Genome-resolved metagenomics uncovers diversity and functional landscapes of the gastrointestinal epithelium-associated microbiome in cattle.

backgroundThe ruminant gastrointestinal epithelium harbors a diverse and functionally critical remains poorly characterized microbial community due to persistent host-derived DNA contamination in metagenomic studies.

resultsWe develop Dilute-MetaSeq (dilution-based metagenomic sequencing), a novel, metagenomic workflow integrating gradient dilution with multiple displacement amplification. Dilute-MetaSeq reduces host DNA interference by 52.4-fold and achieves > 90% microbial sequencing efficiency to assess gastrointestinal epithelium-associated microbiome. This enables the construction of the microbial genome atlas of gastrointestinal epithelium (MGA-GE). This comprehensive resource, comprising 1,907 nonredundant prokaryotic and 5,603 viral genomes, reveals extraordinary microbial diversity and novelty, with 41.4% of prokaryotic and 99.9% of viral genomes representing taxonomically unclassified lineages. Spatial profiling identifies the rumen and reticulum as a biodiversity hotspot dominated by epithelium-adapted Butyrivibrio and methylotrophic Methanomassiliicoccales, while functional annotation uncovers 1,200 biosynthetic gene clusters (primarily RiPPs and NRPSs) and 1,212 viral auxiliary metabolic genes linked to host metabolism modulation. Pangenome analysis of 987 strains, including a novel Butyrivibrio clade with reduced genome sizes, elevated GC content, and butyrate synthesis from amino acid-derived substrates (e.g., glutarate, lysine), highlights metabolic adaptations to the nutrient-scarce epithelial niche compared to digesta-associated microbes.

conclusionsCollectively, the MGA-GE provides transformative insights into host-microbe-virus interactions and establishes a foundation for developing microbiome-based intervention strategies to enhance ruminant health, agricultural productivity, and bioactive discovery.