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

AIMS: To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. METHODS AND RESULTS: 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 ß-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The ß-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 ß-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km  = 2·82 g l(-1) and Vmax  = 4·01 µmoles reducing sugar equivalents × mg(-1)  × min(-1) . CONCLUSION: Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Fructanolytic and saccharolytic enzymes of the rumen bacterium Pseudobutyrivibrio ruminis strain 3--preliminary study.

P. ruminis strain 3 was isolated from the ovine rumen and identified on the basis of comparison of its 16S rRNA gene with GenBank. The bacterium was able to grow on Timothy grass fructan, inulin, sucrose, fructose and glucose as a sole carbon source, reaching absorbance of population in a range of 0.4-1.2. During 1 d the bacteria exhausted 92-97% of initial dose of saccharides except for inulin (its utilization did not exceed 33%). The bacterial cell extract catalyzed the degradation of Timothy grass fructan, inulin and sucrose in relation to carbon source present in growth medium. Molecular filtration on Sephadex G-150, polyacrylamide gel electrophoresis combined with zymography technique and TLC was used to identify enzymes responsible for the digestion of sucrose and both polymers of fructose. Two specific endolevanases (EC 3.2.1.65), nonspecific beta-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7) were detected in cell-free extract from bacteria grown on Timothy grass fructan.

Variability of treponemes in the rumen of ruminants.

Complete 16S rRNA sequences were determined of recently proposed new species of treponemes designated strain S and T. Sequence comparison indicated that both species belong to the Treponema saccharophilum cluster, having thus at least 5 cultivable representatives. Phylogenetic analysis of available GenBank 16S rRNA sequences revealed two phylogenetically distant treponema clusters (T. saccharophilum cluster and T. bryantii cluster). Surprisingly, while among cultivated treponemes dominate T. saccharophilum cluster members, detailed analysis showed that all treponema-like sequences obtained by culture independent 16S rRNA methods belong to the T. bryantii cluster, from which only two cultivable representatives have so far been known. Meta-analysis of available data revealed that treponemes are an infrequent and minor group of bacteria, representing less than 2.4% of total rumen bacteria.

Phosphorolytic cleavage of sucrose by sucrose-grown ruminal bacterium Pseudobutyrivibrio ruminis strain k3.

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90% sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase (EC 2.4.1.7) of molar mass approximately 54 kDa and maximum activity at pH 6.0 and 45 degrees C. The calculated Michaelis constant (Km) for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction (Vlim) were 1.19 and 0.64 micromol/L per mg protein per min, respectively.

Fructanolytic and saccharolytic enzymes of Treponema zioleckii strain kT.

Enzymes in the newly described rumen bacterium, Treponema zioleckii strain kT, capable of digesting Timothy grass fructan, inulin, and sucrose were identified and characterized. Two specific endolevanases and one non-specific beta-fructofuranosidase were found in a cell-free extract. The molecular weight of the endolevanases were estimated to be 60 and 36 kDa, whereas that of beta-fructofuranosidase, 87 kDa. The former of the specific enzymes was associated with the outer membrane, while the latter and the non-specific beta-fructofuranosidase, with the periplasm or cytosol. The K(m) and V(max) for Timothy grass fructan degradation by endolevanase were 0.27% and 15.75 microM fructose equivalents x mg protein(-1) x min(-1), those for sucrose and inulin digestion by beta-fructofuranosidase were 1.35 x 10(-3)M and 1.73 microM hexoses x mg protein(-1) x min(-1) and 1.77% and 1.83 microM hexoses x mg protein(-1) x min(-1), respectively.

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSION: Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.

New species of rumen treponemes.

Three strains of rumen treponemes were isolated and partially characterized. The strains differed significantly one from another in morphology, fermentation characteristics and plasmid profiles. Their genetic variability was assayed using DNA-based molecular approaches. Easily differentiated ARDRA (amplified ribosomal DNA restriction analysis) patterns indicated that the strains represent different bacterial species.

Assessment of the fructanolytic activities in the rumen bacterium Treponema saccharophilum strain S.

AIMS: To characterize the fructose polymer degrading enzymes of rumen bacterium Treponema saccharophilum strain S. METHODS AND RESULTS: Conventional methods were used to examine bacterial growth and enzyme activities. Electrophoretic zymogram under native conditions, and thin layer chromatography, were applied to identify and characterize the enzymes. Treponema saccharophilum utilized Timothy grass fructan, inulin and sucrose but not free fructose. Timothy grass fructan was degraded at a significantly higher rate than sucrose and inulin. Two fructanolytic enzymes were found in the soluble, and one in the membrane fraction of bacterial cell extract. The first degraded each mentioned carbohydrate to monosaccharides. The second released oligosaccharides only from Timothy grass fructan. CONCLUSIONS: The bacterium T. saccharophilum strain S is capable of synthesizing non-specific beta-fructofuranosidases and 2,6-beta-D-fructan fructanohydrolase. The enzymes are of constitutive character. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been stated for the first time that the 2,6-beta-D-fructan fructanohydrolase is synthesized by the rumen bacterium T. saccharophilum. This organism appears to be responsible for grass fructan degradation in the rumen.

Comparison of utilization of pectins from various sources by pure cultures of pectinolytic rumen bacteria and mixed cultures of rumen microorganisms.

Utilization of citrus, lucerne, apple and sugar beet pulp pectins by pure strains of rumen bacteria, Prevotella ruminicola, Lachnospira multiparus and Butyrivibrio fibrisolvens was compared. Additionally, the utilization of pectins by mixed rumen microorganisms was evaluated. The comparison was based on the depletion of galacturonic acid from medium, content of cellular protein in the cultures and the amount of end products of pectin fermentation in cell-free culture fluids. It was found that citrus pectin was utilized best; utilization of lucerne, apple and sugar beet pectins was dependent on the species of bacteria. P. ruminicola and B. fibrisolvens utilized polygalacturonic acid from sugar beet pectins better than that from apple or lucerne pectin, while L. multiparus was capable of significantly better utilization of lucerne pectin than pectin from sugar beet or apple. The source of pectin was less important for mixed cultures of rumen microorganisms than for pure cultures of rumen bacteria. The amount of fermentation products in the culture fluids supported the conclusion that citrus pectin was utilized better than others. Microbial protein content in the cultures was found to be a less sensitive indicator of pectin utilization than the remaining examined parameters. P. ruminicola strains and mixed cultures of rumen microorganisms were shown to have the highest ability to utilize pectins, L. multiparus-moderate, while the B. fibrisolvens strains utilized pectin the least.