Snapshot of the Eukaryotic Gene Expression in Muskoxen Rumen—A Metatranscriptomic Approach

BACKGROUND: Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus), with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA) was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6), GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. CONCLUSIONS/SIGNIFICANCE: The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes

Metagenomics of the Svalbard Reindeer Rumen Microbiome Reveals Abundance of Polysaccharide Utilization Loci

Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation

A Proposed Taxonomy of Anaerobic Fungi (Class Neocallimastigomycetes) Suitable for Large-Scale Sequence-Based Community Structure Analysis

Anaerobic fungi are key players in the breakdown of fibrous plant material in the rumen, but not much is known about the composition and stability of fungal communities in ruminants. We analyzed anaerobic fungi in 53 rumen samples from farmed sheep (4 different flocks), cattle, and deer feeding on a variety of diets. Denaturing gradient gel electrophoresis fingerprinting of the internal transcribed spacer 1 (ITS1) region of the rrn operon revealed a high diversity of anaerobic fungal phylotypes across all samples. Clone libraries of the ITS1 region were constructed from DNA from 11 rumen samples that had distinctly different fungal communities. A total of 417 new sequences were generated to expand the number and diversity of ITS1 sequences available. Major phylogenetic groups of anaerobic fungi in New Zealand ruminants belonged to the genera Piromyces, Neocallimastix, Caecomyces and Orpinomyces. In addition, sequences forming four novel clades were obtained, which may represent so far undetected genera or species of anaerobic fungi. We propose a revised phylogeny and pragmatic taxonomy for anaerobic fungi, which was tested and proved suitable for analysis of datasets stemming from high-throughput next-generation sequencing methods. Comparing our revised taxonomy to the taxonomic assignment of sequences deposited in the GenBank database, we believe that >29% of ITS1 sequences derived from anaerobic fungal isolates or clones are misnamed at the genus level