A. Kasperowicz

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.

Treponema zioleckii sp. nov., a novel fructan-utilizing species of rumen treponemes

During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.

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 β-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.

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 ≈54 kDa and maximum activity at pH 6.0 and 45 °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 μmol/L per mg protein per min, 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.

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.

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.

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.

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.