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Michel Fèvre - One of the best experts on this subject based on the ideXlab platform.
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Transient expression of the beta-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis.
Current genetics, 1997Co-Authors: Roger Durand, Christine Rascle, M. Fischer, Michel FèvreAbstract:The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial β-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for β-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the β-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus.
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A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: Support for the hypothesis that hydrogenosomes are modified mitochondria
Molecular microbiology, 1997Co-Authors: Mark Van Der Giezen, K. Bjo¨rn Rechinger, Ib Svendsen, Roger Durand, Robert P. Hirt, Michel Fèvre, T. Martin Embley, Rudolf A PrinsAbstract:The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD(+)/NADP(+) binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position -2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.
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Sequence of the phosphoenolpyruvate carboxykinase-encoding cDNA from the rumen anaerobic fungus Neocallimastix frontalis: comparison of the amino acid sequence with animals and yeast.
Gene, 1992Co-Authors: P. Reymond, C. Geourjon, B. Roux, Roger Durand, Michel FèvreAbstract:The nucleotide sequence of the cDNA of the phosphoenolpyruvate carboxykinase-encoding gene from the fungus Neocallimastix frontalis, was determined. The deduced amino acid sequence (608 residues) and the predicted protein structure were compared to their counterparts in animals and yeast. Catalytic regions (substrate-binding site and nucleotide-binding domains) are highly conserved among fungal and animal organisms. The yeast sequence showed no similarity to the fungal sequence.
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Molecular cloning of genes from the rumen anaerobic fungus Neocallimastix frontalis: expression during hydrolase induction.
FEMS Microbiology Letters, 1991Co-Authors: P. Reymond, M Hebraud, Roger Durand, Michel FèvreAbstract:Abstract Glycoside and polysaccharide hydrolase production by the rumen anaerobic fungus, Neocallimastix frontalis is induced by the presence of crystalline cellulose. A differential screening of a cDNA library was used to isolate DNA sequences transcribed at high levels under growth conditions which induce enzyme production. Seven clones were isolated that prefentially hybridized to the induced cDNA probe versus the non-induced cDNA probe. Southern analysis showed that a cDNA clone (118) hybridized to a DNA probe encoding part of the exo-cellobiohydrolase I (CBH I) gene of Trichoderma reesei . Northern analysis demonstrated that the cDNA 118 was transcribed to yield a 2.1 kb RNA. This transcript was induced in the presence of cellulose.
Rudolf A Prins - One of the best experts on this subject based on the ideXlab platform.
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A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: Support for the hypothesis that hydrogenosomes are modified mitochondria
Molecular microbiology, 1997Co-Authors: Mark Van Der Giezen, K. Bjo¨rn Rechinger, Ib Svendsen, Roger Durand, Robert P. Hirt, Michel Fèvre, T. Martin Embley, Rudolf A PrinsAbstract:The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD(+)/NADP(+) binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position -2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.
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Metabolic energy generation in hydrogenosomes of the anaerobic fungus Neocallimastix: evidence for a functional relationship with mitochondria
Mycological Research, 1994Co-Authors: Femke D. Marvin-sikkema, Jan C Gottschal, Arnold J. M. Driessen, Rudolf A PrinsAbstract:Anaerobic eukaryotes are often devoid of mitochondria but contain special organelles separated from the cytosol by a single (in fungi) or a double (in protozoa) membrane. Hydrogenosomes from the anaerobic fungus Neocallimastix sp. L2 are thought to catalyse the enzymic steps in the ATP-yielding metabolism of malate into acetate, H 2 and CO 2 . Isolated hydrogenosomes contain a Mg 2+ - or Mn 2+ -dependent ATPase activity. This activity is involved in the maintenance of a pH gradient across the hydrogenosomal membrane, which renders these organelles alkaline inside. ATPase activity and ΔpH generation is sensitive to diethylstilboestrol but not to other known ATPase inhibitors. Typical inhibitors of the mitochondrial ADP/ATP translocase, bongkrekic acid and carboxyatractylate reduced the ATPase activity, suggesting the presence of a nucleotide transporter. Under anaerobic conditions hydrogenosomes produced H 2 and acetate from malate. This process was found to be dependent on the external supply of ATP or ADP and succinate, and was blocked by protonophores, diethylstilboestrol, and the inhibitors bongkrekic acid and carboxyatractylate. These results demonstrate that hydrogenosomes of Neocallimastix sp. L2 perform the essential functions required for the generation of metabolic energy from malate. It is suggested that hydrogenosomes are functionally related to mitochondria but lack an outer membrane.
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Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2
Archives of Microbiology, 1993Co-Authors: Femke D. Marvin-sikkema, Teresa M. Pedro Gomes, Jan C Gottschal, Jean Philippe Grivet, Rudolf A PrinsAbstract: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.
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INFLUENCE OF METRONIDAZOLE, CO, CO2, AND METHANOGENS ON THE FERMENTATIVE METABOLISM OF THE ANAEROBIC FUNGUS Neocallimastix SP STRAIN L2
Applied and environmental microbiology, 1993Co-Authors: Femke D. Marvin-sikkema, Jan C Gottschal, Elizabeth Rees, Marjan N. Kraak, Rudolf A PrinsAbstract:The effects of metronidazole, CO, methanogens, and CO, on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H-2, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H-2 and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO2 in the gas phase resulted in increased H-2 and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.
Roger Durand - One of the best experts on this subject based on the ideXlab platform.
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A double membrane surrounds the hydrogenosomes of the anaerobic fungus Neocallimastix frontalis
FEMS microbiology letters, 1997Co-Authors: Marlene Benchimol, Roger Durand, João Carlos De Aquino AlmeidaAbstract:The structure of hydrogenosomes of the anaerobic fungus Neocallimastix frontalis was analyzed using routine preparations for transmission electron microscopy, freeze-fracture and immunocytochemistry. They appeared as round or elongated structures, always enveloped by two distinct, but tightly apposed membranes. Images of organelle division were very similar to those observed in trichomonad protozoa. These observations suggest that hydrogenosomes are homologous organelles in unrelated species weakening the hypothesis of a polyphyletic origin and supporting the evidence that fungal hydrogenosomes are probably derived from an endosymbiont relationship.
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Transient expression of the beta-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis.
Current genetics, 1997Co-Authors: Roger Durand, Christine Rascle, M. Fischer, Michel FèvreAbstract:The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial β-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for β-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the β-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus.
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A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: Support for the hypothesis that hydrogenosomes are modified mitochondria
Molecular microbiology, 1997Co-Authors: Mark Van Der Giezen, K. Bjo¨rn Rechinger, Ib Svendsen, Roger Durand, Robert P. Hirt, Michel Fèvre, T. Martin Embley, Rudolf A PrinsAbstract:The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD(+)/NADP(+) binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position -2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.
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Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata.
Current microbiology, 1995Co-Authors: André Breton, Roger Durand, Brigitte Gaillard-martinie, C. Gerbi, B. Gomez De Segura, B. KherratiaAbstract:β-d-Glucosidase, β-d-fucosidase β-d-xylosidase, and β-cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. β-d-Galactosidase and α-l-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.
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Sequence of the phosphoenolpyruvate carboxykinase-encoding cDNA from the rumen anaerobic fungus Neocallimastix frontalis: comparison of the amino acid sequence with animals and yeast.
Gene, 1992Co-Authors: P. Reymond, C. Geourjon, B. Roux, Roger Durand, Michel FèvreAbstract:The nucleotide sequence of the cDNA of the phosphoenolpyruvate carboxykinase-encoding gene from the fungus Neocallimastix frontalis, was determined. The deduced amino acid sequence (608 residues) and the predicted protein structure were compared to their counterparts in animals and yeast. Catalytic regions (substrate-binding site and nucleotide-binding domains) are highly conserved among fungal and animal organisms. The yeast sequence showed no similarity to the fungal sequence.
Roger E. Calza - One of the best experts on this subject based on the ideXlab platform.
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Optimization of protein and cellulase secretion in Neocallimastix frontalis EB188
Applied Microbiology and Biotechnology, 1993Co-Authors: Kwang Pin Tsai, Roger E. CalzaAbstract:Variations in culture feeding protocols were used to optimize the secretion of protein and cellulase in Neocallimastix frontalis EB188. High numbers (2000/ml) of zoospores, culture feeding at 55 h using a 1:3 dilution and cotton cellulose [0.25% (w/v) final] as the carbon source increased secretion. Endoglucanase reached 1.6±0.06 IU/ml, exoglucanase reached 0.032±0.006 IU/ml and β-glucosidase reached 0.874±0.090 IU/ml. Medium containing twice the concentration of non-carbon-source components failed to increase secretions. Gel electrophoresis demonstrated that eleven cellulases were present. Two cellulases were secreted only in stationary cultures. rotein and cellulase secretion in N. frontalis EB188 may be dependent on the dilution of fermentation products.
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Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188.
Biochemical Journal, 1991Co-Authors: James V. O'fallon, Raymond W. Wright, Roger E. CalzaAbstract:Primary pathways for glucose metabolism were established in the anaerobic rumen fungus Neocallimastix frontalis EB188. This highly capable cellulolytic organism demonstrated a strict anaerobic integration of metabolic pathways. Glycolysis in N. frontalis EB188 was coupled to malate dehydrogenase, 'malic' enzyme and specified hydrogenosome reactions. Pyruvate, as in most life forms, was a pivotal compound. The major fermentation products of N. frontalis EB188 were acetate, ethanol and lactate, with the concomitant generation of H2. On the basis of its unique characteristics and streamlined fermentation pathways, it was concluded that N. frontalis EB188 should be an important contributor to programs generating energy and selected chemicals from currently intractable biomass.
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Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values
Applied Microbiology and Biotechnology, 1991Co-Authors: Roger E. CalzaAbstract:Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastix frontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel Rf values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases.
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Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima, protease and ion sensitivities
Applied Microbiology and Biotechnology, 1991Co-Authors: Roger E. CalzaAbstract:The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono-δ-lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to β-endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases.
Alan G. Williams - One of the best experts on this subject based on the ideXlab platform.
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The effects of differing concentrations of CO2 and O2 on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix frontalis L2
Canadian Journal of Microbiology, 1998Co-Authors: Elizabeth M. R. Rees, David Lloyd, Alan G. WilliamsAbstract:The effects of decreasing the concentration of CO2 and introducing up to 10% O2 into the headspace gases on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix frontalis L2 were investigated. The relative proportion of metabolites produced by both fungi depended on the concentration of CO2 in the headspace. Under lowered CO2 levels, both fungi produced increased acetate, lactate, and H2, whereas the production of ethanol, formate, and (in the case of N. frontalis L2) succinate decreased. Lowered CO2 concentrations also decreased the rate of glucose utilization and cumulative gas production by both fungal isolates. In addition, decreased CO2 levels resulted in decreases in NAD(P)H ferredoxin oxidoreductase and hydrogenase activities, whereas malate dehydrogenase and pyruvate ferredoxin oxidoreductase activities were increased. Both N. patriciarum and N. frontalis L2 required at least 7% CO2 in the gas phase for growth. Both isolates also showed a degree of aerotolerance as they grew when exposed to 5% O2; they also grew in media lacking a reducing agent, providing that O2 was initially <1% of the total headspace concentration.Key words: rumen fungi, Neocallimastix, metabolism, carbon dioxide, oxygen.
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The effects of differing concentrations of CO2 and O2 on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix frontalis L2
Canadian Journal of Microbiology, 1998Co-Authors: Elizabeth M. R. Rees, David Lloyd, Alan G. WilliamsAbstract:The effects of decreasing the concentration of CO2 and introducing up to 10% O2 into the headspace gases on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix frontalis L2 were investigated. The relative proportion of metabolites produced by both fungi depended on the concentration of CO2 in the headspace. Under lowered CO2 levels, both fungi produced increased acetate, lactate, and H2, whereas the production of ethanol, formate, and (in the case of N. frontalis L2) succinate decreased. Lowered CO2 concentrations also decreased the rate of glucose utilization and cumulative gas production by both fungal isolates. In addition, decreased CO2 levels resulted in decreases in NAD(P)H ferredoxin oxidoreductase and hydrogenase activities, whereas malate dehydrogenase and pyruvate ferredoxin oxidoreductase activities were increased. Both N. patriciarum and N. frontalis L2 required at least 7% CO2 in the gas phase for growth. Both isolates also showed a degree of aerotoler...
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The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2
FEMS microbiology letters, 1995Co-Authors: Elizabeth M. R. Rees, David Lloyd, Alan G. WilliamsAbstract:The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H2 and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.
