Ninety-nine de novo assembled genomes from the moose (Alces alces) rumen microbiome provide new insights into microbial plant biomass degradation

Abstract

The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

Figure 1

Locations where samples were collected and area characteristics (a). Overall taxonomic composition based on 16S and 18S rRNA reads (b). Prokaryotic taxonomic composition based on 16S rRNA reads and MAG phylogeny (c). Eukaryotic taxonomic composition based on 18S rRNA reads (d).

Figure 2

Phylogeny and CAZome profiles of MAGs. A phylogenomic tree of the 99 moose rumen MAGs (a). Triangles represent genus-level clades based on average amino-acid identity levels of encoded proteins. For Bacteroidetes, family Prevotellaceae and Rikenellaceae are indicated, as well as classifications based on 16S genes for selected MAGs. Included reference genomes are from isolates except the Cyanobacteria/Melainabacteria genomes (Di Rienzi et al., 2013), the MGS00153 Alphaproteobacteria genome (Laczny et al., 2016) and the SRM-1 Bacteroidetes genome (Pope et al., 2012) that were assembled from metagenomes and the SR1 (Campbell et al., 2013) and TM7 genomes (Podar et al., 2007) that were sequenced from single cells. Heatmaps showing counts of selected GHs involved in plant degradation (b) and of cohesins (COHs), dockerins (DOCs) and polysaccharide utilizing loci (PULs) (c) in the MAGs. Relative abundances of the MAGs in the different moose samples (d), expressed as average coverage depth per million read pairs.

Figure 3

Different examples of gene organizations in a subset of the PULs identified in MAGs of phylum Bacteroidetes. PULs absent from CAZymes that rather contained genes encoding proteins of unknown function (a, b). PULs exhibiting similar organization and CAZyme content as experimentally characterized PULs, where (c) is similar to xylan-degrading PULs in B. ovatus ATCC 8483 (Rogowski et al., 2015) and (d) is similar to starch-degrading PULs in B. thetaiotaomicron VPI-5482 (Foley et al., 2016). A PUL with GH5 and GH26 genes (e). PULs exhibiting combinations of GH9 genes with various other GH-encoding genes (f). A PUL shared in four Prevotellaceae MAGs that harbored dockerin-like modules (g). PULs, containing dockerin modules, that are likely involved in pectin or starch breakdown (h). The red horizontal bars indicate contig starts or ends.