Invited review: Application of meta-omics to understand the dynamic nature of the rumen microbiome and how it responds to diet in ruminants.

Ruminants are unique among livestock due to their ability to efficiently convert plant cell wall carbohydrates into meat and milk. This ability is a result of the evolution of an essential symbiotic association with a complex microbial community in the rumen that includes vast numbers of bacteria, methanogenic archaea, anaerobic fungi and protozoa. These microbes produce a diverse array of enzymes that convert ingested feedstuffs into volatile fatty acids and microbial protein which are used by the animal for growth. Recent advances in high-throughput sequencing and bioinformatic analyses have helped to reveal how the composition of the rumen microbiome varies significantly during the development of the ruminant host, and with changes in diet. These sequencing efforts are also beginning to explain how shifts in the microbiome affect feed efficiency. In this review, we provide an overview of how meta-omics technologies have been applied to understanding the rumen microbiome, and the impact that diet has on the rumen microbial community.

Characterization of the rumen and fecal microbiome in bloated and non-bloated cattle grazing alfalfa pastures and subjected to bloat prevention strategies.

Frothy bloat is an often fatal digestive disorder of cattle grazing alfalfa pastures. The aim of this study was to investigate ruminal and fecal microbiota dynamics associated with development of alfalfa-induced frothy bloat and to further explore how bloat prevention strategies influence the composition of these microbial communities. In a 3 × 3 crossover experiment, twelve rumen-cannulated steers were sequentially subjected to: (1) pure alfalfa pasture, (2) pure alfalfa pasture supplemented with the pluronic detergent ALFASURE, and (3) alfalfa - sainfoin mixed pasture. Eleven out of 12 steers in pure alfalfa pasture developed clinical bloat, whereas ALFASURE treatment prevented the development of bloat in all 12 steers and alfalfa - sainfoin prevented bloat in 5 out of 11 steers. Development of bloat was associated with considerable shifts in the microbiota profile of rumen contents. In particular, the microbiota of solid rumen contents from bloated steers contained higher species richness and diversity. Streptococcus, Succinivibrio and unclassified Myxococcales were enriched in the rumen microbiota of bloated steers, whereas Fibrobacter and Ruminococcus were overrepresented in the rumen contents of non-bloated steers. Our results provide novel insights into bloat-associated shifts in the composition and predicted functional properties of the rumen microbiota of cattle grazing alfalfa pasture.

Isolation of High Quality RNA for Metatranscriptomic Analysis of Lignocellulose Digestion in the Rumen.

Metatranscriptomics can be used to examine both the composition of a microbial community as well as its metabolic activity under a particular set of conditions and complement metagenomic studies. The availability of low-cost, high-throughput next-generation sequencing has led to a rapid increase in the number of metatranscriptomic studies being undertaken. One of the primary difficulties when conducting transcriptomics is the ability to isolate high-quality RNA from samples of interest. The application of metatranscriptomics in rumen microbiology is still relatively novel but there is a significant push toward applying this technology in this field. In this protocol, we outline the method that is used routinely in our laboratory to purify high quality RNA from rumen contents that are suitable for metatranscriptomic sequencing using RNA-seq.

Biochemical and kinetic characterization of the multifunctional ß-glucosidase/ß-xylosidase/α-arabinosidase, Bgxa1.

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, ß-glucosidase/ß-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of ß-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-ß-D-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-α-D-arabinofuranoside (pNPAf), suggesting it is a multifunctional ß-glucosidase/ß-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of ß-glucosidase activity was 100× greater than ß-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0-10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.