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J P Grivet

Publications and source records attributed to J P Grivet.

At least 19 recordsLinked to original sources
PubMed2001

NMR and microorganisms.

This article is an introduction to the use of NMR for the investigation of microbial physiology and metabolism. NMR parameters which determine the sensitivity and resolving power of the method are reviewed. A broad survey of current applications follows. Qualitative uses are described first; they include compound identification and localisation. Quantitative aspects, such as pH, concentration and flux measurements are then examined, as well as the corresponding experimental constraints. The review ends with suggestions of possible future developments in instrument capabilities aimed at improving sensitivity: higher fields, spectroscopic and imaging microprobes.

Cell Membrane↗
PubMed1997

Physiology and development of Pectinatus cerevisiiphilus and Pectinatus frisingensis, two strict anaerobic beer spoilage bacteria.

The genus Pectinatus was isolated recently and the deposited strains were classified as beer spoilage bacteria producing propionate as a major fermentation product. A recent investigation of this genus demonstrated the existence of two species: Pectinatus cerevisiiphilus, the type strain and Pectinatus frisingensis, a new species with a different pattern of growth substrates. Different culture media tested for both species demonstrated a higher specific growth rate for P. cerevisiiphilus. However, final biomass production was in every case around 20% higher in P. frisingensis. A 400% decrease of final biomass production was measured when the species were cultivated on poor culture medium; this decrease was found to be broadly proportional to amounts of acetate excreted in the medium. Both species produced CO2 from glucose; however, no significant modifications of biomass and volatile fatty acid production were demonstrated when varying head space composition with regard to CO2 levels. Growth experiments on glucose with increasing amounts of ethanol added in the culture, revealed a higher sensitivity of P. cerevisiiphilus to ethanol inhibition. Ethanol concentrations over 1.7 M resulted in a complete inhibition of growth for both Pectinatus species. Combined effects of culture medium pH, lactate, and glucose concentrations, demonstrated the prevailing role of glucose in the development of the bacteria. However, these three parameters had a different influence on growth characteristics of both Pectinatus species. P cerevisiiphilus grew very poorly with a glucose concentration of 5 mM for pH values below 4.1. This species had optimal pH for growth between 6 and 6.2 and it excreted increasing amounts of acetate with increase of pH, glucose or lactate in the culture medium. P. frisingensis showed a wide range of pH allowing a good growth. For glucose concentrations below 20 mM, highest final biomass productions were measured in the culture for pH values around 4.9; this also corresponded to a minimum in acetate excretion. The above results pointed at P. frisingensis as the prevailing species of Pectinatus in beer spoilage.

Bacteria, Anaerobic↗
PubMed1997

13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colon.

Glucose fermentation by four acetogenic species (two Clostridium strains, one Streptococcus strain and Ruminococcus hydrogenotrophicus) isolated from the human colon was of a mixed-acid type, whereas pyruvate metabolism was characterised by homoacetogenesis. Acetate formation from [1-13C] and [2-13C]glucose was consistent with the formation of acetyl-SCoA from pyruvate generated by the Embden-Meyerhof-Parnas pathway. Labelling of lactate and ethanol demonstrated that these metabolites were formed by reduction of pyruvate and acetyl-SCoA, respectively. In contrast, the reductive pathway of acetate formation was the preferential means of re-oxidising cofactors formed during [1-13C]pyruvate catabolism.

Acetates↗
PubMed1996

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe.

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of L-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the L-lactate produced originates from endogenous L-malate and 25% from exogenous L-malate. Moreover, although a small amount of exogenous L-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded L-malate was converted into L-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades L-malate. Total malolactic fermentation was obtained in this strain, with most of the L-malate converted into L-lactate and CO2. Moreover, L-malate was used preferentially by the malolactic enzyme in this strain also.

Base Sequence↗
PubMed1996

3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species.

Glycerol fermentation by Enterobacter agglomerans revealed that both growth and 1,3-propanediol production ceased after consumption of about 430 mM glycerol, irrespective of the initial glycerol content. This phenomenon was assigned to the production of 3-hydroxypropionaldehyde, which was identified by proton nuclear magnetic resonance and which showed a bacteriostatic effect. The accumulation during glycerol fermentation was also observed with two other enterobacterial species, i.e., Klebsiella pneumoniae and Citrobacter freundii.

Aldehydes↗
PubMed1994

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora.

Washed bacterial suspensions obtained from the pig hindgut were incubated under 13CO2 in a buffer containing NaH13CO3 and carbohydrates. Incorporation of 13C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H2 added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H2 markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H2 significantly increased the incorporation of 13CO2 into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H2 partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO2 and H2 (represented mostly by double labelling). A highly significant interaction between BES and H2 concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.

Acetates↗
PubMed1993

Interactions of basic amphiphilic peptides with dimyristoylphosphatidylcholine small unilamellar vesicles: optical, NMR, and electron microscopy studies and conformational calculations.

The interactions of DMPC small unilamellar vesicles with four amphiphilic polypeptides [(LKKL)n, (LRRL)n, (LKKL)4, and (YKKY)n] have been investigated by circular and infrared dichroism, turbidimetry, electron microscopy, and fluorescence, 1H, and 31P nuclear magnetic resonance spectroscopy. The main results obtained are the following: (1) Well-defined complexes are formed by the association of one amino acid residue with approximately two lipid molecules. (2) In the presence of polypeptides fusions are observed between SUVs when the molar ratio p is less than 0.05, and a clearance effect is observed when p is higher than 0.05. (3) The anchoring sites of the polypeptides on DMPC molecules are the negative phosphate groups through electrostatic interactions with the terminal NH3+ of lysine residues. (4) The polypeptides adopt an alpha-helical conformation with their axis parallel to the membrane surface. The hydrophobic part of the amphiphilic alpha helix can penetrate the outer lipid leaflet down to the C5 position. (5) Choline methyl groups are not involved in the interactions between lipid molecules and amino acid residues. (6) Phosphorus atom mobility around the P-O-glycerol bond is strongly reduced whereas that of methylene groups is progressively weakened when going up from C13 to C1. Finally, using modeling and energy calculations a model of possible Ac(LKKL)4NHEt-DMPC SUV complexes is presented.

Amino Acid Sequence↗
PubMed1993

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2.

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.

Adenylate Kinase↗
PubMed1992

A double-tuned probe for metabolic NMR studies.

A double-tuned single coil probe for high field metabolic NMR studies has been designed and tested. The resonant coil is a single-turn of copper foil wound on a quartz cylinder. It is fed by a short length of parallel strip line made of the same material; the feeder is also used as a tuning capacitance. Electrical parameters (quality factors, efficiencies at proton and carbon frequencies) and magnetic resonance data (90 degrees pulse lengths, minimum decoupler power) for lossy samples have been measured. The probe allows for the optimal use of the limited high homogeneity region of a vertical magnet. It shows good sensitivity and efficient decoupling.

Copper↗
PubMed1992

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway.

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-13C] ethanol and CO2 to [2-13C] propionate, [1-13C] acetate and [2-13C] propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.

Acrylates↗
PubMed1991

Biochemical events occurring during the respiratory burst of macrophages: A 31P and 13C NMR study.

In a previous study, 31P NMR revealed that an intracellular acidification occurred during the respiratory burst of P388 D1 macrophages, but this NMR technique could not provide information about the localization of this event in cells. However, using a fluorescent pH-dependent probe, it was confirmed that this transient pH decrease does not occur in the cytosol but more probably occurs in relation to the function of endocytic vesicles. A 31P NMR study allowed us to evidence a transient increase in ADP phosphorylation at the beginning of the respiratory burst, possibly in connection with the initiation of the oxidase complex involved in superoxide anion production. A 13C NMR study of perchloric acid extracts from in vivo primed cells revealed an increase in glucose consumption due to Con A triggering. Sugar phosphates, which must be considered markers of the hexose monophosphate shunt involved in the respiratory burst, were also observed upon this activation process.

Adenosine Diphosphate↗
PubMed1991

Glucose and lactate catabolism by bacteria of the pig large intestine and sheep rumen as assessed by 13C nuclear magnetic resonance.

The fermentation of [1-13C] glucose and [3-13C]lactate by bacteria isolated from sheep rumen and pig large intestine was compared by the nuclear magnetic resonance (NMR) technique. Washed cell suspensions were incubated directly in the NMR spectrometer and spectra were recorded every 10 min after injection of the labelled substrates. The results showed large differences in the fermentation patterns between rumen and hindgut bacteria. The latter pattern indicated a greater ability for formation and fermentation of lactate than that of the rumen. Moreover, with both substrates the amount of propionate formed via the acrylate pathway was always greater with hindgut than with rumen bacteria, 50% and 20% of the total, respectively.

Animals↗
PubMed1990

31P NMR study of intracellular pH during the respiratory burst of macrophages.

The metabolic events occurring during the respiratory burst of macrophages previously primed in vivo with lipopolysaccharide were studied by 31P nuclear magnetic resonance, using the P388D1 cell line as a model of the mature macrophages. Using perchloric acid extracts, the presence of phosphocreatine was shown in the primed cells, indicating that in control P388 D1 macrophages, in which no phosphocreatine was seen, in vivo maturation was incomplete. The cells primed in vivo exhibited greater maturation than the control cells, as well as greater creatine kinase activity. Perfusion of gel-embedded macrophages allowed the monitoring of phosphorylated metabolite peak intensities and of the intracellular pH. After the respiratory burst of the primed macrophages had been triggered by concanavalin A, these intensities did not alter significantly, but the intracellular pH decreased. 31P NMR spectra reflected transient acidification in the primed cells, possibly due to the formation of endocytic vesicles and their fusion with lysosomes.

Animals↗
PubMed1990

NMR study of 13CO2 incorporation into short-chain fatty acids by pig large-intestinal flora.

The nuclear magnetic resonance technique was used to study carbon dioxide reduction by the pig large-intestinal flora. Washed bacterial cell suspensions were incubated for 6 and 15 h under 13CO2 and H2 as the gas phase and with a buffer containing NaH13CO3 and cellobiose and amino acids (casein hydrolysate) as substrates. Methane was produced in all incubation media. Significant amounts of single- as well as multiple-labelled acetate and butyrate were formed, demonstrating synthesis of acetate from H2 + CO2. Propionate was labelled mainly on the carboxyl group, which was attributed to an enzymatic exchange of the carboxyl group of propionate with 13CO2. These results indicate that the reduction of CO2 to acetate may be an important pathway for microbial production of acetate in the pig large intestine even in the presence of methanogenesis.

Acetates↗
PubMed1989

In vivo 13C NMR studies of glucose catabolism by isolated rumen bacteria.

Nuclear magnetic resonance (NMR) was used to study the metabolism of the rumen bacterial ecosystem. The fermentation of 1-13C-glucose was investigated. This system showed a transient build-up and decay of lactate labelled at the C3 position. The degradation of lactate gave rise to volatile fatty acids. These results agree with in vivo data on diets rich in available carbohydrates.

Animals↗
PubMed1989

Decrease in internal H+ and positive inotropic effect of heptaminol hydrochloride: a 31P n.m.r. spectroscopy study in rat isolated heart.

1. The cardiotonic effect of heptaminol hydrochloride (Hept-a-myl, Delalande) was studied using 31P-nuclear magnetic resonance (n.m.r.) spectroscopy and left ventricular pressure (LVP) measurements in rat isolated hearts. The possibility of this effect being mediated by an intracellular realkalinisation was tested. 2. Isolated hearts were perfused at 10 ml min-1 by the Langendorff method with Krebs-Henseleit solution at 37 degrees C and stimulated at 5 Hz. Mechanical activity was measured as variations of left ventricular pressure (LVP). 31P-n.m.r. spectra were recorded every 2 min. Changes in cardiac adenosine triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate (Pi) were followed and intracellular pH (pHi) was estimated from the chemical shift of Pi. 3. The effects of heptaminol were tested in different conditions: normoxia, moderate ischaemia, severe ischaemia, and moderate ischaemia in the presence of amiloride or guanidinium chloride as inhibitors of the Na-H exchange. 4. In normoxia, heptaminol induced a cyclic increase of systolic LVP, associated with an increase in Pi. No significant effect on pHi was observed. In changing from normoxia to moderate ischaemia, PCr and systolic LVP decreased; a mild intracellular acidification (pHi 6.96) was obtained. Heptaminol induced a restoration of pHi and increased LVP. In severe ischaemia, the realkalinization effect and the restoration of LVP induced by heptaminol were no longer observed. During moderate ischaemia, Na-H exchange inhibitors decreased pHi and LVP. Heptaminol applied in the presence of these inhibitors was unable to restore pHi and LVP. In severe ischaemia, the realkalinization effect and the restoration of LVP induced by heptaminol were no longer observed. During moderate ischaemia, Na-H exchange inhibitors decreased pHi and LVP. Heptaminol applied in the presence of these inhibitors was unable to restore pHi and LVP. 5. These results suggest that the positive inotropic effect of heptaminol during moderate ischaemia could be related to a restoration of internal pH, possibly mediated by a stimulation of the Na-H exchange.

Alkalosis↗
PubMed1987

The binding of N-trifluoroacetyl chito-oligosaccharides to wheat-germ agglutinin: a fluorescence investigation.

We describe the synthesis of N-trifluoroacetyl chito-oligosaccharides and their use as ligands to probe the binding sites of wheat-germ agglutinin, a lectin specific for N-acetylglucosamine. The binding is monitored using intrinsic protein fluorescence, which is due to tryptophan side-chains. We present arguments purporting to show the presence of a fluorophore close to each of the four sites. The binding of chito-oligosaccharides to wheat-germ agglutinin is complex and can only be approximately described by an independent and equivalent sites model. This model applies when the ligand concentration range is restricted to higher values. The possible role of ligand-mediated protein aggregation and of site inequivalence is discussed. We find that the affinity of trifluoroacetylated chito-oligosaccharides for wheat-germ agglutinin is higher than that of the N-acetylated parent compounds, the difference increasing with chain length. Our results are in agreement with a model of the binding site previously proposed by Clegg et al. (Biochemistry 22 (1983) 4797-4804).

Algorithms↗