Characterization of the Lipids of Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens strain D-1 was grown on a lipid-free chemically defined medium. The lipids were extracted with chloroform-methanol and separated into nonpolar and polar fractions by silicic acid column chromatography. Further separations were made by preparative thin-layer chromatography. The lipid fractions were identified by specific staining reactions and R(F) values, by phosphorus and nitrogen determinations, by chromatography of hydrolysis products, and by the use of infrared spectroscopy. The major nonpolar lipid was free fatty acid. Four major polar lipids were identified: phosphatidylethanolamine, phosphatidyl glycerol, lipoaminoacid, and glycolipid. The lipoaminoacid contained alanine, leucine, and isoleucine. The glycolipid contained galactose. The major fatty acids identified were C16:0 and C18:1. The significance of the presence of lipoaminoacid is discussed.     

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens.

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of Butyrivibrio fibrisolvens: a gram-positive bacterium.

The cells of bacteria of the genus Butyrivibrio are universally described as being gram negative, and they produce an unequivocal gram-negative reaction in the standard staining procedure. However, their cell walls contain derivatives of teichoic acid, which are characteristic of gram-positive cells. In this study, the cell walls of two representative strains of Butyrivibrio were of the gram-positive morphological type, as seen by electron microscopy, but they were very thin (12 to 18 nm). The thinness of these cell walls may account for the tendency of these cells to stain gram negatively in the standard staining procedure. Ruthenium red staining revealed an extracellular structure surrounding cells of Butyrivibio sp. (strain C3). This structure was composed of individual "knobs" that sometimes mediated cell-to-cell adhesion in the culture.     

Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens.

A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains.     

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens

A method of obtaining clones of Tetrahymena pyriformis on solid medium has been developed. The medium consists of a basal layer of 1.5% agar topped with 2 ml of 0.3% agar in sterile, plastic petri plates (100 by 15 mm). Both agar layers contain either 2% proteose peptone and 0.1% liver extract (complex medium) or defined medium supplemented with proteose peptone. After drying, 0.5 ml of liquid culture is spread evenly over the top agar, and the plates are then sprinkled lightly and evenly with autoclaved dry Sephadex G-25 (fine). Cell colonies can be observed after 5 days of incubation either by viewing with a microscope or without the aid of a microscope after staining. Plating efficiency is high on either complex or defined medium with a number of strains of Tetrahymena, both micronucleate and amicronucleate. Colonies can be picked and transferred to liquid culture for further growth. The existence of clones was demonstrated by plating a mixture of two different drug-resistant mutants. The method should prove useful in selective procedures for the isolation of mutants and for determining survival after treatments such as ultraviolet irradiation.     

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a beta-glucosidase

The cloning, expression and nucleotide sequence of a 3.74 kb DNA segment on pLS215 containing a beta-glucosidase gene (bglA) from Butyrivibrio fibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a beta-glucosidase of 830 amino acid residues with a calculated Mr of 91,800. In Escherichia coli C600(pLS215) cells the beta-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent Mr of approximately 94,000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the beta-glucosidase showed greater than 40% similarity with a domain of 237 amino acids present in the beta-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens beta-glucosidase hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.     

Enzymology of butyrate formation by Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens is a major butyrate-forming species in the bovine and ovine rumen. The enzymology of butyrate formation from pyruvate was investigated in cell-free extracts of B. fibrisolvens D1. Pyruvate owas oxidized to acetylcoenzyme A (CoA) in the presence of CoA.SH and benzyl viologen or flavin nucleotides. The bacterium uses thiolase, beta-hydroxybutyryl-CoA dehydrogenase, crotonase, and crotonyl-CoA reductase to form butyryl-CoA from acetyl-CoA. Reduction of acetoacetyl-CoA to beta-hydroxybutyryl-CoA was faster with NADH than with NADPH. Crotonyl-CoA was reduced to butyryl-CoA by NADH, but not by NADPH, only in the presence of flavin nucleotides. Reduction of flavin nucleotides by NADH was much slower than the flavin-dependent reduction of crotonyl-CoA. This indicates that flavoproteins rather than free flavin participated in the reduction of crotonyl-CoA. Butyryl-CoA was converted to butyrate by phosphate butyryl transferase and butyrate kinase.     

Purification and Characterization of an alpha-l-Arabinofuranosidase from Butyrivibrio fibrisolvens GS113

An alpha-l-arabinofuranosidase (EC 3.2.1.55) was purified from the cytoplasm of Butyrivibrio fibrisolvens GS113. The native enzyme had an apparent molecular mass of 240 kDa and was composed of eight polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.0, a pH optimum of 6.0 to 6.5, a pH stability of 4.0 to 8.0, and a temperature optimum of 45 degrees C and was stable to 55 degrees C. The K(m) and V(max) for p-nitrophenyl-alpha-l-arabinofuranoside were 0.7 mM and 109 mumol/min/mg of protein, respectively. The enzyme was specific for the furanoside configuration and also readily cleaved methylumbelliferyl-alpha-l-arabinofuranoside but had no activity on a variety of other nitrophenyl- or methylumbelliferyl glycosides. When the enzyme was incubated with cellulose, carboxymethyl cellulose, or arabinogalactan, no release of sugars was found. Arabinose was found as the hydrolysis product of oatspelt xylan, corn endosperm xylan, or beet arabinan. No activity was detected when either coumaric or ferulic acid ester linked to arabinoxylobiose was used as substrates, but arabinoxylobiose was degraded to arabinose and xylobiose. Since B. fibrisolvens GS113 possesses essentially no extracellular arabinofuranosidase activity, the major role of the purified enzyme is apparently in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.     

Cloning, sequencing and expression of a gene encoding a 73 kDa xylanase enzyme from the rumen anaerobe Butyrivibrio fibrisolvens H17c

Summary The cloning, expression and nucleotide sequence of a 3 kb DNA segment on pLS206 containing a xylanase gene (xynB) from Butyrivibrio fibrisolvens H17c was investigated. The open reading frame (ORF) of 1905 by encoded a xylanase of 635 amino acid residues (M_r 73156). At least 850 by at the 3 end of the gene could be deleted without loss of xylanase activity. The deduced amino acid sequence was confirmed by purifying the enzyme and subjecting it to N-terminal amino acid sequence analysis. In Escherichia coli C600 (pLS206) cells the xylanase was localized in the cytoplasm. Its optimum pH for activity was between pH 5.4 and 6, and optimum temperature 55° C. The primary structure of the xylanase showed a significant level of identity with a cellobiohydrolase/endoglucanase of Caldocellum saccharolyticum, as well as with the xylanases of the alkaliphilic Bacillus sp. strain C-125, B. fibrisolvens strain 49, and Pseudomonas fluorescens subsp. cellulosa.Abbreviations ORF open reading frame - pNPCase p-nitrophen-yl--d-cellobiosidase - (xynB) gene coding for XynB - XynB xylanase     

Isolation and Characterization of Xylan-Degrading Strains of Butyrivibrio fibrisolvens from a Napier Grass-Fed Anaerobic Digester

Six new xylanolytic bacterial strains have been isolated from a Napier grass-fed anaerobic digester. These strains were identified as Butyrivibrio fibrisolvens and were similar in many respects to ruminal isolates described previously. The new isolates exhibited a high degree of DNA homology with several ruminal strains of B. fibrisolvens. Xylan or xylose was required to induce the production of enzymes for xylan degradation, xylanase and xylosidase. Production of these enzymes was repressed in the presence of glucose. Xylanase activity was predominantly extracellular, while that of xylosidases was cell associated. The new isolates of B. fibrisolvens grew well in defined medium containing xylan as the sole carbon source and did not produce obvious slime or capsular layers. These strains may be useful for future genetic investigations.     

Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities.

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.     

Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis

We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.     

Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.     

The Butyrivibrio fibrisolvens tet(W) gene is carried on the novel conjugative transposon TnB1230, which contains duplicated nitroreductase coding sequences

The Butyrivibrio fibrisolvens tet(W) gene is located on the conjugative transposon TnB1230. TnB1230 encodes transfer proteins with 48 to 67% identity to some of those encoded by Tn1549. tet(W) is flanked by directly repeated sequences with significant homology to oxygen-insensitive nitroreductases. The 340 nucleotides upstream of tet(W) are strongly conserved and are required for tetracycline resistance.