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Evelyne Forano - One of the best experts on this subject based on the ideXlab platform.

  • In Vivo Competitions between Fibrobacter Succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses.
    mBio, 2021
    Co-Authors: Carl J. Yeoman, Evelyne Forano, Christopher J. Fields, Pascale Lepercq, Philippe Ruiz, Bryan A. White, Pascale Mosoni
    Abstract:

    ABSTRACT Fibrobacter Succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. Succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. Succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. Succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. Succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response. IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter Succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. Succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.

  • Production of oligosaccharides and cellobionic acid by Fibrobacter Succinogenes S85 growing on sugars, cellulose and wheat straw
    Applied Microbiology and Biotechnology, 2009
    Co-Authors: Régis Nouaille, Maria Matulova, Vladimír Pätoprstý, Anne-marie Delort, Evelyne Forano
    Abstract:

    Extracellular culture fluid of Fibrobacter Succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1 P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. Succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.

  • Carbohydrate metabolism in Fibrobacter Succinogenes: What NMR tells us
    Microbial Ecology in Health & Disease, 2008
    Co-Authors: Evelyne Forano, Anne-marie Delort, Maria Matulova
    Abstract:

    Fibrobacter Succinogenes is a major rumen fibrolytic bacterium found in high numbers when ruminants are fed cellulose-rich diets. It produces a very efficient fibrolytic system comprising numerous enzymes, but the organization and catalytic features of this system remain unclear. We were using nuclear magnetic resonance (NMR) to study carbohydrate metabolism by Fibrobacter spp. well before the term ‘metabolomics’ was coined. We first analyzed in detail the NMR spectra arising from resting cells of F. Succinogenes provided with glucose, cellobiose or cellulose as a substrate. We were able to show carbohydrate cycling and reversibility of some steps. The use of 1D and 2D 1 H and 13 C NMR showed the synthesis by the cells of unexpected oligosaccharides: maltodextrins, maltodextrin-1-phosphate, and other glucose derivatives. We also showed that maltodextrins and maltodextrins-1-phosphate were synthesized and excreted out of the cell, and could originate from exogenous sugars or endogenous glycogen. According to the substrate and the physiological state of the bacterium (resting or growing cells), metabolic deviations toward the synthesis of these oligosaccharides were observed. We have also used liquid- and solid-state NMR to monitor wheat straw degradation by F. Succinogenes . The originality of this NMR approach was to investigate the activity of an entire fibrolytic system on an intact complex substrate (wheat straw). No preferential degradation of amorphous versus crystalline cellulose was observed, nor of cellulose versus hemicelluloses. We showed for the first time a sequential activity of some enzymes of F. Succinogenes S85 on wheat straw. In conclusion, our studies illustrate the utility of various NMR approaches to study and better understand sugar and polysaccharide metabolism in rumen bacteria. Key words: ruminants, NMR, Fibrobacter Succinogenes, polysaccharide metabolism, cellulose

  • Oligosaccharide synthesis in Fibrobacter Succinogenes S85 and its modulation by the substrate
    FEBS Journal, 2005
    Co-Authors: Régis Nouaille, Maria Matulova, Anne-marie Delort, Evelyne Forano
    Abstract:

    In this article we compared the metabolism of phosphorylated and unphosphorylated oligosaccharides (cellodextrins and maltodextrins) in Fibrobacter Succinogenes S85 resting cells incubated with the following substrates: glucose; cellobiose; a mixture of glucose and cellobiose; and cellulose. Intracellular and extracellular media were analysed by 1H-NMR and by TLC. The first important finding is that no cellodextrins were found to accumulate in the extracellular media of cells, regardless of the substrate; this contrasts to what is generally reported in the literature. The second finding of this work is that maltodextrins of degree of polymerization > 2 are synthesized regardless of the substrate, and can be used by the bacteria. Maltotriose plays a key role in this metabolism of maltodextrin. Maltodextrin-1-phosphate was detected in all the incubations, and a new metabolite, corresponding to a phosphorylated glucose derivative, was produced in the extracellular medium when cells were incubated with cellulose. The accumulation of these phosphorylated sugars increased with the degree of polymerization of the substrate

  • 23Na NMR study of Fibrobacter Succinogenes S85: comparison of three chemical shift reagents and calculation of sodium concentration using ionophores.
    Analytical biochemistry, 2002
    Co-Authors: Anne-marie Delort, Genevieve Gaudet, Evelyne Forano
    Abstract:

    Abstract In order to measure intracellular sodium concentrations in resting cells of Fibrobacter Succinogenes S85 by 23Na NMR spectrometry, two methodological aspects were studied. First, three different shift reagents (Dy(PPPi)7−2, Tm(DOTP)5−, and Dy(TTHA)3−) were tested for their ability to separate internal and external 23Na NMR resonances. Their toxicity toward F. Succinogenes cells was evaluated by in vivo13C NMR experiments. Tm(DOTP)5− was found to be the most efficient shift reagent while being nontoxic. Second, a new methodology was developed to calculate intracellular sodium concentration in F. Succinogenes by using ionophores. This approach avoided the problem of intracellular volume measurement and that of sodium visibility determination.

Cecil W. Forsberg - One of the best experts on this subject based on the ideXlab platform.

  • Genomic Differences between Fibrobacter Succinogenes S85 and Fibrobacter intestinalis DR7, Identified by Suppression Subtractive Hybridization
    Applied and environmental microbiology, 2007
    Co-Authors: Karen E. Nelson, Sean C. Daugherty, William C. Nelson, Ioana R. Hance, Mark Morrison, Cecil W. Forsberg
    Abstract:

    Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter Succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. Succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. Succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. Succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. Succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B12 for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.

  • Characterization and Synergistic Interactions of Fibrobacter Succinogenes Glycoside Hydrolases
    Applied and Environmental Microbiology, 2007
    Co-Authors: Meng Qi, Cecil W. Forsberg
    Abstract:

    The objectives of this study were to characterize Fibrobacter Succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. Succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. Succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

  • Outer Membrane Proteins of Fibrobacter Succinogenes with Potential Roles in Adhesion to Cellulose and in Cellulose Digestion
    Journal of bacteriology, 2007
    Co-Authors: Hyun-sik Jun, Emmanuel E. Egbosimba, Joshua Gong, Cecil W. Forsberg
    Abstract:

    Comparative analysis of binding of intact glucose-grown Fibrobacter Succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. Succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. Succinogenes.

  • Fibrobacter Succinogenes, a Dominant Fibrolytic Ruminal Bacterium: Transition to the Post Genomic Era
    Asian-Australasian Journal of Animal Sciences, 2007
    Co-Authors: H. S. Jun, Cecil W. Forsberg
    Abstract:

    Fibrobacter Succinogenes, a Gram-negative, anaerobic ruminal bacterium is a major fibre digesting species in the rumen. It intensively degrades plant cell walls by an erosion type of mechanism, burrowing its way through the complex matrix of cellulose and hemicellulose with the release of digestible and undigested cell wall fragments. The enzymes involved in this process include a combination of glucanases, xylanases, arabinofuranosidase(s) and esterases. The genome of the bacterium has been sequenced and this has revealed in excess of 100 putative glycosyl hydrolase, pectate lyase and carbohydrate esterase genes, which is greater than the numbers reported present in other major cellulolytic organisms for which genomes have been sequenced. Modelling of the amino acid sequences of two glycanases, CedA and EGB, by reference to crystallized homologs has enabled prediction of the major features of their tertiary structures. Two dimensional gel electrophoresis in conjunction with mass spectroscopy has permitted the documentation of proteins over expressed in F. Succinogenes grown on cellulose, and analysis of the cell surfaces of mutant strains unable to bind to cellulose has enabled the identification of candidate proteins with roles in adhesion to the plant cell wall substrate, the precursor to cellulose biodegradation.

  • Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter Succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan
    Canadian journal of microbiology, 2005
    Co-Authors: Dong Keun Kam, Hyun-sik Jun, G. Douglas Inglis, Cecil W. Forsberg
    Abstract:

    Acetylxylan esterase genes axe6A and axe6B located adjacent to one another on a Fibrobacter Succinogenes chromosome have been separately cloned and their properties characterized. The corresponding esterases contained an N-terminal carbohydrate esterase family 6 catalytic domain (CD) and a C-terminal family 6 carbohydrate-binding module (CBM). The amino acid sequences of the CDs and CBMs were found to exhibit 52% and 40% amino acid similarity, respectively. The CDs of the two esterases exhibited the highest similarity to CDs of acetylxylan esterases: AxeA from the ruminal fungi Orpinomyces sp. and BnaA from Neocallimastix patriciarum. Axe6A and Axe6B were optimally active at neutral pH and had low K(m) values of 0.084 and 0.056 mmol x L(-1), respectively. Axe6A and Axe6B were shown to bind to insoluble cellulose and xylan and to soluble arabinoxylan. Axe6A deacetylated acetylated xylan at the same initial rate in the presence and absence of added Xyn10E xylanase from F. Succinogenes, but the action of the xylanase on acetylated xylan was dependent upon the initial activity of Axe6A. The capacity of acetylxylan esterases to bind to plant cell wall polymers and to independently deacetylate xylan enabling xylanase to release xylooligo saccharides, documents the central role these enzymes have to improve access of F. Succinogenes to cellulose.

Shu-hua Lee - One of the best experts on this subject based on the ideXlab platform.

  • mutational and structural studies of the active site residues in truncated Fibrobacter Succinogenes 1 3 1 4 β d glucanase
    Acta Crystallographica Section D-biological Crystallography, 2008
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Hsiaochuan Huang, Chinghua Hsiao, Yuanneng Chiang, Yushiun Lin, Shu-hua Lee
    Abstract:

    1,3–1,4-β-d-Glucanases (EC 3.2.1.73) specifically hydrolyze β-1,4-glycosidic bonds located prior to β-1,3-glycosidic linkages in lichenan or β-d-glucans. It has been suggested that truncated Fibrobacter Succinogenes 1,3–1,4-β-d-glucanase (TFsβ-glucanase) can accommodate five glucose rings in its active site upon enzyme–substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower kcat/Km for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsβ-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.

  • mutational and structural studies of the active site residues in truncated Fibrobacter Succinogenes 1 3 1 4 β d glucanase
    Acta Crystallographica Section D-biological Crystallography, 2008
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Hsiaochuan Huang, Chinghua Hsiao, Yuanneng Chiang, Yushiun Lin, Shu-hua Lee
    Abstract:

    1,3-1,4-beta-D-Glucanases (EC 3.2.1.73) specifically hydrolyze beta-1,4-glycosidic bonds located prior to beta-1,3-glycosidic linkages in lichenan or beta-D-glucans. It has been suggested that truncated Fibrobacter Succinogenes 1,3-1,4-beta-D-glucanase (TFsbeta-glucanase) can accommodate five glucose rings in its active site upon enzyme-substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower k(cat)/K(m) for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsbeta-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.

  • Crystal structure of truncated Fibrobacter Succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose.
    Journal of molecular biology, 2005
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Yi-sheng Cheng, Shu-hua Lee
    Abstract:

    Fibrobacter Succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.

Li-chu Tsai - One of the best experts on this subject based on the ideXlab platform.

  • mutational and structural studies of the active site residues in truncated Fibrobacter Succinogenes 1 3 1 4 β d glucanase
    Acta Crystallographica Section D-biological Crystallography, 2008
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Hsiaochuan Huang, Chinghua Hsiao, Yuanneng Chiang, Yushiun Lin, Shu-hua Lee
    Abstract:

    1,3–1,4-β-d-Glucanases (EC 3.2.1.73) specifically hydrolyze β-1,4-glycosidic bonds located prior to β-1,3-glycosidic linkages in lichenan or β-d-glucans. It has been suggested that truncated Fibrobacter Succinogenes 1,3–1,4-β-d-glucanase (TFsβ-glucanase) can accommodate five glucose rings in its active site upon enzyme–substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower kcat/Km for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsβ-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.

  • mutational and structural studies of the active site residues in truncated Fibrobacter Succinogenes 1 3 1 4 β d glucanase
    Acta Crystallographica Section D-biological Crystallography, 2008
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Hsiaochuan Huang, Chinghua Hsiao, Yuanneng Chiang, Yushiun Lin, Shu-hua Lee
    Abstract:

    1,3-1,4-beta-D-Glucanases (EC 3.2.1.73) specifically hydrolyze beta-1,4-glycosidic bonds located prior to beta-1,3-glycosidic linkages in lichenan or beta-D-glucans. It has been suggested that truncated Fibrobacter Succinogenes 1,3-1,4-beta-D-glucanase (TFsbeta-glucanase) can accommodate five glucose rings in its active site upon enzyme-substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower k(cat)/K(m) for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsbeta-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.

  • Crystal structure of truncated Fibrobacter Succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose.
    Journal of molecular biology, 2005
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Yi-sheng Cheng, Shu-hua Lee
    Abstract:

    Fibrobacter Succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.

  • crystallization and preliminary x ray diffraction analysis of the 1 3 1 4 β d glucanase from Fibrobacter Succinogenes
    Acta Crystallographica Section D-biological Crystallography, 2001
    Co-Authors: Li-chu Tsai, Lie-fen Shyur, Sushiang Lin, Hanna S Yuan
    Abstract:

    The truncated 1,3-1,4-β-glucanase (1,3-1,4-β-d-glucan 4-glucanohydrolase; E.C. 3.2.1.73) from Fibrobacter Succinogenes was crystallized in four different forms by the vapour-diffusion method. Form A crystals have the largest trigonal P321 unit cell, diffracting to 3.0 A resolution with four to six molecules per asymmetric unit. Form B and C crystals belong to the same monoclinic space group P21, but the form B unit cell is twice as large as the unit cell of form C. Form B crystals diffract to 2.5 A resolution and contain four molecules per asymmetric unit. Form C crystals diffract to 2.1 A resolution and contain two molecules per asymmetric unit. Form D crystals have the smallest orthorhombic P212121 unit cell, containing only one molecule per asymmetric unit, and diffract beyond 2.1 A resolution. The crystallization conditions for form B and C crystals are almost identical, except that form C crystals were grown in the presence of 2 mM Ca2+ ions. It is likely that Ca2+ directly binds to the glucanase, leading to unit-cell shrinkage as observed in other Bacillus glucanase crystals. A self-rotation search identified non-crystallographic twofold axes that combine with the crystallographic twofold dyads to give 222 symmetry for both form A and form B crystals, indicating that the glucanase has a tendency to pack in 222 symmetry.

Genevieve Gaudet - One of the best experts on this subject based on the ideXlab platform.

  • 23Na NMR study of Fibrobacter Succinogenes S85: comparison of three chemical shift reagents and calculation of sodium concentration using ionophores.
    Analytical biochemistry, 2002
    Co-Authors: Anne-marie Delort, Genevieve Gaudet, Evelyne Forano
    Abstract:

    Abstract In order to measure intracellular sodium concentrations in resting cells of Fibrobacter Succinogenes S85 by 23Na NMR spectrometry, two methodological aspects were studied. First, three different shift reagents (Dy(PPPi)7−2, Tm(DOTP)5−, and Dy(TTHA)3−) were tested for their ability to separate internal and external 23Na NMR resonances. Their toxicity toward F. Succinogenes cells was evaluated by in vivo13C NMR experiments. Tm(DOTP)5− was found to be the most efficient shift reagent while being nontoxic. Second, a new methodology was developed to calculate intracellular sodium concentration in F. Succinogenes by using ionophores. This approach avoided the problem of intracellular volume measurement and that of sodium visibility determination.

  • In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter Succinogenes S85.
    Applied and environmental microbiology, 2001
    Co-Authors: Véronique Schwaab, Anne-marie Delort, Christelle Matheron, Genevieve Gaudet, Evelyne Forano
    Abstract:

    Sodium gradients (ΔpNa) were measured in resting cells of Fibrobacter Succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5− [thulium(III) 1,4,7,10-tetraazacyclododecane-N′,N′′,N′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ΔpNa of −55 to −40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).

  • Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter Succinogenes S85 as identified by 2D NMR spectroscopy.
    European journal of biochemistry, 2001
    Co-Authors: Maria Matulova, Régis Nouaille, Anne-marie Delort, Genevieve Gaudet, Evelyne Forano
    Abstract:

    1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter Succinogenes S85 when incubated with [1-13C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was ≤ 4. Both labelled (1-13C and 6-13C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. Succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.

  • Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter Succinogenes S85 grown on different substrates.
    Biochimica et biophysica acta, 2000
    Co-Authors: Christel Béra Maillet, Genevieve Gaudet, Evelyne Forano
    Abstract:

    Abstract The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter Succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (β-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. Succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endAFS genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. Succinogenes.

  • Characterisation of endoglucanases EGB and EGC from Fibrobacter Succinogenes
    Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2000
    Co-Authors: Christel Béra Maillet, Genevieve Gaudet, Véronique Broussolle, Peter Pristas, Jean-pierre Girardeau, Evelyne Forano
    Abstract:

    The enzymatic properties of two endoglucanases from Fibrobacter Succinogenes, EGB and EGG, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection in F. Succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K-m and V-max were respectively 13.6 mg/ml and 46 mu mol/min mg protein for EGB, and 7 mg/ml and 110 mu mol/min mg protein for EGG. The reactivity of the antipeptide and the anti-EGG sera with F. Succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. Succinogenes cultures. Expression of endB and endC genes in F. Succinogenes was confirmed by RT-PCR.