Succinate Production by Corynebacterium glutamicum

2014 ◽  
Vol 80 (10) ◽  
pp. 3015-3024 ◽  
Author(s):  
Dušica Radoš ◽  
David L. Turner ◽  
Luís L. Fonseca ◽  
Ana Lúcia Carvalho ◽  
Bastian Blombach ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Organic Acid ◽  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Fold Increase ◽  
Flux Analysis ◽  
Organic Acid Production ◽  
Content Type ◽  
Nuclear Magnetic

ABSTRACTWild-typeCorynebacterium glutamicumproduces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense ofl-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2was estimated by using13C-labeled glucose and13C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured byin vivoNMR, and the stoichiometry (H+:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate withC. glutamicumwithout genetic manipulation.

scholarly journals Interactions between Pyruvate and Lactate Metabolism in Propionibacterium freudenreichii subsp.shermanii: In Vivo 13C Nuclear Magnetic Resonance Studies

2000 ◽  
Vol 66 (5) ◽  
pp. 2012-2020 ◽  
Author(s):  
Catherine Deborde ◽  
Patrick Boyaval
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Tricarboxylic Acid Cycle ◽  
Dry Weight ◽  
Resonance Spectroscopy ◽  
Propionibacterium Freudenreichii ◽  
Pyruvate Metabolism ◽  
13C Nuclear Magnetic Resonance ◽  
Nuclear Magnetic

ABSTRACT In vivo 13C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of Propionibacterium freudenreichiisubsp. shermanii. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 μmol · min−1 · g−1 (cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 μmol · min−1 · g−1when it was cometabolized with pyruvate. P. shermanii used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO2, succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.

scholarly journals Pyrrolamide DNA Gyrase Inhibitors: Fragment-Based Nuclear Magnetic Resonance Screening To Identify Antibacterial Agents

2011 ◽  
Vol 56 (3) ◽  
pp. 1240-1246 ◽  
Author(s):  
Ann E. Eakin ◽  
Oluyinka Green ◽  
Neil Hales ◽  
Grant K. Walkup ◽  
Shanta Bist ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Antibacterial Agents ◽  
Dna Gyrase ◽  
Mouse Lung ◽  
Infection Model ◽  
Lead Compound ◽  
Content Type ◽  
Gyrase Inhibitors ◽  
Nuclear Magnetic

ABSTRACTDNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC50) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated inStaphylococcus aureusby plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of thegyrBgenes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated againstStreptococcus pneumoniaein a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

scholarly journals Catabolism of Glucose and Lactose in Bifidobacterium animalis subsp. lactis, Studied by13C Nuclear Magnetic Resonance

2013 ◽  
Vol 79 (24) ◽  
pp. 7628-7638 ◽  
Author(s):  
Irene González-Rodríguez ◽  
Paula Gaspar ◽  
Borja Sánchez ◽  
Miguel Gueimonde ◽  
Abelardo Margolles ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Metabolic Pathways ◽  
Transcriptional Analysis ◽  
Content Type ◽  
Bifidobacterium Animalis ◽  
Metabolic Flux Distribution ◽  
Nuclear Magnetic ◽  
In Cells

ABSTRACTBifidobacteria are widely used as probiotics in several commercial products; however, to date there is little knowledge about their carbohydrate metabolic pathways. In this work, we studied the metabolism of glucose and lactose in the widely used probiotic strainBifidobacterium animalissubsp.lactisBB-12 byin vivo13C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-13C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with13C on carbon 1 of the glucose or the galactose moiety was determined in suspensions of cells grown in lactose. These experiments allowed the quantification of some intermediate and end products of the metabolic pathways, as well as determination of the consumption rate of carbon sources. Additionally, the labeling patterns in metabolites derived from the metabolism of glucose specifically labeled with13C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-13Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-13Cgalactose]lactose), and [1-13C]glucose in lactose-grown cells were determined in cell extracts by13C NMR. The NMR analysis showed that the recovery of carbon was fully compatible with the fructose 6-phosphate, or bifid, shunt. The activity of lactate dehydrogenase, acetate kinase, fructose 6-phosphate phosphoketolase, and pyruvate formate lyase differed significantly between glucose and lactose cultures. The transcriptional analysis of several putative glucose and lactose transporters showed a significant induction of Balat_0475 in the presence of lactose, suggesting a role for this protein as a lactose permease. This report provides the firstin vivoexperimental evidence of the metabolic flux distribution in the catabolic pathway of glucose and lactose in bifidobacteria and shows that the bifid shunt is the only pathway involved in energy recruitment from these two sugars. On the basis of our experimental results, a model of sugar metabolism inB. animalissubsp.lactisis proposed.

scholarly journals Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

2015 ◽  
Vol 14 (11) ◽  
pp. 1064-1072 ◽  
Author(s):  
Courtney Reichhardt ◽  
Jose A. G. Ferreira ◽  
Lydia-Marie Joubert ◽  
Karl V. Clemons ◽  
David A. Stevens ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Extracellular Matrix ◽  
Solid State ◽  
Magnetic Resonance ◽  
Aspergillus Fumigatus ◽  
Fungal Infections ◽  
Resonance Spectroscopy ◽  
Chronic Infections ◽  
Content Type ◽  
Nuclear Magnetic

ABSTRACTAspergillus fumigatusis commonly responsible for lethal fungal infections among immunosuppressed individuals.A. fumigatusforms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of theA. fumigatusextracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The13C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional15N and31P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that theA. fumigatusECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in theA. fumigatusECM and will enable future comparisons with other organisms and withA. fumigatusgrown under alternate conditions.

scholarly journals Metabolism of Lactate in Cultured GABAergic Neurons Studied by 13C Nuclear Magnetic Resonance Spectroscopy

1998 ◽  
Vol 18 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Helle S. Waagepetersen ◽  
Inger J. Bakken ◽  
Orla M. Larsson ◽  
Ursala Sonnewald ◽  
Arne Schousboe
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Resonance Spectroscopy ◽  
Acid Cycle ◽  
Cell Extracts ◽  
Gaba Synthesis ◽  
Nuclear Magnetic

Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), γ-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).

Novel self-coloured polymers based on new fluorescent naphthalimide derivatives: synthesis, characterisation and photophysical properties

2017 ◽  
Vol 46 (3) ◽  
pp. 244-250 ◽  
Author(s):  
Hanieh Shaki ◽  
Alireza Khosravi ◽  
Kamaladin Gharanjig
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Polymer Chain ◽  
Fluorescent Dyes ◽  
Proton Nuclear Magnetic Resonance ◽  
Quantum Yields ◽  
The Novel ◽  
Acrylic Polymer ◽  
Content Type ◽  
Nuclear Magnetic

Purpose In this study, two novel fluorescent dyes, based on naphthalimide derivatives have been synthesised from acenaphthene as a starting material. The ability of the dyes to graft to polymer chain was then demonstrated. The novel synthesised dyes and self-coloured polymers were characterised by a variety of techniques. Design/methodology/approach The novel dyes were prepared through by halogenation, oxidation, imidation and amination reactions. All steps of these processes were monitored by thin layer chromatography. The fluorescent dyes and their intermediates were characterised by differential scanning calorimeter, fourier transform infrared spectroscopy (FTIR), Proton Nuclear Magnetic Resonance (1H-NMR) and carbon-13 nuclear magnetic resonance (13-CNMR) spectroscopic techniques. The molar extinction coefficients and absorption maximum wavelength were obtained by examining the dyes and polymer solutions in Dimethylformamide (DMF) and toluene solvents. The fluorescency of novel dyes and self-coloured polymers was evaluated. Their quantum yields and Stokes shift values were determined as DMF and toluene solutions. The percentage of the covalently bounded dyes into the polymer chain was calculated. Findings The characterisation of the synthesised dyes and self-coloured polymers verified their structural correctness. The results of reaction dyes with resin demonstrated that the dyes were covalently bonded to the chain of an acrylic polymer (resin) containing carboxylic acid groups giving self-coloured polymers. The extent of fluorescence of the synthesised dyes and their polymers showed that compounds containing functional amino group in C-4 position of naphthalimide ring have high fluorescence properties. Originality/value This study is original. Self-coloured polymers based on acrylic were synthesised by novel naphthalimide dyes with acrylic resin for the first time, successfully. The novel dyes and their self-coloured polymers exhibit good and acceptable fluorescent activity.

Synthesis of N-substituted arylhydrazones: applying to polyester fabric as disperse dyes

2017 ◽  
Vol 46 (6) ◽  
pp. 449-457 ◽  
Author(s):  
Abdel-Zaher A. Elassar ◽  
Saleh M. Al-Mousawi ◽  
Maher Helmi Helal ◽  
Mohamed E. Elgazzar
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Polyester Fabric ◽  
Proton Nuclear Magnetic Resonance ◽  
Disperse Dyes ◽  
Polyester Fabrics ◽  
Content Type ◽  
Temperature Method ◽  
Synthetic Reaction ◽  
Nuclear Magnetic

Purpose The purpose of this paper is to prepare new disperse dyes and apply for dying polyester fabrics. Design/methodology/approach The synthetic reaction was carried out through two steps: preparation of arylhydrazones and alkylation using enaminone and dimethylaminovinyl-pyridazine. The high temperature method was used to apply these dyes to polyester fibres. Findings The study revealed that there is a significant effect of the new prepared disperse dyes on polyester fabrics. The structures of the prepared dyes were established based on elemental analysis and spectral data (infra red (IR), mass spectrometry (MS) and proton nuclear magnetic resonance (1H-NMR), carbon 13th nuclear magnetic resonance (13C-NMR)). Research limitations/implications Disperse dyes containing heterocyclic moiety have attracted great academic and industrial attention owing to their significant. The potential of using disperse dyes easily prepared from arylhydrazones are promise broad applications for these dyes. Practical implications The presence of N-thienyl and N-pyridazinyl in the structure of the synthesised disperse dyes would be expected to add the bioactivity advantage. Also, it can be used in formulating the antimicrobial fabrics. Social implications The N-thienyl and N-pyrdiazinyl derivatives of azo dyes are expected to be superior to in the application for fabrics. It may be useful for other applications like painting. Originality/value This paper helps to synthesise novel thiophene or pyridazine-based dyestuff for application in dying properties on polyester fabric and study their fastness properties.

scholarly journals Use of1H Nuclear Magnetic Resonance To Measure Intracellular Metabolite Levels during Growth and Asexual Sporulation in Neurospora crassa

2011 ◽  
Vol 10 (6) ◽  
pp. 820-831 ◽  
Author(s):  
James D. Kim ◽  
Kayla Kaiser ◽  
Cynthia K. Larive ◽  
Katherine A. Borkovich
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Neurospora Crassa ◽  
Nutrient Sensing ◽  
Wild Type ◽  
Content Type ◽  
Asexual Sporulation ◽  
Significant Difference ◽  
High Sucrose ◽  
Nuclear Magnetic

ABSTRACTConidiation is an asexual sporulation pathway that is a response to adverse conditions and is the main mode of dispersal utilized by filamentous fungal pathogens for reestablishment in a more favorable environment. Heterotrimeric G proteins (consisting of α, β, and γ subunits) have been shown to regulate conidiation in diverse fungi. Previous work has demonstrated that all three of the Gα subunits in the filamentous fungusNeurospora crassaaffect the accumulation of mass on poor carbon sources and that loss ofgna-3leads to the most dramatic effects on conidiation. In this study, we used1H nuclear magnetic resonance (NMR) to profile the metabolome ofN. crassain extracts isolated from vegetative hyphae and conidia from cultures grown under conditions of high or low sucrose. We compared wild-type and Δgna-3strains to determine whether lack ofgna-3causes a significant difference in the global metabolite profile. The results demonstrate that the global metabolome of wild-type hyphae is influenced by carbon availability. The metabolome of the Δgna-3strain cultured on both high and low sucrose is similar to that of the wild type grown on high sucrose, suggesting an overall defect in nutrient sensing in the mutant. However, analysis of individual metabolites revealed differences in wild-type and Δgna-3strains cultured under conditions of low and high sucrose.

scholarly journals In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by15N Nuclear Magnetic Resonance

1999 ◽  
Vol 65 (3) ◽  
pp. 1099-1109 ◽  
Author(s):  
M. Tesch ◽  
A. A. de Graaf ◽  
H. Sahm
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Corynebacterium Glutamicum ◽  
Ammonium Assimilation ◽  
Equation Model ◽  
Wild Type ◽  
In The Wild ◽  
High Fraction ◽  
Nuclear Magnetic

ABSTRACT Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicumstrains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4 + was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.

scholarly journals Comparative Survey of Rumen Microbial Communities and Metabolites across One Caprine and Three Bovine Groups, Using Bar-Coded Pyrosequencing and1H Nuclear Magnetic Resonance Spectroscopy

2012 ◽  
Vol 78 (17) ◽  
pp. 5983-5993 ◽  
Author(s):  
Hyo Jung Lee ◽  
Ji Young Jung ◽  
Young Kyoon Oh ◽  
Sang-Suk Lee ◽  
Eugene L. Madsen ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
16S Rrna ◽  
Microbial Communities ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Dietary Shift ◽  
Metabolite Profiles ◽  
Nuclear Magnetic

ABSTRACTPyrosequencing of 16S rRNA genes (targetingBacteriaandArchaea) and1H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla—Bacteroidetes,Firmicutes,Fibrobacteres, andProteobacteria—in all ruminants. However, interestingly, the bacterial reads belonging toFibrobactereswere present at a very low abundance (<0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen.

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