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.