Functional and Phylogenetic Characterization of Bacteria in Bovine Rumen Using Fractionation of Ruminal Fluid

Abstract

Cattle productivity depends on our ability to fully understand and manipulate the fermentation process of plant material that occurs in the bovine rumen, which ultimately leads to the improvement of animal health and increased productivity with a reduction in environmental impact. An essential step in this direction is the phylogenetic and functional characterization of the microbial species composing the ruminal microbiota. To address this challenge, we separated a ruminal fluid sample by size and density using a sucrose density gradient. We used the full sample and the smallest fraction (5%), allowing the enrichment of bacteria, to assemble metagenome-assembled genomes (MAGs). We obtained a total of 16 bacterial genomes, 15 of these enriched in the smallest fraction of the gradient. According to the recently proposed Genome Taxonomy Database (GTDB) taxonomy, these MAGs belong to Bacteroidota, Firmicutes_A, Firmicutes, Proteobacteria, and Spirochaetota phyla. Fifteen MAGs were novel at the species level and four at the genus level. The functional characterization of these MAGs suggests differences from what is currently known from the genomic potential of well-characterized members from this complex environment. Species of the phyla Bacteroidota and Spirochaetota show the potential for hydrolysis of complex polysaccharides in the plant cell wall and toward the production of B-complex vitamins and protein degradation in the rumen. Conversely, the MAGs belonging to Firmicutes and Alphaproteobacteria showed a reduction in several metabolic pathways; however, they have genes for lactate fermentation and the presence of hydrolases and esterases related to chitin degradation. Our results demonstrate that the separation of the rumen microbial community by size and density reduced the complexity of the ruminal fluid sample and enriched some poorly characterized ruminal bacteria allowing exploration of their genomic potential and their functional role in the rumen ecosystem.

Keywords: MAGs; fractionation; new species; ruminal fluid; small-sized bacteria.

FIGURE 1

Clustering of the high-quality metagenome-assembled genomes (MAGs) found in the 5% fraction of the total sample of ruminal fluid of the Bon and Holstein animals according to MASH-ANI distances.

FIGURE 2

Bootstrapped maximum-likelihood tree based on concatenated ribosomal marker genes including representative MAGs found in this study placed onto the phylogenetic tree proposed by Hug et al. (2016). Clades are colored based on both domain and order, but only orders that are relevant to the MAGs are presented in this study, as shown by gray boxes.

FIGURE 3

Absolute abundance of the subsystem feature counts found in the functional categories established by RAST (rapid annotation using subsystem technology) for each of the representative MAGs.

FIGURE 4

Absolute abundance of the subsystem feature counts found in the carbohydrate category established by RAST (rapid annotation using subsystem technology) for each of the representative MAGs.

FIGURE 5

Absolute abundance of the subsystem feature counts found in the vitamins and co-factors category established by RAST (rapid annotation using subsystem technology) for each of the representative MAGs.

FIGURE 6

Absolute abundance of the subsystem feature counts found in the protein degradation category established by RAST (rapid annotation using subsystem technology) for each of the representative MAGs.

FIGURE 7

Glycoside hydrolases found in the representative MAGs using dbCAN2 carbohydrate-active enzyme (CAZy) domain HMM database. (A) Gene counts per each MAG of enzymes degrading substrates such as cellulose, hemicellulose, and pectin, according to Seshadri et al. (2018). (B) Gene counts for other glycoside hydrolases.