Advances In The Understanding of The Commensal Eukaryota And Viruses Of The Herbivore Gut

Cover image for research topic "Advances In The Understanding of The Commensal Eukaryota And Viruses Of The Herbivore Gut"
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Carbohydrate-active enzymes employed by anaerobic fungi during biomass conversion. Anaerobic fungi (AF) deploy various strategies for the degradation of plant biomass. It has been suggested that their ability to produce secreted free CAZymes, cell-bound multi-enzyme complexes (cellulosomes), as well as free cellulosomes might provide the AF with the competitive advantage over the CAZyme repertoire produced by anaerobic bacteria (Henske et al., 2017).
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Original Research
29 April 2020
Rumen Protozoa Play a Significant Role in Fungal Predation and Plant Carbohydrate Breakdown
Cate L. Williams
4 more and 
Sharon A. Huws
Comparative phylogenetic tree of all putative xylanase (GH10 or GH11) genes detected within the protozoal metatranscriptome. Fourteen representative protozoal sequences from Epidinium ecaudatum (4), Polyplastron multivesiculatum (5), uncultured bovine rumen ciliates (3) Epidinium caudatum (1) and Eudiplodinium maggii (1), three fungal cellulases produced by Neocallimastix sp., Piromyces sp., Orpinomyces sp. and Orpinomyces joyonii and two bacterial cellulases from Bacteroides ruminicola and Butyrivibrio fibrisolvens are given for comparison. Target sequences from the metatranscriptome are shown in bold with a diamond shape node marker, the most highly expressed enzyme is marked with a square, protozoal sequences are given.

The rumen protozoa, alongside fungi, comprise the eukaryotic portion of the rumen microbiome. Rumen protozoa may account for up to 50% of biomass, yet their role in this ecosystem remains unclear. Early experiments inferred a role in carbohydrate and protein metabolism, but due to their close association with bacteria, definitively attributing these functions to the protozoa was challenging. The advent of ‘omic technologies has created opportunities to broaden our understanding of the rumen protozoa. This study aimed to utilize these methods to further our understanding of the role that protozoa play in the rumen in terms of their metabolic capacities, and in doing so, contribute valuable sequence data to reduce the chance of mis or under-representation of the rumen protozoa in meta’omic datasets. Rumen protozoa were isolated and purified using glucose-based sedimentation and differential centrifugation, extracted RNA was Poly(A) fraction enriched and DNase treated before use in a phage-based, cDNA metatranscriptomic library. Biochemical activity testing of the phage library showed 6 putatively positive plaques in response to carboxymethyl cellulose agar (indicative of cellulose activity), and no positive results for tributyrin (indicative of esterase/lipase activity) or egg yolk agar (indicative of proteolysis). Direct sequencing of the cDNA was also conducted using the Illumina HiSeq 2500. The metatranscriptome identified a wealth of carbohydrate-active enzymes which accounted for 8% of total reads. The most highly expressed carbohydrate-active enzymes were glycosyl hydrolases 5 and 11, polysaccharide lyases and deacetylases, xylanases and enzymes active against pectin, mannan and chitin; the latter likely used to digest rumen fungi which contain a chitin-rich cell membrane. Codon usage analysis of expressed genes also showed evidence of horizontal gene transfer, suggesting that many of these enzymes were acquired from the rumen bacteria in an evolutionary response to the carbohydrate-rich environment of the rumen. This study provides evidence of the significant contribution that the protozoa make to carbohydrate breakdown in the rumen, potentially using horizontally acquired genes, and highlights their predatory capacity.

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