Lactate dehydrogenase gene variability among predominant lactate utilizing ruminal bacteria.

The inter- and intraspecies variability of lactate dehydrogenase (ldh) gene was determined among the predominant ruminal lactate utilizing bacteria. Nearly complete nucleotide sequences of ldh gene, encoding NAD-dependent lactate dehydrogenase of three Megasphaera elsdenii and six Selenomonas ruminantium strains, were obtained and compared. Phylogenetic analyses revealed a limited variability between the ldh sequences studied. The majority of differences observed were silent mutations at the 3rd position of codons. Surprisingly, the intraspecies diversity of the ldh gene among S. ruminantium isolates was higher than the interspecies level between S. ruminantium and M. elsdenii, which strongly suggests the possibility of acquisition of this gene by horizontal gene transfer.

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.

Genetic variability of rumen Selenomonads.

Molecular diversity of rumen bacteria belonging to the species Selenomonas ruminantium was evaluated by biochemical and PCR analyses targeted at the 16S rRNA operon and lactate dehydrogenase gene. While extremely variable in metabolic characteristics, two different RISA (ribosomal intergenic spacer analysis), and five lactate dehydrogenase gene RFLP profiles were observed among the twelve strains studied. The strains showed very limited variability ARDRA ( amplified ribosomal DNA restriction analysis) when two different profiles were observed only. 16S rDNA sequence comparisons indicate complex genetic structure within S.ruminantium population.

Genome organization of Treponema zioleckii.

Genome analysis of Treponema zioleckii proved that, in this bacterium, besides chromosomal DNA, a relatively small extrachromosomal DNA element is present. This element was shown to be a double-stranded circular plasmid DNA of approximately 7 kbp; it was designated as pKT. The plasmid was characterized by molecular and bioinformatic analysis. No pKT homologous DNA sequences were detected in other rumen Treponema strains. The overall G+C content of the pKT plasmid is approximately 56 %, which is higher than in other Treponema plasmids or genomes. The Rep module of the pKT plasmid consisting of the rep gene and the region of repeats was identified within a 1.6-kbp fragment. The putative rep gene encodes the replication protein belonging to the pfam04796 RepA_C family of proteins with the highest similarity (25 % within 249 amino acids) to the RepA protein from the green sulfur bacterium Prosthecochloris aestuarii.

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

Limited genetic variability in Megasphaera elsdenii strains.

Levels of phenotypic and genotypic diversity among seven Megasphaera elsdenii strains recovered from rumen contents of cattle, sheep and lambs were determined by a combination of antibiotic-resistance analysis and PCR fingerprint techniques targeted both to the ribosomal RNA operon (ARDRA, RISA) and the whole genome (ERIC-PCR, RAPD-PCR). Despite exhibiting different antibiotic resistance profiles, the tested strains represent genetically nearly identical isolates. Close genetic relatedness was found among M. elsdenii isolates that originated from vastly different habitats worldwide, as revealed by the comparison of 16S rDNA sequences.

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.

Occurrence of restriction-modification systems in ruminal butyrate-producing bacteria.

Thirty-five strains of ruminal bacteria belonging to the former Butyrivibrio fibrisolvens species were screened for the presence of site-specific restriction endonuclease and modification methyltransferase activities. Seven strains possessed endonuclease activities detectable in crude cell extracts. The recognition sequences and optimal reaction conditions for seven of them were determined. Five enzymes were found to be isoschizomers of type II endonucleases (EcoRV, NsiI, AseI (2x) and SauI), one was type IIS (FokI) and two remained unknown. The optimal reaction buffer was found to be a low ionic strength buffer and all enzymes possessed sufficient activity at 39 degrees C. The presence of DNA modification among all strains was also determined. Most of the methylation activities correlated with restriction activities, yet some strains possessed unaccompanied modification methyltransferases.

GATC-specific restriction--modification systems in ruminal bacteria.

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.

GATC-specific restriction and modification systems in treponemes.

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.