Profiling human gut bacterial metabolism and its kinetics using [U-13C]glucose and NMR.

This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR.

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR.

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Transport and compartmentation of phosphite in higher plant cells--kinetic and P nuclear magnetic resonance studies.

Phosphite (Phi, H(2)PO(3)(-)), being the active part of several fungicides, has been shown to influence not only the fungal metabolism but also the development of phosphate-deficient plants. However, the mechanism of phosphite effects on plants is still widely unknown. In this paper we analysed uptake, subcellular distribution and metabolic effects of Phi in tobacco BY-2 cells using in vivo(31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy. Based on the kinetic properties of the phosphate transport system of tobacco BY-2 cells, it was demonstrated that phosphite inhibited phosphate uptake in a competitive manner. To directly follow the fate of phosphate and phosphite in cytoplasmic and vacuolar pools of tobacco cells, we took advantage of the pH-sensitive chemical shift of the Phi anion. The NMR studies showed a distinct cytoplasmic accumulation of Phi in Pi-deprived cells, whereas Pi resupply resulted in a rapid efflux of Phi. Pi-preloaded cells shifted Phi directly into vacuoles. These studies allowed for the first time to follow Phi flux processes in an in vivo setting in plants. On the other hand, the external Pi nutrition status and the metabolic state of the cells had a strong influence on the intracellular compartmentalization of xenobiotic Phi.

Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing.

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.

Microaerobic and anaerobic metabolism of a Methylocystis parvus strain isolated from a denitrifying bioreactor.

An obligate methanotrophic bacterium, strain MTS, was isolated from a methane-fed microaerobic denitrifying bioreactor. 16S rRNA and DNA-DNA hybridization analysis revealed that this organism was most closely related to Methylocystis parvus, a Type II methanotroph, belonging to the α-subclass of the Proteobacteria. The metabolism of the bacterium under microaerobic and anaerobic conditions was studied by (13) C-NMR. (13) C-labelled poly-ß-hydroxybutyrate (PHB) formation occurred in cell suspensions incubated with (13) C-labelled methane at low (5-10%) oxygen concentration. Under these conditions low levels of succinate, acetate and 2,3-butanediol were formed and excreted into the culture medium. Intracellular PHB degradation was observed in intact cells under anaerobic conditions in the absence of an exogenous carbon source during a long-term incubation of 90 days. Multiple (13) C-labelled ß-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were identified as products in in vivo(13) C-NMR spectra and in the spectra of culture medium during the dynamic PHB degradation. The isolated obligate methanotroph clearly shows a fermentative metabolism of PHB under anaerobic conditions. The excreted products may serve as substrates for denitrifying bacteria.

Archaeoglobus fulgidus couples CO oxidation to sulfate reduction and acetogenesis with transient formate accumulation.

The genome sequence of Archaeoglobus fulgidus VC16 encodes three CO dehydrogenase genes. Here we explore the capacity of A. fulgidus to use CO as growth substrate. Archaeoglobus fulgidus VC16 was successfully adapted to growth medium that contained sulfate and CO. In the presence of CO and sulfate the culture OD(660) increased to 0.41 and sulfide, carbon dioxide, acetate and formate were formed. Accumulation of formate was transient. Similar results, except that no sulfide was formed, were obtained when sulfate was omitted. Hydrogen was never detected. Under the conditions tested, the observed concentrations of acetate (18 mM) and formate (8.2 mM) were highest in cultures without sulfate. Proton NMR spectroscopy indicated that CO2, and not CO, is the precursor of formate and the methyl group of acetate. Methylviologen-dependent formate dehydrogenase activity (1.4 micromol formate oxidized min(-1) mg(-1)) was detected in cell-free extracts and expected to have a role in formate reuptake. It is speculated that formate formation proceeds through hydrolysis of formyl-methanofuran or formyl-tetrahydromethanopterin. This study demonstrates that A. fulgidus can grow chemolithoautotrophically with CO as acetogen, and is not strictly dependent on the presence of sulfate, thiosulfate or other sulfur compounds as electron acceptor.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).

Biochemical adaptations of two sugar kinases from the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden-Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase. In the present study, we report the characterization of a P. furiosus galactokinase (GALK) and its comparison with the P. furiosus ADP-GLK. The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity. Both enzymes are specific kinases with an optimal activity at approx. 90 degrees C. Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP. There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P. furiosus.