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Rumen Protozoa Play a Significant Role in Fungal Predation and Plant Carbohydrate Breakdown.

ABSTRACT: 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.

SUBMITTER: Williams CL

PROVIDER: S-EPMC7200989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mitigating the effects of global warming has become the main challenge for humanity in recent decades. Livestock farming contributes to greenhouse gas emissions, with an important output of methane from enteric fermentation processes, mostly in ruminants. Because ruminal microbiota is directly involved in digestive fermentation processes and methane biosynthesis, understanding the ecological relationships between rumen microorganisms and their active metabolic pathways is essential for reducing emissions. This study analysed whole rumen metagenome using long reads and considering its compositional nature in order to disentangle the role of rumen microbes in methane emissions. The β-diversity analyses suggested a subtle association between methane production and overall microbiota composition (0.01 < R2 < 0.02). Differential abundance analysis identified 36 genera and 279 KEGGs as significantly associated with methane production (Padj < 0.05). Those genera associated with high methane production were Eukaryota from Alveolata and Fungi clades, while Bacteria were associated with low methane emissions. The genus-level association network showed 2 clusters grouping Eukaryota and Bacteria, respectively. Regarding microbial gene functions, 41 KEGGs were found to be differentially abundant between low- and high-emission animals and were mainly involved in metabolic pathways. No KEGGs included in the methane metabolism pathway (ko00680) were detected as associated with high methane emissions. The KEGG network showed 3 clusters grouping KEGGs associated with high emissions, low emissions, and not differentially abundant in either. A deeper analysis of the differentially abundant KEGGs revealed that genes related with anaerobic respiration through nitrate degradation were more abundant in low-emission animals. Methane emissions are largely associated with the relative abundance of ciliates and fungi. The role of nitrate electron acceptors can be particularly important because this respiration mechanism directly competes with methanogenesis. Whole metagenome sequencing is necessary to jointly consider the relative abundance of Bacteria, Archaea, and Eukaryota in the statistical analyses. Nutritional and genetic strategies to reduce CH4 emissions should focus on reducing the relative abundance of Alveolata and Fungi in the rumen. This experiment has generated the largest ONT ruminal metagenomic dataset currently available.

Project description:Myxococcus xanthus is a soil myxobacterium that exhibits a complex lifecycle with two multicellular stages: cooperative predation and development. During predation, myxobacterial cells produce a wide variety of secondary metabolites and hydrolytic enzymes to kill and consume the prey. It is known that eukaryotic predators, such as ameba and macrophages, introduce copper and other metals into the phagosomes to kill their prey by oxidative stress. However, the role of metals in bacterial predation has not yet been established. In this work, we have addressed the role of copper during predation of M. xanthus on Sinorhizobium meliloti. The use of biosensors, variable pressure scanning electron microscopy, high-resolution scanning transmission electron microscopy, and energy dispersive X ray analysis has revealed that copper accumulates in the region where predator and prey collide. This accumulation of metal up-regulates the expression of several mechanisms involved in copper detoxification in the predator (the P1 B-ATPase CopA, the multicopper oxidase CuoA and the tripartite pump Cus2), and the production by the prey of copper-inducible melanin, which is a polymer with the ability to protect cells from oxidative stress. We have identified two genes in S. meliloti (encoding a tyrosinase and a multicopper oxidase) that participate in the biosynthesis of melanin. Analysis of prey survivability in the co-culture of M. xanthus and a mutant of S. meliloti in which the two genes involved in melanin biosynthesis have been deleted has revealed that this mutant is more sensitive to predation than the wild-type strain. These results indicate that copper plays a role in bacterial predation and that melanin is used by the prey to defend itself from the predator. Taking into consideration that S. meliloti is a nitrogen-fixing bacterium in symbiosis with legumes that coexists in soils with M. xanthus and that copper is a common metal found in this habitat as a consequence of several human activities, these results provide clear evidence that the accumulation of this metal in the soil may influence the microbial ecosystems by affecting bacterial predatory activities.

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Rumen Protozoa Play a Significant Role in Fungal Predation and Plant Carbohydrate Breakdown.

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