High-fidelity long-read metagenomics reveals the metabolic functions of dominant termite gut bacteria

Abstract

Many termites digest lignocellulosic wood using diverse gut microbiota, yet functions are rarely linked to specific symbionts because complete genomes are scarce. We used PacBio HiFi long reads to reconstruct circular single-contig bacterial metagenome-assembled genomes (MAGs) from the gut microbiota of the wood-feeding termite Nasutitermes takasagoensis. From 13.9 Gb of HiFi data, we obtained highly contiguous assemblies, including 11 Breznakiellaceae (Spirochaetota), two Candidatus Fibrimonas (Fibrobacterota), and one Chitinispirillaceae genome—three dominant lineages in this gut ecosystem. The reconstructed genomes encoded extensive carbohydrate-active enzyme repertoires and anaerobic fermentation pathways consistent with lignocellulose degradation. They also carried genes for essential amino acid biosynthesis, while nitrogen-fixation capacity was present in representatives of all three lineages but varied among closely related strains. One Breznakiellaceae genome possessed a complete Wood–Ljungdahl pathway potentially supporting reductive acetogenesis in the presence of external formate, whereas related strains carried incomplete variants likely involved in anabolic C□ metabolism. Differences in CAZymes, C□ metabolism, and nitrogen fixation among closely related strains reveal functional diversification that fragmented MAGs would not resolve. The mapping of metatranscriptomic reads from three biological replicates of the fiber-associated community to the reconstructed genomes provided independent evidence that key predicted metabolic functions are transcriptionally active. Our results demonstrate the power of HiFi metagenomics to recover highly contiguous genomes from complex insect microbiomes and uncover lineage-specific metabolic roles of dominant termite gut symbionts. IMPORTANCE Termites are among the most efficient degraders of plant biomass in nature and rely on specialized gut microbes to convert lignocellulose into nutrients. However, most termite gut bacteria remain uncultivated, limiting our understanding of their metabolic capabilities and ecological roles. Using PacBio HiFi long-read metagenomics, we reconstructed highly contiguous genomes of dominant termite gut bacteria, including the first complete genome from termite-associated Candidatus Fibrimonas. These genomes revealed complementary metabolic functions involved in lignocellulose degradation, reductive acetogenesis, nitrogen metabolism, and energy conservation. Metatranscriptomic data further showed that key predicted metabolic functions were transcriptionally active in the fiber-associated community. Our study shows how long-read metagenomics combined with transcriptomic evidence can resolve the metabolic roles and functional diversification of previously uncultivated microorganisms in complex microbial communities.