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Abstract Background Despite rapid advances in genomic-resolved metagenomics and remarkable explosion of metagenome-assembled genomes (MAGs), the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain largely uncertain, which has profound implications in biotechnology and biogeochemistry. Results In this study, we combined long-read sequencing and metatranscriptomics-guided metabolic reconstruction to provide a genome-wide perspective of carbon mineralization flow from polymers to methane in an anaerobic bioreactor. Our results showed that incorporating long reads resulted in a substantial improvement in the quality of metagenomic assemblies, enabling the effective recovery of 132 high-quality genomes meeting stringent criteria of minimum information about a metagenome-assembled genome (MIMAG). In addition, hybrid assembly obtained 51% more prokaryotic genes in comparison to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction unveiled the remarkable metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acid oxidation through syntrophic relationships with dominant methanogens Methanoregulaceae bin.74 and Methanothrix sp. bin.206. The activity of bin.206 preferring acetate as substrate exceeded that of bin.74 with increasing loading, reinforcing the substrate determinantal role. Conclusion Overall, our study uncovered some key active anaerobic lineages and their metabolic functions in this complex anaerobic ecosystem, offering a framework for understanding carbon transformations in anaerobic digestion. These findings advance the understanding of metabolic activities and trophic interactions between anaerobic guilds, providing foundational insights into carbon flux within both engineered and natural ecosystems. Video Abstract

ABSTRACT To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.IMPORTANCECulture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

ABSTRACT Each prokaryotic domain, Bacteria and Archaea, contains a large and diverse group of organisms characterized by their ultrasmall cell size and symbiotic lifestyles (potentially commensal, mutualistic, and parasitic relationships), namely, Candidatus Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) and DPANN archaea, respectively. Cultivation-based approaches have revealed that Ca. Patescibacteria and DPANN symbiotically interact with bacterial and archaeal partners and hosts, respectively, but that cross-domain symbiosis and parasitism have never been observed. By amending wastewater treatment sludge samples with methanogenic archaea, we observed increased abundances of Ca. Patescibacteria (Ca. Yanofskybacteria/UBA5738) and, using fluorescence in situ hybridization (FISH), discovered that nearly all of the Ca. Yanofskybacteria/UBA5738 cells were attached to Methanothrix (95.7 ± 2.1%) and that none of the cells were attached to other lineages, implying high host dependency and specificity. Methanothrix filaments (multicellular) with Ca. Yanofskybacteria/UBA5738 attached had significantly more cells with no or low detectable ribosomal activity (based on FISH fluorescence) and often showed deformations at the sites of attachment (based on transmission electron microscopy), suggesting that the interaction is parasitic. Metagenome-assisted metabolic reconstruction showed that Ca. Yanofskybacteria/UBA5738 lacks most of the biosynthetic pathways necessary for cell growth and universally conserves three unique gene arrays that contain multiple genes with signal peptides in the metagenome-assembled genomes of the Ca. Yanofskybacteria/UBA5738 lineage. The results shed light on a novel cross-domain symbiosis and inspire potential strategies for culturing CPR and DPANN. IMPORTANCE One highly diverse phylogenetic group of Bacteria, Ca. Patescibacteria, remains poorly understood, but, from the few cultured representatives and metagenomic investigations, they are thought to live symbiotically or parasitically with other bacteria or even with eukarya. We explored the possibility of symbiotic interactions with Archaea by amending wastewater treatment sludge samples that were rich in Ca. Patescibacteria and Archaea with an isolate archaeon that is closely related to a methanogen population abundant in situ (Methanothrix). This strategic cultivation successfully established enrichment cultures that were mainly comprised of Ca. Patescibacteria (family level lineage Ca. Yanofskybacteria/UBA5738) and Methanothrix, in which we found highly specific physical interactions between the two organisms. Microscopic observations based on transmission electron microscopy, target-specific fluorescence in situ hybridization, and metagenomic analyses showed evidence that the interaction is likely parasitic. The results show a novel cross-domain parasitism between Bacteria and Archaea and suggest that the amendment of host Archaea may be an effective approach in culturing novel Ca. Patescibacteria.