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At least 19 recordsLinked to original sources

PubMed1991

Degradation of wheat straw and alkaline hydrogen peroxide-treated wheat straw by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1.

Degradation of wheat straw (WS) and alkaline hydrogen peroxide (AHP)-treated wheat straw (AHPWS) by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1 was determined by measuring the growth (OD600) of each bacterium and determining DM disappearance (DMD) of the substrate. Complex medium and defined medium with or without the addition of phenylpropanoic acid (PPA) and phenylacetic acid (PAA) were used. Tubes were incubated at 39 degrees C for 8 d. Both OD600 and DMD indicated that AHPWS was degraded to a much greater extent by either bacterium (R. flavefaciens FD-1, 60.8 +/- 1.8% and R. albus 8, 42.3 +/- 3.5%) vs untreated WS (R. flavefaciens FD-1, 16.5 +/- 1.8% and R. albus 8, 8.6 +/- 6%) in the complex medium. Most degradation occurred between d 1 and 4. With the complex medium, addition of PPA and PAA did not stimulate degradation by either bacterium. When the defined medium was used, the addition of PPA and PAA enhanced (P less than .05) degradation of AHPWS (39.6 +/- 2.6%) vs AHPWS with no added PPA and PAA (24.9 +/- 7.6%) by R. albus 8. There was no synergistic effect on degradation when the two species were co-cultured with either WS or AHPWS as the substrate. No effect of PPA and PAA on disappearance of AHPWS was observed for R. flavefaciens FD-1 or when the two bacteria were grown together. Dry matter disappearance analysis showed that R. flavefaciens FD-1 degraded AHPWS more rapidly (6.1 mg/d) than R. albus 8 did (4.2 mg/d) in complex medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1977

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.

Acetates↗

PubMed1991

Sequence of a cellulase gene from the rumen anaerobe Ruminococcus flavefaciens 17.

A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.

Amino Acid Sequence↗

PubMed1978

Adhesion of Bacteroides succinogenes in pure culture and in the presence of Ruminococcus flavefaciens to cell walls in leaves of perennial ryegrass (Lolium perenne).

Bacteroides succinogenes and Ruminococcus flavefaciens are two of the most important cellulolytic bacteria in the rumen. Adhesion of B. succinogenes in pure culture, and in mixed culture with R. flavefaciens, to the various types of cell walls in sections of perennial ryegrass (Lolium perenne L. cultivar S24) leaves was examined by transmission and scanning electron microscopy. B. succinogenes adhered to the cut edges of most plant cell walls except those of the meta- and protoxylem. It also adhered, though in much smaller numbers, to the uncut surfaces of mesophyll, epidermal, and phloem cell walls. In mixed culture, both species adhered in significant numbers to the cut edges of most types of plant cell wall, but R. flavefaciens predominated on the epidermis, phloem, and sclerenchyma cell walls. B. succinogenes predominated on the cut edges and on the uncut surfaces of the mesophyll cell walls, and its ability to adhere to uncut surfaces of other cell walls was not affected by the presence of the ruminococcus. Both organisms rapidly digested the epidermal, mesophyll, and phloem cell walls. Zones of digestion were observed around bacteria of both species when attached to the lignified cell walls of the sclerenchyma, but not when attached to the lignified xylem vessels.

Adhesiveness↗

PubMed1990

Nucleotide sequence of the Ruminococcus albus SY3 endoglucanase genes celA and celB.

The complete nucleotide sequences of Ruminococcus albus genes celA and celB coding for endoglucanase A (EGA) and endoglucanase B (EGB), respectively, have been determined. The celA structural gene consists of an open reading frame of 1095 bp. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified EGA. The celB structural gene consists of an open reading frame of 1227 bp; 7 bp upstream of the translational start codon of celB is a typical gram-positive Shine-Dalgarno sequence. The deduced N-terminal region of EGB conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete celB gene, cloned into pUC vectors, caused lethality in Escherichia coli. In contrast, celA cloned in pUC18, under the control of lacZp, directed high-level synthesis of EGA in E. coli JM83. EGA in cell-free extract, purified to near homogeneity by ion-exchange chromatography, had a Mr of 44.5 kDa. Gene deletion and subcloning studies with celA revealed that EGA hydrolysed both CMC and xylan, and did not contain discrete functional domains. EGA and EGB showed considerable homology with each other, in addition to exhibiting similarity with Eg1 (R. albus), EGE (Clostridium thermocellum) and End (Butyrivibrio fibrisolvens).

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1.

The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) from Ruminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CelA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other organisms. Two lysozyme-type active sites were found in the amino-terminal third of the enzyme. In E. coli the cloned Cel A protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposon phoA mutagenesis experiments indicated that CelA is secreted by a mechanism other than a leader peptide.

Amino Acid Sequence↗

PubMed1990

Mode of action and substrate specificity of a purified exo-1,4-beta-D-glucosidase cloned from the cellulolytic bacterium Ruminococcus albus AR67.

A gene encoding exo-1,4-beta-D-glucosidase, from Ruminococcus albus AR67, was cloned in Escherichia coli, restriction mapped, and shown to be expressed from sequences within the insert that function as a promoter in E. coli. The cloned enzyme was located predominantly in the cytoplasm (40%) and attached to insoluble cell components (48%). After purification to homogeneity, the enzyme (Mr = 64,000, monomeric) was specific for substrates with beta-D-glucopyranosyl configuration and was inactive against alpha-glucosides, lactosides and xylosides. Km values of the enzyme decreased with increasing chain length (G2-G5). Glucose was the major product of hydrolysis from cellodextrins. Preference for longer chain cellodextrins is consistent with exo-1,4-beta-D-glucan glucohydrolase mode of action [E.C. 3.2.1.74].

Chromatography, Gel↗

PubMed1992

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation.

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.

DNA, Bacterial↗

PubMed1992

Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC.

Heparin-agarose chromatography was used to isolate a restriction endonuclease (ENase) from the cellulolytic Gram+ anaerobe, Ruminococcus albus 8. The enzyme, Ral8I, was eluted from the column using 230-310 mM Na+. However, the preparation was active only with DNA substrates that were not Dam-methylated. Moreover, the restriction fragment pattern generated from simian virus 40 (SV40) DNA was not consistent with the expected number of Dam-methylation sites. Alignment of the Dam-methylation sites in SV40 DNA indicated that Ral8I may actually recognize the asymmetric sequence, GGATC. This was confirmed by nucleotide (nt) sequence analysis and, further, Ral8I was found to cause cleavage of the DNA approx. 5 nt downstream from the recognition sequence. Ral8I can therefore be classified as a type-IIS restriction endonuclease and is an isoschizomer of AlwI, BinI and BthII.

Animals↗

PubMed1991

Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains.

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

Amino Acid Sequence↗

PubMed1991

Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13.

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in lambda EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5.5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and beta-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.

Animals↗

PubMed1992

A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine-rich sequence.

The nucleotide sequence of the xynA gene of Ruminococcus flavefaciens 17 was determined and found to consist of a 2862bp open reading frame beginning with a TTG start codon. The predicted product, XYLA, consisted of distinct amino-terminal (A) and carboxy terminal (C) domains (248 amino acids, including a putative signal sequence, and 332 amino acids, respectively) linked by a repetitive sequence (B, 374 amino acids) extraordinarily rich in asparagine (45%) and glutamine (26%) residues. Domains A and C were shown to be capable of expressing xylanase activity independently of each other when suitably truncated derivatives of the xynA coding region were expressed as lacZ fusions. The activities associated with the two domains were shown to differ with respect to the average size of hydrolysis products formed from oat-spelt xylan, with domain C releasing relatively more xylose and domain A more xylo-oligosaccharides. The amino acid sequence of domain A of XYLA closely resembled that of the Bacillus pumilus xynA enzyme (45% identical residues). On the other hand domain C showed significant similarity (33% to 40% identical residues) to a different group of bacterial xylanases and exoglucanases exemplified by the Caldocellum saccharolyticum xynA and celB products. The xynA product is, therefore, a bifunctional enzyme having two dissimilar catalytic domains capable of acting on xylan.

Amino Acid Sequence↗

PubMed1977

Tetrahydrofolate and other growth requirements of certain strains of Ruminococcus flavefaciens.

Two strains of Ruminococcus flavefaciens were studied. Each grew in a chemically defined minimal medium containing: minerals; ammonium sulfate as a nitrogen source; amino acids as a nitrogen source, a growth promotant(s) or as both; cellobiose as an energy and carbon source; isobutyric acid, isovaleric acid, carbonic acid, and bicarbonate as additional carbon sources; and biotin, thiamine, and tetrahydrofolic acid as vitamins. Tetrahydrofolic acid (5 ng/ml) served as a replacement for rumen fluid that was required in previous media tested for the growth of these bacteria. The present bacteria differ from many of the ruminococci previously studied in that they do not require either p-amino-benzoic acid or folic acid but do require tetrahydrofolic acid for maximum growth. Dihydrofolic acid and 5-methyltetrahydrofolic acid can substitute for tetrahydrofolic acid in minimal chemically defined medium. Thus, there must be extensive metabolic interaction between the microbes inhabitating the rumen, because the R. flavefaciens isolated had complex requirements for growth and yet was among the predominant bacteria in the rumen of cattle fed a simple vitamin B-deficient, nonprotein nitrogen, high-fiber, purified diet.

Acetates↗

PubMed1991

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens.

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucan 1,3-beta-Glucosidase↗

PubMed1991

Cellobiose uptake and metabolism by Ruminococcus flavefaciens.

The cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 utilizes cellobiose but not glucose as a substrate for growth. Cellobiose uptake by R. flavefaciens FD-1 was measured under anaerobic conditions (N2), using [G-3H]cellobiose. The rate of cellobiose uptake for early- or late-log-phase cellobiose-grown cells was 9 nmol/min per mg of whole-cell protein. Cellobiose uptake was inhibited by electron transport inhibitors, iron-reactive compounds, proton ionophores, sulfhydryl inhibitors, N,N-dicyclohexylcarbodiimide, and NaF, as well as lasalocid and monensin. The results support the existence of an active transport system for cellobiose. Transport of [U-14C]glucose was not detected with this system. Phosphorylation of cellobiose was not by a phosphoenolpyruvate-dependent system. Cellobiose phosphorylase activity was detected by both a coupled spectrophotometric assay and a discontinuous assay. The enzyme was produced constitutively in cellobiose-grown cells at a specific activity of 329 nmol/min per mg of cell-free extract protein.

Antimetabolites↗

PubMed1992

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.

Adenine↗

PubMed1992

Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.

Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).

Animals↗

PubMed1992

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.

Bacteriological Techniques↗