Aims: To examine the diversity of protozoa in the rumen contents of cow. Methods and Results: Protozoa that inhabit the rumen were detected by PCR using protozoan-specific primers. Libraries of protozoan rDNA sequences were constructed from rumen fluid, solid tissues and epithelium. Twentythree clones isolated from rumen fluid fell into two genera identified as Entodinium (69AE6% of clones) and Epidinium (31AE4% of clones). Of the clones isolated from rumen fluid, a moderate number were unidentifiable (30AE4%). Conclusions: The predominant protozoan genus identified in the whole rumen belonged to the Entodinium group (81AE1%). Protozoa were not detected in the rumen epithelium. Significance and Impact of the Study: These findings suggest that rumen fluid and solid tissues contain different protozoan populations that may play specific roles in rumen function. Quantitative PCR techniques and a more specific set of phylogenetic probes that distinguish between protozoan species are needed to determine the significance of newly identified groups and to determine the distribution of identified protozoan clusters in rumen microbial communities.
Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative β-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Serx-x-Lys motif that is conserved among all β-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme is a serine protein and was active against p-nitrophenyl esters of C 2 , C 4 , C 8 , and C 10 . The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity of 55% remained at up to 5°C with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by Cd 2+ , Cu 2+ , and Hg 2+ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.
The composition of yeast communities in the rumen of cattle was investigated using comparative DNA sequence analysis of yeast 26S rDNA genes. 26S rDNA libraries were constructed from rumen fluid (FF), rumen solid (FS) and rumen epithelium (FE). A total of 97 clones, containing a partial 26S rDNA sequence of 0·6 kb length, were sequenced and subjected to an on-line similarity search.The 41 FF clones could be divided into five classes. The largest class was affiliated with Pezizomycotina class (85·4% of clones), and the remaining classes were related with the Urediniomycotina (2·4%), Hymenomycetes (4·9%), Ustilaginomycetes (4·9%) and Saccharomycotina (2·4%) classes. The 26 FE clones could be divided into three classes and the Saccharomycetes class (92·4% of clones) was the largest group. The remaining classes were related with either Pezizomycotina (3·8%) or Ustilaginomycetes (3·8%). The 30 FS clones were all affiliated with Saccharomycotina. Saccharomycotina were predominant in rumen epithelium and rumen solid while Pezizomycotina were predominant in rumen fluid. Yeast belonging to the Saccharomycotina class was predominant in the rumen as a whole (57%). One clone (FF34) had less than 90% similarity to any sequence in the database and was thus apparently unrelated to any previously described yeast.
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