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PubMed2001

Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide-lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85.

The polysaccharides from the outer membrane of the Gram-negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size-exclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated 'glycolipid' and a high-molecular-mass 'capsular polysaccharide' fraction. Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures: : : where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except L-rhamnose had a D-configuration. In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.

Animals↗
PubMed2001

Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy.

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-(13)C]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 < or = 4. Both labelled (1-(13)C and 6-(13)C) 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.

Animals↗
PubMed1997

Production of a cell wall-specific monoclonal antibody to Fibrobacter succinogenes.

A monoclonal antibody (mAb) was raised against Fibrobacter succinogenes and produced after fusion as ascites in BALB/c mice. An ELISA was used to test for specificity and sensitivity of the mAb to detect F. succinogenes. The mAb BD1 was tested for sensitivity and cross-reactivity in detecting F. succinogenes with ELISA. The lower limits for F. succinogenes detection in pure and mixed culture-using mAb BD1 with ELISA was 10(5) cells ml-1. Twenty-six other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA but none was detected. Electron micrographs of F. succinogenes cells with immunogold labelling showed that the mAb BD1 reacted exclusively with cell wall epitopes but not intracellular material, as confirmed by ELISA.

Animals↗
PubMed2001

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability.

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.

Amino Acid Sequence↗
PubMed1991

The involvement of transcriptional read-through from internal promoters in the expression of a novel endoglucanase gene FSendA, from Fibrobacter succinogenes AR1.

Two distinct mRNA transcripts were synthesized in Escherichia coli during expression of FSendA, an endoglucanase gene from Fibrobacter succinogenes AR1. Expression of FSendA required a ribosomal frameshift between open reading frame 1 (ORF1) and ORF2 to allow contiguous translation of a 453 amino acid protein (1). The primary transcript initiated upstream of ORF1 and the secondary transcript from within ORF1. Both transcripts terminated downstream of ORF2 and termination was essential for endoglucanase expression. Deletion of the primary transcript promoter region allowed read-through of the secondary transcript beyond the terminator region, indicating that a component of the intact FSendA gene allowed efficient transcription termination. The possibility of autogenous regulation by translation products is suggested.

Amino Acid Sequence↗
PubMed2000

Characterization of function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85.

Xylanase C from the ruminant bacterium Fibrobacter succinogenes is comprised of two catalytic domains, A and B, and a third domain, C, of unknown function. The DNA coding for domains A and B of xylanase C were separately cloned and expressed in Escherichia coli as fusion proteins with glutathione-S:-transferase. The fusion proteins were isolated by affinity chromatography on glutathione-Sepharose 4B, cleaved with thrombin and the released xylanase C catalytic domains A and B were purified to apparent homogeneity by anion-exchange chromatography on Mono Q. Electrospray mass spectrometry provided a molecular mass of 27 818 Da (expected, 27 820 Da) for domain B. The pH and temperature optima for activity of domain B on oat spelt xylan were 5.0 and 52 degrees C, respectively. A kinetic analysis of the activity of the catalytic domain A on oat spelt xylan, birch wood xylan and xylooligomers at pH 6.5 and 37 degrees C provided data significantly different to those obtained previously with a protease-derived form of the enzyme [Zhu et al. (1994) J. Bacteriol. 176, 3885-3894]. The isolated domain A was more active on barley-glucan than the protease-derived form and its affinity for birch wood xylan was enhanced resulting in greater overall catalytic efficiency as reflected by k(cat)/K:(M) values. Likewise, significant differences in the Michaelis-Menten parameters K:(M), k(cat) and k(cat)/K:(M) were obtained with domain B compared with values previously reported with this domain attached to domain C. In general, the presence of domain C appeared to decrease the overall efficiency of domain B 7- and 36-fold with birch wood xylan and xylopentaose as substrates, respectively, as reflected by values of k(cat)/K:(M). The removal of domain C also affected the mode of action of domain B such that it more closely resembled that of catalytic domain A. However, no change in either pH and temperature optima or stability were found with domain B compared with the combined domains B and C. The function of domain C remains unknown, but hydrophobic cluster analysis indicated that it may belong to a class of dockerin domains involved in the protein-protein interactions of cellulolytic and xylanolytic complexes.

Amino Acid Sequence↗
PubMed1993

Analysis of bacterial phospholipid markers and plant monosaccharides during forage degradation by Ruminococcus flavefaciens and Fibrobacter succinogenes in co-culture.

Marker components of the phospholipids of Ruminococcus flavefaciens and Fibrobacter succinogenes were identified for studies on the degradation of forage by these bacteria growing in mixed culture. The principal fatty acid methyl esters and dimethyl acetals detected varied between strains and were influenced by the addition of a mixture of higher volatile fatty acids and vitamins to the medium, but these effects were small compared to the differences between the species. When two strains of R. flavefaciens were grown on a mixture of clover and ryegrass, and on barley straw in the presence or absence of two strains of F. succinogenes, the solubilization of plant material tended to be lowered by the presence of F. succinogenes. R. flavefaciens was the predominant bacterium among colonies recovered from roll tubes, and the phospholipids were primarily those of R. flavefaciens. Analysis of the culture supernatant liquids showed that F. succinogenes produced greater amounts of free and bound xylose from both clover and straw than did R. flavefaciens. With both forages, cultures containing the two species produced more soluble free arabinose, and less soluble-bound arabinose, than either species grown alone.

Animal Feed↗
PubMed2001

Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo.

DNase A is a non-specific endonuclease of Fibrobacter succinogenes. The enzyme was purified to homogeneity and its properties studied both in vitro and in vivo. Magnesium but not calcium was essential for nucleolytic activity. Manganese ions substituted for magnesium but were less stimulatory. DNase A activity was markedly inhibited by either NaCl or KCl at concentrations greater than 75 mM. The enzyme had a temperature optimum of 25 degrees C and a pH optimum of about 7.0. Values for K:(m) and K:(cat) were determined to be 61 microM and 330 s(-1) respectively, with a catalytic efficiency approximately threefold greater than bovine pancreatic DNase I, but 10-fold less than the Serratia marcescens NucA. DNase A was localized to the periplasm and probably exists as a monomeric species. The enzyme possessed one or more disulfide bonds. In the reduced form it had an apparent mass of 33 kDa, while in the oxidized form it was 29 kDa as estimated by SDS-PAGE. Reduction of the disulfide bonds by dithiothreitol with or without subsequent alkylation by iodoacetamide strongly inactivated the enzyme. DNase A accumulated in vivo had an apparent mass of 29 kDa, indicating that it was in an oxidized form. This is the first indication in a strict anaerobe of a functional periplasmic disulfide bond forming system, phenotypically similar to Dsb systems in facultative and aerobic bacteria.

Animals↗
PubMed2001

The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga-Flavobacterium-Bacteroides division.

The phylogenetic placement of the rumen bacterium Fibrobacter succinogenes was determined using a signature sequence approach that allows determination of the relative branching order of the major divisions among Bacteria [Gupta, R. S. (2000) FEMS Microbiol Rev 24, 367-402]. For this purpose, segments of the Hsp60 (groEL), Hsp70 (dnaK), CTP synthase and alanyl-tRNA synthetase genes, which are known to contain signature sequences that are useful for phylogenetic deterministic purposes, were cloned. Using degenerate oligonucleotide primers for highly conserved regions in these proteins, 1.4 kb, 0.75 kb, 401 bp and 171 bp fragments of the Hsp70, Hsp60, CTP synthase and alanyl-tRNA synthetase genes respectively were amplified by PCR, and these fragments were cloned and sequenced. These primers, because of their high degree of conservation, could also be used for cloning these genes from other bacterial species. The Hsp70 homologues from different Gram-negative bacteria contain a 21-23 aa insert that is not found in any Gram-positive bacteria. The presence of this insert in the F. succinogenes Hsp70 supports its placement within the Gram-negative group of bacteria. A conserved insert in F. succinogenes Hsp60 that is commonly present in all bacterial species, except various Gram-positive bacteria, Deinococcus-Thermus groups and green non-sulphur bacteria, provides evidence that F. succinogenes does not belong to these taxa. A particularly useful signature consisting of a 4 aa insert is found in Ala-tRNA synthetase. This insert is present in all proteobacterial homologues as well as in homologues from species belonging to the Chlamydia and Cytophaga-Flavobacterium- Bacteroides (CFB) groups, but it is not found in homologues from any other groups of bacteria. The presence of this insert in F. succinogenes Ala-tRNA synthetase provides evidence that this species is related to these groups. However, two other signatures in CTP synthase and Hsp70 proteins, that are distinctive of the proteobacterial species, are not present in the F. succinogenes homologues. These results provide evidence that F. succinogenes does not belong to the proteobacterial division and thus should be placed in a similar position as the Chlamydia and CFB groups of species.

Alanine-tRNA Ligase↗
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of the 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes.

The truncated 1,3-1,4-beta-glucanase (1,3-1,4-beta-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 P2(1), 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 P2(1)2(1)2(1) 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 Ca(2+) ions. It is likely that Ca(2+) 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.

Bacteria↗
PubMed1992

Two beta-glucosidase activities in Fibrobacter succinogenes S85.

Few bacteria are capable of degrading crystalline cellulose but there is considerable interest in the properties of enzyme systems with this capability. In the bovine and ovine rumen the principal cellulolytic bacterium is Fibrobacter (formerly Bacteroides) succinogenes. The cellulase system of this organism is composed of multiple enzyme components, including a constitutive and cell-associated beta-glucosidase active against cellobiose. The properties of the beta-glucosidase activity have been investigated with the chromogenic substrate p-nitrophenyl beta-D-glucoside (pNPG). Hydrolytic activity against pNPG was located primarily in the cytoplasm and the cytoplasmic membrane but showed a gradual migration to the periplasm during growth on either glucose or cellobiose. Activity against cellobiose was found in the periplasm in significant amounts in all growth phases. Of the beta-glucosides tested, only cellobiose and pNPG were hydrolysed by crude cell extracts. In the presence of cellobiose, however, the rate of hydrolysis of pNPG was stimulated up to 10-fold, and extracts hydrolysed methylumbelliferyl beta-D-glucoside, 5-bromo-4-chloro-3-indolyl beta-D-glucoside, arbutin and aesculin. Activities against pNPG in the presence and absence of cellobiose displayed similar instability in the presence of oxygen; both were stabilized by dithiothreitol and the temperature and pH optima were identical. A significant proportion of the membrane-associated beta-glucosidase was released by treatment with 0.3 mol/1 KCl, and fractionation by chromatography on CM-cellulose showed the presence of two activities against pNPG, only one of which was stimulated by cellobiose.

Cellobiose↗
PubMed1992

Futile cycling of glycogen in Fibrobacter succinogenes as shown by in situ 1H-NMR and 13C-NMR investigation.

Glycogen was synthesized during all the growth phases in the rumen anaerobic cellulolytic bacterium Fibrobacter succinogenes. Glycogen synthesis and degradation were monitored using in situ 13C and 1H-NMR spectroscopy in resting cells of F. succinogenes. The cells were incubated at 37 degrees C under anaerobic conditions with [1-13C]glucose and [2-13C]glucose. 1H-NMR spectra were used to quantify enrichment by 13C of metabolism products. Glucose was utilized for energy requirements of the bacterium, essentially via the Embden-Meyerhof pathway, leading to the synthesis of succinate and acetate, while glycogen was stored. From [1-13C]glucose, labeling occurred on C2 of succinate and acetate, and on both C1 and C6 of glycogen, the labeling on C1 being predominant. The C6-labeling of glycogen may be explained by scrambling and reversal of the glycolytic pathway at the triose-phosphate and fructose 1,6-bisphosphate level. When the bacteria were incubated first with [1-13C]glucose, then washed and incubated with [2-13C]glucose, the pattern of 13C labeling in the products of the metabolism, as shown by 13C and 1H-NMR spectra, indicated that glycogen was degraded at the same time as it was being stored, suggesting futile cycling of glycogen. The hydrolysis of previously stored glycogen can provide, in the presence of glucose, up to 30% of the carbon source for the bacteria.

Bacterial Proteins↗
PubMed1996

Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product.

A gene encoding an enzyme which degrades cellulose (end-1) was isolated from a library of Fibrobacter succinogenes SD35 DNA fragments and expressed in pUC18. The product of end-1 showed significant activity against carboxymethylcellulose but relatively minor activity against lichenan, xylan and avicel. The nucleotide sequence indicated a product of 388 amino acids with a molecular mass of 50.2 kDa. This was in agreement with the molecular size estimated by gel electrophoresis. No significant DNA sequence similarity was identified with any published endoglucanase.

Amino Acid Sequence↗
PubMed1994

Gene sequence and analysis of protein domains of EGB, a novel family E endoglucanase from Fibrobacter succinogenes S85.

The endoglucanase gene (endB) of Fibrobacter succinogenes S85 encodes a protein of 555 amino acids (EGB) with a M(r) of 62,500. EGB shows homology with cellulases belonging to family E. Residues involved in the catalytic activity of CelD from Clostridium thermocellum are also found in EGB. Structure predictions suggest that EGB, like CelD, comprises a large alpha-helical catalytic domain plus a beta-strand domain of unknown function located in the N-terminal part of the protein. Construction of a phylogenetic tree of family E catalytic domains revealed that EGB is closest to a cellodextrinase from Butyrivibrio fibrisolvens.

Amino Acid Sequence↗
PubMed1996

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein 1 (CBP1) of Fibrobacter succinogenes S85.

The nucleotide sequence of the gene encoding the Fibrobacter succinogenes S85 cellulose-binding protein 1 (CBP1) has been determined. The gene encodes a protein of 1054 amino acids with a molecular mass of 118614. The deduced amino acid sequence of CBP1 showed an extensive similarity to the cellulose-binding domain of an endoglucanase (EGCCD) from Clostridium cellulolyticum and contained the reiterated regions. The cloned gene was inserted into an expression vector, pRSETA, and was expressed in E. coli as a fused protein with the peptide consisting of six consecutive histidine residues. The fused protein was detected by immunoblotting using antiserum against CBP1, and exhibited the cellulose-binding activities.

Amino Acid Sequence↗
PubMed2000

Identification of the cellulose-binding domain of Fibrobacter succinogenes endoglucanase F.

The cellulose-binding domain (CBD) of Fibrobacter succinogenes endoglucanase F (EGF) has been determined. The gene encoding EGF (celF) and its derivatives were expressed in Escherichia coli. We were able to obtain eight recombinant proteins and examine their cellulose-binding ability and endoglucanase activity. Because four recombinant proteins, which contain the first N-terminal reiterated region of EGF, bound to cellulose, the region has been identified as the CBD. Although the CBD did not show significant sequence similarity with any other CBDs, it did show significant similarity with a part of endoglucanase J (CelJ) of Clostridium thermocellum F1. Moreover, a large part of the C-terminal catalytic region of EGF showed sequence similarity with alpha-L-arabinofuranosidases of glycosyl hydrolase family 51.

Amino Acid Sequence↗
PubMed2001

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens.

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.

Animals↗
PubMed2002

Properties of cellulose-binding modules in endoglucanase F from Fibrobacter succinogenes S85 by means of surface plasmon resonance.

Properties of the recombinant proteins derived from Fibrobacter succinogenes endoglucanase F (EGF), AD2 and AD4, were characterized using surface plasmon resonance. Because AD2, which contains two reiterated regions, showed stronger affinity to immobilized carboxymethylcellulose (CMC) than did AD4, which contains only the first reiterated region, it has been assumed that the reiterated regions of EGF are cellulose-binding modules. While calcium enhanced the binding of AD2 to the immobilized CMC, it did not enhance the binding of AD4. Moreover, the results obtained from experiments using cellooligosaccharides showed that the binding sites of AD4 and AD2 span approximately four and nine glucosyl units, respectively.

Bacteria↗