| dc.description.abstracteng | The mammalian gut harbors a complex and specialized microbiota, which includes all tree domains of life (Archaea, Bacteria, and Eukarya). Bacteria are the most abundant group within the gut system, and they take part in metabolic processes that expand the hosts metabolic potential. One of the most beneficial traits of this relationship is that bacteria help to degrade recalcitrant plant materials. In this study, the microbiome of three Eurasian beaver gut systems (male and female subadult, and male juvenile) were analysed by applying metagenomics using 16S rRNA gene marker-based and direct sequencing-based approaches, to gain insights into the diversity, structure and function of the gut-inhabiting bacterial communities and genes involved in cellulose breakdown.
Metagenomic DNA was isolated from the entire gut system of three Eurasian beavers, covering different compartments (stomach, small intestine, cecum, and colon). The taxonomic compositions of the bacterial communities within these compartments were assessed using the hypervariable regions V3-V4 of the 16S rRNA gene, amplified from the isolated DNA by PCR. Subsequently, amplicon-based analysis of 2,599,870 high-quality paired end reads revealed 277 unique OTUs in the entire dataset. The bacterial diversity in the cecum and colon was higher in the male subadult beaver than in the female subadult and male juvenile beaver. The gut bacterial community was dominated by Firmicutes and Actinobacteria, followed by Proteobacteria, Verrucomicrobia, Fusobacteria, Bacteroidetes, and Tenericutes. A bacterial community shift from the juvenile to subadult beaver was indicated by the decrease of members of Bacteroidetes and an increase in Verrucomicrobia members. In addition, the presence and abundance of some phyla could be associated with sex, e.g. Fusobacteria, were detected in both male beavers but not in the female. However, further studies of Eurasian beaver gut microbial communities are necessary to confirm these trends. The presence of Clostridiaceae 1, Lachnospiraceae, and Ruminococcaceae in the cecum and colon of the beaver indicated that plant cell wall breakdown is mainly performed in these compartments. The predicted functional profiles showed an increased relative abundance in genes necessary for cellulose breakdown and uptake of degradation products, which is in accordance to the presence of potential cellulolytic bacterial species. The abundance of unclassified Clostridiaceae 1, Lachnospiraceae, and Ruminococcaceae in the cecum and colon of the beaver as well as unclassified Bacteroidaceae in juvenile beaver suggest the presence of novel species exhibiting cellulolytic activity. In comparison to its North American relative, the Eurasian beaver has a higher relative abundance of Actinobacteria and Verrucomicrobia, and a lower abundance of Bacteroidetes in the gut system. In addition, high relative abundances of Ruminococcaceae and Lachnospiraceae were detected in the cecum and colon compartments of the Eurasian North American beaver (NAGrun). In general, the bacterial community structure in the cecum was similar to that of the colon in both the Eurasian and North American beavers.
In-depth analysis of the microbiome of the back cecum and lower colon of both subadult beavers was performed through direct metagenome sequencing. A total of 6,200,436 high-quality paired-end reads were obtained. Metagenome assembly resulted in a total of 101,060 contigs with the largest contig of 198,219 bp. The bacterial community structure derived from the metagenome sequencing differed to that based on amplicon sequencing. The relative abundance of Proteobacteria was higher in the metagenome sequences. However, Proteobacteria, Firmicutes, and Actinobacteria remained the dominant phyla. The functional analysis of the metagenomes indicates the metabolic ability of the Eurasian beaver gut microbiome to neutralize toxic compounds from plants and heavy metals, as well as resistance to pathogens and antibiotics. Functional analysis revealed a high diversity of CAZymes, especially glycoside hydrolases (GHs) for the degradation of polysaccharides. A high relative abundance of genes associated to the cellulolytic process, especially GH3 and GH5 family members, was detected in the metagenomes. In addition, the CAZyme family involved in lignin breakdown, AA2, and an annotated DyP-like protein responsible for lignin degradation were also present in the gut microbiome of the beaver. Through combination of marker gene community analysis and metagenome analysis, a novel cellulase from GH family 5 was identified and isolated. The beaver cellulase candidate 33 (BC33) was characterized and exhibit optimal catalytic activity at pH 4 and 60 °C, with high affinity towards barley glucan (Km 0.205 ± 0.022 µM/min, Vmax 0.732 ± 0.026 µM/min).
Since Eurasian beaver gut system is mainly an anoxic environment, the anaerobic cellulolytic bacteria, e.g. Clostridia could also produce cellulosomes. Parts of the building block for cellulosomes such as dockerin and cohesin were present in the Eurasian beaver gut metagenomes. Further studies are needed to confirm the existence of cellulosome system and to identify which CAZymes families are bound to the cellulosome, thus, enabling a better understanding of plant material digestion in the Eurasian beaver gut. | de |