Butyrivibrio Fibrisolvens Strain Research Articles

The fructanolytic abilities of the rumen bacterium Butyrivibrio fibrisolvens strain 3071.

To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. Strain 3071 utilized 97, 89, 85 and 60% of sucrose, timothy grass fructan, inulin oligosaccharides and inulin, respectively, in the growth medium. A cell extract from timothy grass fructan-grown bacteria was used for identification of fructanolytic enzymes by anion exchange chromatography, gel filtration, zymography and thin-layer chromatography. The bacterium synthesizes a specific endolevanase and a nonspecific β-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The β-fructofuranosidase was not able to digest long-chain inulin or timothy grass fructan, but degraded inulin oligosaccharides and sucrose. Addition of 1,4-dithioerythritol to an enzyme solution did not affect the activity of endolevanase(s), but increased the ability of β-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km =2·82gl(-1) and Vmax =4·01μmoles reducing sugar equivalents×mg(-1) ×min(-1) . Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Tannin-tolerant bacteria from crossbred Holstein x Zebu cows

The objective of this work was to isolate and characterize tannin-tolerant ruminal bacteria from crossbred Holstein x Zebu cows fed a chopped mixture of elephant grass (Pennisetum purpureum), young stems of "angico-vermelho" (Parapiptadenia rigida), and banana tree (Musa sp.) leaves. A total of 117 bacteria strains were isolated from enrichment cultures of rumen microflora in medium containing tannin extracts. Of these, 11 isolates were able to tolerate up to 3 g L-1 of tannins. Classical characterization procedures indicated that different morphological and physiological groups were represented. Restriction fragments profiles using Alu1 and Taq1 of 1,450 bp PCR products from the 16S rRNA gene grouped the 11 isolates into types I to VI. Sequencing of 16S rRNA PCR products was used for identification. From the 11 strains studied, seven were not identifiable by the methods used in this work, two were strains of Butyrivibrio fibrisolvens, and two of Streptococcus bovis.

Sucrose phosphorylase of the rumen bacteriumPseudobutyrivibrio ruminisstrain A

To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose. Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis. Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6.0 and temperature 45 degrees C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K(m) for glucose-1-P formation and fructose release were 3.88 x 10(-3) and 5.56 x 10(-3) mol l(-1) sucrose, respectively - while the V(max) of the reactions were -0.579 and 0.9 mumol mg protein(-1) min(-1). The enzyme also released free glucose from glucose phosphate. Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage. Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.

Control of rumen methanogenesis by inhibiting the growth and activity of methanogens with hydroxymethylglutaryl-SCoA inhibitors

The CH 4-forming bacteria in the rumen are Archaea. All Archaea have unique membrane lipids that contain glycerol joined by ether linkages to long chain isoprenoid alcohols. Mevalonate is a key precursor for isoprenoid synthesis by methanogens and is also essential for the production of cholesterol by humans. Mevalonate is produced by reduction of hydroxymethylglutaryl-SCoA (HMG-CoA). HMG-CoA reductase is a target of HMG-CoA inhibitors (statins) used to inhibit human cholesterol synthesis. Statins would be expected to specifically inhibit growth of rumen methanogenic bacteria by inhibiting their synthesis of mevalonate. Rumen fermentative bacteria would not be inhibited because they are eubacteria and most of their lipids are glycerol esters of long chain fatty acids. These predictions are supported by the results of our study of the effect of statins on the growth of pure cultures of rumen methanogenic and fermentative bacteria. The HMG-CoA reductase inhibitors, mevastatin and lovastatin (ca. 10 nM), inhibited the growth of strains of rumen Methanobrevibacter. They did not inhibit the growth of strains of Ruminococcus albus, R. flavefaciens, Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Selenomonas ruminantium. These eubacterial species are essential for ruminal fermentation of cellulose, starch and other plant polysaccharides.

The effect of condensed tannins from Lotus corniculatus on the proteolytic activities and growth of rumen bacteria

Eleven strains of ruminal bacteria ( Streptococcus bovis NCFB 2476 and B315, Butyrivibrio fibrisolvens strains WV1 and C211, Prevotella ruminicola 23, Prevotella-like strain C21a, Eubacterium spp. C12b and C124b, Clostridium proteoclasticum B316 T, Ruminococcus albus 8, and Fibrobacter succinogenes S85) were used to determine the effect of condensed tannins (CTs) from Lotus corniculatus (LC) on the proteolysis of the large (LSU) and small (SSU) subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) protein extracted from white clover ( Trifolium repens), and on bacterial growth in vitro. The effects of CTs were determined with and without polyethylene glycol (PEG; 1.7 mg PEG/mg CT), which binds and inactivates CTs. Proteolysis of the LSU and the SSU of Rubisco (with 1.5 mg CT/ml) was determined in vitro and measured using a sodium dodecylsulphate polyacrylamide electrophoresis (SDS-PAGE) procedure. Bacterial growth in a plant protein medium containing 0, 50, 100, 200, 400, and 600 μg CT/ml was also measured after 0, 2, 4, 8, 12 and 24 h of incubation. Proteolytic bacterial cultures without CTs or with CTs + PEG degraded LSU and SSU rapidly (6–8%/h), but proteolysis varied markedly between species. Cultures of P. ruminicola C21a degraded both the LSU (11.3%/h) and SSU (9.8%/h) when grown in the presence of 1.5 mg CT/ml. Bacterial strains S. bovis B315 (11.5%/h) and strain NCFB 2476 (10.6%/h), Eubacterium sp. C12b (5.1%/h) and C124b (5.5%/h) appeared to degrade SSU more effectively than the LSU (2.6, 2.3, 3.8, and 2.6%/h, respectively) in the presence of 1.5 mg CT/ml. F. succinogenes S85, R. albus 8, P. ruminicola 23, C. proteoclasticum B316 T, B. fibrisolvens WV1 and C211a had low (0.3%/h) to moderate (3–4%/h) rates of proteolysis when CT was included in the medium. In the absence of CT, all bacterial strains showed typical growth. Addition of 200, 400 and 600 μg CT/ml significantly ( P < 0.05) reduced the growth rate and maximum optical density of most bacterial strains tested compared to the minus CT controls. Some strains ( C. proteoclasticum B316 T; P < 0.05 and R. albus 8; P = 0.09), however, showed transient increases in their growth rate at low (50–100 μg/ml), but not at high (>200 μg/ml) concentrations of CT. It was concluded that CTs from LC reduced the rate of proteolysis and inhibited the growth of proteolytic rumen micro-organisms.

The use of PCR for the identification and characterisation of bacteriocin genes from bacterial strains isolated from rumen or caecal contents of cattle and sheep

PCR primers were designed to amplify the gene that encodes bovicin 255 from Streptococcus gallolyticus LRC0255 and the bacteriocin genes from Butyrivibrio fibrisolvens strains AR10 and OR79A ( bviD and bvi79A) in order to screen for their incidence in rumen and caecal B. fibrisolvens and Streptococcus bovis-like isolates from New Zealand and North American ruminants. None of the B. fibrisolvens-like strains ( n=34) isolated from New Zealand or North America had the genes encoding for butyrivibriocins AR10 ( bviD) or OR79 ( bvi79A). However, seven S. bovis isolates from New Zealand ruminants and three from North American animals had the bovicin 255 gene. Sequence comparison of cloned bovicin 255 PCR products indicated a 92.9–95.7% similarity to that of the corresponding bovicin 255 gene sequence of S. gallolyticus. Four of the New Zealand bovicin 255 positive S. bovis isolates were from the caecal contents of the same sheep and had identical PFGE profiles. Two other S. bovis isolates sharing the same PFGE profile were isolated from a separate animal from the same flock. PFGE analysis of the North American strains indicated that all three were closely related as two of three had identical PFGE profiles with the remaining isolate differing only by a single band position. The 16S rRNA gene sequences of the 10 isolates were at least 99.8% identical to S. bovis. All 10 S. bovis isolates having the gene for bovicin 255 produced bacteriocin activity that inhibited the growth of Peptostreptococcus anaerobius D1 in a deferred antagonism plating (DAP) assay. Certain S. bovis isolates obtained from ruminants have bacteriocin activity associated with a distinct bovicin 255 gene sequence but it appears that bacteriocin production by the rumen anaerobe B. fibrisolvens may be uncommon in strains isolated from cattle and sheep in New Zealand.

Structural studies of the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b

The extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b, when grown under strictly anaerobic conditions with glucose as carbohydrate source, has been studied by chemical and spectroscopic techniques. The results demonstrate that the polysaccharide consists of hexasaccharide repeating units with the following structure: The isolated polysaccharide was found to be ∼65% acetylated at O-2 of the 3- O-[( S)-1-carboxyethyl]-β- d-Glc p residue. The absolute configuration of the 1-carboxyethyl groups was determined by circular dichroism.

Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens.

To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.

Phenotypic and genetic data supporting reclassification of Butyrivibrio fibrisolvens isolates.

Among 55 Butyrivibrio fibrisolvens strains five ribotypes of B. fibrisolvens were described on the basis of RFLP profiles of 16S rDNA regions obtained with restriction endonuclease HaeIII. In the phylogenetic tree, these ribotypes were located in the XIVa cluster of Gram-positive bacteria. Phenotypic differences of selected ribotype groups became the basis for further reclassification of B. fibrisolvens.

Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens.

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.

Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria

We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens. Donor strains were postulated to carry two TcR genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet(O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible TcR gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet(W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet(W) genes with almost identical sequences are also shown to be present in TcR strains of B. fibrisolvens from Australian sheep and in TcR strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus. This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet(W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus.

Genetically modified ruminal bacteria protect sheep from fluoroacetate poisoning.

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10(6) to 10(7) cells ml-1 in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.

Butyrivibriospp. and Other Xylanolytic Microorganisms From the Rumen have Cinnamoyl Esterase Activity

High concentrations of hydroxycinnamic acids in the hemicellulosic fraction of dry season tropical grasses may influence the rate of microbial degradation of arabinoxylans by ruminant animals. The ability of 22 strains ofButyrivibrio fibrisolvens, other ruminal bacteria (Ruminococcus albusSY3,Ruminococcus flavefaciensRF1,Prevotella ruminicolaAR20) and the ruminal phycomyceteNeocallimastix patriciarumCX to digest the tropical grassHeteropogon contortus(spear grass) and hydrolyse esterified ferulic and p-coumaric acid was examined. Significant digestion (8–36%) of spear grass occurred with theB. fibrisolvensstrains H17c, A38, LP92-1-1, 49,R. albusSY3 andN. patriciarum. Hydrolysis of ester-linked ferulic and p-coumaric acid occurred with all organisms exceptB. fibrisolvensstrains GS113, OB156 and LP1028 andP. ruminicolaAR20. The ratio of ferulic to p-coumaric acid hydrolysed by different strains ofButyrivibriospp. varied markedly from 0.96 for AR 51 to 0.16 for A38. Butyrivibrios which were fibrolytic (H17c and A38) had higher extracellular cinnamoyl esterase activity than bacteria that did not digest spear grass fibre (LP 91-4-1 and AR 20) which had low activities or only produced cell associated enzyme. Cell associated and extracellular esterase activity were induced whenButyrivibriospp. strains H17c, A38 and E14 and theRuminococcusspp. were grown on birchwood xylan but induction did not occur to the same extent withN. patriciarum. This is the first reported observation of cinnamoyl esterase activity in the genusRuminococcus. The fungusN. patriciarumhad significantly higher digestibility of spear grass and solubilisation of phenolic acids than the bacteria. The study shows that high levels of extracellular cinnamoyl esterases are characteristic of a selection of fibre-degrading ruminal bacteria and fungi which probably indicates that these enzymes are common amongst xylanolytic ruminal microorganisms.

High-frequency transfer of a naturally occurring chromosomal tetracycline resistance element in the ruminal anaerobe Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens strains resistant to tetracycline were isolated from the bovine rumen. Two of three Tcr B. fibrisolvens tested were able to donate tetracycline resistance at frequencies ranging from 10(-7) to 10(-1) per donor cell in anaerobic filter matings to a rifampin-resistant mutant of the type strain of B.fibrisolvens, 2221R. The recipient strain 2221R exhibited rapid autoaggregation, which might be a factor in the high transfer rates observed. Tcr transconjugants of B. fibrisolvens 2221R were also capable of further transferring tetracycline resistance to a fusidic acid-resistant mutant, 2221F. Comparison of genomic DNAs by pulsed-field gel electrophoresis demonstrated altered band profiles in transconjugants, consistent with the acquisition of a large mobile chromosomal element. The transferable elements from the two B. fibrisolvens donors 1.23 and 1.230 (TnB123 and TnB1230, respectively) showed the same preferred insertion site in the B. fibrisolvens 2221R chromosome and are likely to be similar, or identical, elements. Hybridization experiments showed no close relationship between TnB1230 and int-xis regions from Tn916 or Tn5253. Although DNA from the B. fibrisolvens donor strains hybridized with probes carrying tet(M) or tet(O) sequences, transconjugants were found to have acquired a distinct band that hybridized only weakly with these probes, suggesting that a second, distantly related Tcr determinant had been transferred.

Lack of methionine biosynthesis de novo in Butyrivibrio fibrisolvens strain E14

Butyrivibrio fibrisolvens strain E14 has an absolute requirement for methionine. Metabolism of L-[ β-14C]-serine to methionine occurred in the methionine-independent B. fibrisolvens strain H17c but not in strain E14. The absolute requirement for methionine in strain E14 could be met by addition of S-adenosylmethionine to the medium, but incorporation was not due to the presence of free methionine in the S-adenosylmethionine preparation. The results show that B. fibrisolvens strain E14 is unable to synthesize methionine de novo, probably due to a lack of methionine synthase. Butyrivibrio fibrisolvens may also possess an alternative pathway of methionine biosynthesis from S-adenosylmethionine.

Structural studies of the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3

The structure of the Butyrivibrio fibrisolvens strain CF3 capsular polysaccharide has been investigated mainly by sugar and methylation analyses, Smith degradation, NMR spectroscopy, and mass spectrometry. The results indicate that the polysaccharide is composed of pentasaccharide repeating units having the following structure: ▪

Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios.

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.

16S rDNA analysis of Butyrivibrio fibrisolvens: phylogenetic position and relation to butyrate-producing anaerobic bacteria from the rumen of white-tailed deer.

Complete 16S rDNA sequences of six strains of Butyrivibrio fibrisolvens, including the type strain (ATCC 19171), were determined. The type strain was found to have less than 89% sequence similarity to the other isolates that were examined. The five plasmid-bearing strains formed a closely related cluster and three of these strains (OB156, OB157 and OB192) were very highly related (> 99%), indicating that they are isolates of the same genomic species. The phylogenetic position of Butyrivibrio was found to be within the subphylum Clostridium, of Gram-positive bacteria. The closest relatives to the type strain were Eubacterium cellulosolvens and Cl. xylanolyticum and the closest relatives to the separately clustered strains were Roseburia ceciola, Lachnospira pectinoschiza and Eubacterium rectale.

Structural studies of the extracellular polysaccharide from Butyrivibrio fibrisolvens strain 49

The structure of Butyrivibrio fibrisolvens strain 49 capsular polysaccharide has been investigated mainly by sugar and methylation analysis, partial chemical degradations. NMR spectroscopy, and mass spectrometry. The results suggest that the polysaccharide is composed of pentasaccharide repeating units having the following structure. ▪ The polysaccharide contains O-acetyl groups, one of which is substituted to O-3 of the 4-substituted α- d-Gal p residue, while others occur in non-stoichiometric amounts at other locations.

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5′ and 3′ termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.