Metatranscriptomics Reveals the Active Bacterial and Eukaryotic Fibrolytic Communities in the Rumen of Dairy Cow Fed a Mixed Diet

Abstract

Ruminants have a unique ability to derive energy from the degradation of plant polysaccharides through the activity of the rumen microbiota. Although this process is well studied in vitro, knowledge gaps remain regarding the relative contribution of the microbiota members and enzymes in vivo. The present study used RNA-sequencing to reveal both the expression of genes encoding carbohydrate-active enzymes (CAZymes) by the rumen microbiota of a lactating dairy cow and the microorganisms forming the fiber-degrading community. Functional analysis identified 12,237 CAZymes, accounting for 1% of the transcripts. The CAZyme profile was dominated by families GH94 (cellobiose-phosphorylase), GH13 (amylase), GH43 and GH10 (hemicellulases), GH9 and GH48 (cellulases), PL11 (pectinase) as well as GH2 and GH3 (oligosaccharidases). Our data support the pivotal role of the most characterized fibrolytic bacteria (Prevotella, Ruminocccus and Fibrobacter), and highlight a substantial, although most probably underestimated, contribution of fungi and ciliate protozoa to polysaccharide degradation. Particularly these results may motivate further exploration of the role and the functions of protozoa in the rumen. Moreover, an important part of the fibrolytic bacterial community remains to be characterized since one third of the CAZyme transcripts originated from distantly related strains. These findings are used to highlight limitations of current metatranscriptomics approaches to understand the functional rumen microbial community and opportunities to circumvent them.

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

“…Not unexpectedly, the well-studied fibrolytic bacteria Fibrobacter succinogenes represented the bacterial species with the highest number of detected proteins (switchgrass: 349; rumen fluid: 210), followed by two strains of Prevotella ruminicola (ranging from 173 to 213 proteins, of which the majority of the detection proteins were homologues of the two strains) and P. brevis (switchgrass: 129; rumen fluid: 168), highlighting their overall importance in the carbohydrate metabolisms in the rumen. This is consistent with previous studies involving functional analysis of the rumen microbiome, demonstrating that a majority of the plant cell wall polysaccharide degradation is carried out by species related to Fibrobacter, Ruminococcus and Prevotella 24,25,36 . Although our metaproteome data suggested that these aforementioned characterized prokaryotes were amongst the most active (i.e.…”

“…and the two Piromyces species, had the highest LFQlevel of all cellulases. While GH48 have been absent or only detected at very low levels in previous rumen metagenomes 31,38 , members of this GH family have been observed in rumen metatranscriptomes from mixed rumen populations, reportingly expressed by Ruminococcus and rumen fungi 24,25,39 . It is also worth noting that while members of the GH48 family were the most abundant CAZymes affiliated to protein sequences of Orpinomyces sp.…”

“…The impact of fungi in the rumen ecosystem was already demonstrated in the early 1990s by Gordon and Phillips who reported a significant decrease in fiber digestion within the rumen after anaerobic fungi had been removed by the administration of fungicides 22 . The importance of rumen fungi for biomass degradation has since the been supported by in vivo studies [23][24][25] , and recently reinforced in transcriptome studies revealing that the fungi express a range of CAZymes when grown on different carbon sources 9,26 . Although enzymes of fungal origin have been regularly explored for their remarkable capacity to degrade lignocellulosic fiber 12,27,28 , their functional role in native anaerobic habitats and within the biomass-degrading enzyme repertoire of the rumen microbiome remains unclear.…”

Identification of bacteria involved in the decomposition of lignocellulosic biomass treated with cow rumen fluid by metagenomic analysis

“…Shotgun metagenomic analysis randomly sequencies the DNAs present in environmental samples using a next-generation sequencer, which provides an effective method to determine microbial composition and general function from uncultivated microorganisms [33]. Several studies have conducted metagenomic analysis in cow rumen and revealed the several microbes that are involved in the decomposition of the plant cell wall decomposition [3, 5]. However, the pretreatment process is an unbuffered closed system that treats a single substrate.…”

Differential gene expression associated with fungal trophic shifts along the senescence gradient of the moss Dicranum scoparium

“…Transcripts with E values < 1e-6 were kept. The E-value cutoff was selected based on previous studies Quandt et al, 2015;Comtet-Marre et al, 2017;Meiser et al, 2017;Gonzalez et al, 2018) and after manual examination of a subset of sequences and their BLASTX results. To gain more taxonomic information and to confirm that the selected transcripts really belong to fungi, transcripts were first translated into proteins with Transdecoder (Haas et al, 2013) and an additional round of BLASTP against the NCBI Non-Redundant (NR) protein database was performed, saving the top five hits.…”