Accounting for only 8% of Earth's land cover, freshwater wetlands remain the foremost contributors to global methane emissions. Yet the microorganisms and processes underlying methane emissions from wetland soils remain poorly understood. Over a five-year period, we surveyed the microbial membership and in situ methane measurements from over 700 samples in one of the most prolific methane-emitting wetlands in the United States. We constructed a catalog of 2,502 metagenome-assembled genomes (MAGs), with more than half of the 70 bacterial and archaeal phyla sampled containing novel lineages. Integration of these data with 133 soil metatranscriptomes provided a genome-resolved view of the biogeochemical specialization and versatility expressed over wetland soil spatial and temporal gradients. Centimeter-scale depth differences best explained patterns of microbial community structure and transcribed functionalities, even more than land cover or temporal information. Moreover, while extended flooding restructured soil redox, this perturbation failed to reconfigure the transcriptional profiles of methane-cycling microorganisms, contrasting with theoretically expected responses to hydrological perturbations. Co-expression analyses, coupled with depth-resolved methane measurements, revealed the metabolisms and trophic structures most predictive of methane hotspots. Mapping the spatiotemporal transcriptional patterns on this compendium of biogeochemically classified soil-derived genomes begins to untangle the microbial carbon, energy, and nutrient processing contributing to wetland methane production.IMPORTANCESoil microbial ecology is increasingly recognized as essential to climate mitigation, but realizing its full potential requires shifting from static genome inventories to dynamic assessments of microbial activity. This study shows that methane-cycling microbes exhibit stable, depth-stratified expression patterns, even in response to major redox and flooding shifts, undermining assumptions that water-table manipulations common in wetland management can alone reduce methanogenesis. Instead, methane cycling is shaped by spatially organized, transcriptionally active networks involving not only methanogens but also methanotrophs, fermenters, and iron reducers. These findings expose the limitations of genome-only models and highlight the need for soil diagnostics that capture in situ activity. Together, we provide a foundation for developing activity-based microbiome tools, embedding microbial functions into Earth system models, and designing interventions that move beyond "single-lever" strategies and instead work with the structure and dynamics of microbial communities as complex, layered systems.

Methane mitigation is regarded as a critical strategy to combat the scale of global warming. Currently, about 40% of methane emissions originate from microbial sources, which is causing strategies to suppress methanogens, either through direct toxic effects or by diverting their substrates and energy, to gain traction. Problematically, current microbial methane mitigation knowledge derives from rumen studies and lacks detailed microbiome-centered insights, limiting translation across ecosystems. Here we utilize genome-resolved metatranscriptomes and metabolomes to assess the impact of a proposed methane inhibitor, catechin, on greenhouse gas emissions for high-methane-emitting peatlands. In microcosms, catechin drastically reduced methane emissions by 72-84% compared to controls. Longitudinal sampling allowed for reconstruction of a novel catechin degradation pathway involving Actinomycetota and Clostridium, which break down catechin into smaller phenolic compounds within the first 21 days, followed by degradation of phenolic compounds by Pseudomonas_E from days 21 to 35. These genomes also co-expressed hydrogen-uptake genes, suggesting that hydrogenases may act as a hydrogen sink during catechin degradation, depriving methanogens of substrates. This was supported by decreased gene expression in hydrogenotrophic and hydrogen-dependent methylotrophic methanogens under catechin treatment. We also saw reduced gene expression from genomes inferred to be functioning syntrophically with hydrogen-utilizing methanogens. We propose that catechin metabolic redirection effectively starves hydrogen-utilizing methanogens, offering a potent avenue for curbing methane emissions across diverse environments including ruminants, landfills, and constructed or managed wetlands.

Current estimates of wetland contributions to the global methane budget carry high uncertainty, particularly in accurately predicting emissions from high methane-emitting wetlands. Microorganisms mediate methane cycling, yet knowledge of their conservation across wetlands remains scarce. To address this, we integrated 1,118 16S rRNA amplicon datasets (116 new), 305 metagenomes (20 new) that yielded 4,745 medium and high-quality metagenome assembled genomes (MAGs; 617 new), 133 metatranscriptomes, and annual methane flux data across 9 wetlands to create the Multi-Omics for Understanding Climate Change (MUCC) v2.0.0 database. This new resource was leveraged to link microbiome compositional profiles to encoded functions and emissions, with specific focus on methane-cycling populations and the microbial carbon decomposition networks that fuel them. We identified eight methane-cycling genera that were conserved across wetlands, and deciphered wetland specific metabolic interactions across marshes, revealing low methanogen-methanotroph connectivity in high-emitting wetlands. Methanoregula emerged as a hub methanogen across networks and was a strong predictor of methane flux, demonstrating the potential broad relevance of methylotrophic methanogenesis in these ecosystems. Collectively, our findings illuminate trends between microbial decomposition networks and methane flux and provide an extensive publicly available database to advance future wetland research.

Climate change thaws permafrost, which releases greenhouse gases partly from dormant microorganisms awakening and metabolizing organic matter. Though DNA viruses that infect these soil microbes have been studied, little is known on soil RNA viruses, which typically infect microeukaryotes. Here we identify and characterize 2,651 RNA viruses from a 4-year time series of bulk soil metatranscriptomes derived from the climatically fragile Stordalen Mire ecosystem - a long-studied permafrost peatland. RNA virus diversity was structured by habitat (palsa, bog, and fen), and these patterns correlated with pH and carbon dioxide and methane emissions. Further, host prediction, virus-encoded metabolite-transforming and information-processing functions suggested roles in ecosystem-scale carbon fluxes and contribute to greenhouse gases emissions. Together, these RNA virus ecogenomic data in permafrost provide essential baseline information for integration into predictive models to support hypothesis testing. ### Competing Interest Statement The authors have declared no competing interest.

With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

ABSTRACTWhile wetlands are major sources of biogenic methane (CH4), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH4 emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site’s methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; for Methanosarcinales and Methanobacteriales MAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while for Methanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats.IMPORTANCEWetlands are the biggest natural source of atmospheric methane (CH4) emissions, yet we have an incomplete understanding of the suite of microbial metabolism that results in CH4 formation. Specifically, methanogenesis from methylated compounds is excluded from all ecosystem models used to predict wetland contributions to the global CH4 budget. Though recent studies have shown methylotrophic methanogenesis to be active across wetlands, the broad climatic importance of the metabolism remains critically understudied. Further, some methylotrophic bacteria are known to produce methanogenic by-products like acetate, increasing the complexity of the microbial methylotrophic metabolic network. Prior studies of Stordalen Mire have suggested that methylotrophic methanogenesis is irrelevant in situ and have not emphasized the bacterial capacity for metabolism, both of which we countered in this study. The importance of our findings lies in the significant advancement toward unraveling the broader impact of methylotrophs in wetland methanogenesis and, consequently, their contribution to the terrestrial global carbon cycle.

Although river ecosystems constitute a small fraction of Earth's total area, they are critical modulators of microbially and virally orchestrated global biogeochemical cycles. However, most studies either use data that is not spatially resolved or is collected at timepoints that do not reflect the short life cycles of microorganisms. To address this gap, we assessed how viral and microbial communities change over a 48-hour period by sampling surface water and pore water compartments of the wastewater-impacted River Erpe in Germany. We sampled every 3 hours resulting in 32 samples for which we obtained metagenomes along with geochemical and metabolite measurements. From our metagenomes, we identified 6,500 viral and 1,033 microbial metagenome assembled genomes (MAGs) and found distinct community membership and abundance associated with each river compartment (e.g., Competibacteraceae in surfacewater and Sulfurimonadaceae in pore water). We show that 17% of our viral MAGs clustered to viruses from other ecosystems like wastewater treatment plants and rivers. Our results also indicated that 70% of the viral community was persistent in surface waters, whereas only 13% were persistent in the pore waters taken from the hyporheic zone. Finally, we predicted linkages between 73 viral genomes and 38 microbial genomes. These putatively linked hosts included members of the Competibacteraceae, which we suggest are potential contributors to river carbon and nitrogen cycling via denitrification and nitrogen fixation. Together, these findings demonstrate that members of the surface water microbiome from this urban river are stable over multiple diurnal cycles. These temporal insights raise important considerations for ecosystem models attempting to constrain dynamics of river biogeochemical cycles.

Life can be stressful. One way to deal with stress is to simply wait it out. Microbes do this by entering a state of reduced activity and increased resistance commonly called 'dormancy'. But what is dormancy? Different scientific disciplines emphasize distinct traits and phenotypic ranges in defining dormancy for their microbial species and system-specific questions of interest. Here, we propose a unified definition of microbial dormancy, using a broad framework to place earlier discipline-specific definitions in a new context. We then discuss how this new definition and framework may improve our ability to investigate dormancy using multi-omics tools. Finally, we leverage our framework to discuss the diversity of genomic mechanisms for dormancy in an extreme environment that challenges easy definitions - the permafrost.

ABSTRACT Despite the promise of the gut microbiome to predict human health, few studies expose the molecular-scale processes underpinning such forecasts. We mined over 200,000 gut-derived genomes from cultivated and uncultivated microbial lineages to inventory the gut microorganisms and their gene content that control trimethylamine-induced cardiovascular disease. We assigned an atherosclerotic profile to the 6,341 microbial genomes that encoded metabolisms associated with heart disease, creating the Methylated Amine Gene Inventory of Catabolism database (MAGICdb). From microbiome gene expression data sets, we demonstrate that MAGICdb enhanced the recovery of disease-relevant genes and identified the most active microorganisms, unveiling future therapeutic targets. From the feces of healthy and diseased subjects, we show that MAGICdb predicted cardiovascular disease status as effectively as traditional lipid blood tests. This functional microbiome catalog is a public, exploitable resource, designed to enable a new era of microbiota-based therapeutics and diagnostics. IMPORTANCE One of the most-cited examples of the gut microbiome modulating human disease is the microbial metabolism of quaternary amines from protein-rich foods. By-products of this microbial processing promote atherosclerotic heart disease, a leading cause of human mortality globally. Our research addresses current knowledge gaps in our understanding of this microbial metabolism by holistically inventorying the microorganisms and expressed genes catalyzing critical atherosclerosis-promoting and -ameliorating reactions in the human gut. This led to the creation of an open-access resource, the Methylated Amine Gene Inventory of Catabolism database, the first systematic inventory of gut methylated amine metabolism. More importantly, using this resource we deliver here, we show for the first time that these gut microbial genes can predict human disease, paving the way for microbiota-inspired diagnostics and interventions.

Despite the promise of the gut microbiome to forecast human health, few studies expose the microbial functions underpinning such predictions. To comprehensively inventory gut microorganisms and their gene content that control trimethylamine induced cardiovascular disease, we mined over 200,000 gut-derived genomes from cultivated and uncultivated microbial lineages. Creating MAGICdb (Methylated Amine Gene Inventory of Catabolism database), we designated an atherosclerotic profile for 6,341 microbial genomes that encoded metabolisms associated with heart disease. We used MAGICdb to evaluate diverse human fecal metatranscriptome and metaproteome datasets, demonstrating how this resource eases the recovery of methylated amine gene content previously obscured in microbiome datasets. From the feces of healthy and diseased subjects, we show MAGICdb gene markers predicted cardiovascular disease as effectively as traditional blood diagnostics. This functional microbiome catalog is a public, exploitable resource, enabling a new era of microbiota-based therapeutics. ### Competing Interest Statement The authors have declared no competing interest.

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent Km for γ-butyrobetaine and higher apparent Vmax, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.

ABSTRACT Rapidly thawing permafrost harbors ∼30 to 50% of global soil carbon, and the fate of this carbon remains unknown. Microorganisms will play a central role in its fate, and their viruses could modulate that impact via induced mortality and metabolic controls. Because of the challenges of recovering viruses from soils, little is known about soil viruses or their role(s) in microbial biogeochemical cycling. Here, we describe 53 viral populations (viral operational taxonomic units [vOTUs]) recovered from seven quantitatively derived (i.e., not multiple-displacement-amplified) viral-particle metagenomes (viromes) along a permafrost thaw gradient at the Stordalen Mire field site in northern Sweden. Only 15% of these vOTUs had genetic similarity to publicly available viruses in the RefSeq database, and ∼30% of the genes could be annotated, supporting the concept of soils as reservoirs of substantial undescribed viral genetic diversity. The vOTUs exhibited distinct ecology, with different distributions along the thaw gradient habitats, and a shift from soil-virus-like assemblages in the dry palsas to aquatic-virus-like assemblages in the inundated fen. Seventeen vOTUs were linked to microbial hosts (in silico), implicating viruses in infecting abundant microbial lineages from Acidobacteria, Verrucomicrobia, and Deltaproteobacteria, including those encoding key biogeochemical functions such as organic matter degradation. Thirty auxiliary metabolic genes (AMGs) were identified and suggested virus-mediated modulation of central carbon metabolism, soil organic matter degradation, polysaccharide binding, and regulation of sporulation. Together, these findings suggest that these soil viruses have distinct ecology, impact host-mediated biogeochemistry, and likely impact ecosystem function in the rapidly changing Arctic. IMPORTANCE This work is part of a 10-year project to examine thawing permafrost peatlands and is the first virome-particle-based approach to characterize viruses in these systems. This method yielded >2-fold-more viral populations (vOTUs) per gigabase of metagenome than vOTUs derived from bulk-soil metagenomes from the same site (J. B. Emerson, S. Roux, J. R. Brum, B. Bolduc, et al., Nat Microbiol 3:870–880, 2018, https://doi.org/10.1038/s41564-018-0190-y). We compared the ecology of the recovered vOTUs along a permafrost thaw gradient and found (i) habitat specificity, (ii) a shift in viral community identity from soil-like to aquatic-like viruses, (iii) infection of dominant microbial hosts, and (iv) carriage of host metabolic genes. These vOTUs can impact ecosystem carbon processing via top-down (inferred from lysing dominant microbial hosts) and bottom-up (inferred from carriage of auxiliary metabolic genes) controls. This work serves as a foundation which future studies can build upon to increase our understanding of the soil virosphere and how viruses affect soil ecosystem services.

Since the enactment of the 1977 Clean Water Act, biologists have created numerous multimetric assessment tools to evaluate the biological integrity of water resources, using biological criteria. The integrity of Big Walnut Creek, Ohio, has been in flux since the 1955 construction of Hoover Dam, and while current water quality is high, mussel communities in the creek have yet to recover to historical levels. This study sought to determine the cause of the decline in the mussels in the lower section of the creek below the dam. Historical creek data, including a fish-based index of biotic integrity (f-IBI), invertebrate community index (ICI), qualitative habitat evaluation index (QHEI) and modified index of well-being (MIWB), were compared to a recent mussel-IBI for the creek. These data were compared to urbanization data (i.e. percent plant cover) describing land use in 1 km sample regions around each biometric data collection site. Data were analyzed using linear region and student t-tests to understand any correlative relationships with the status of mussel communities. Mussel-IBI data were compared to percent land developed (r=0.225, p>0.01), percent plant cover (r=0.1 p>0.01), road density (r=0.007, p>0.01), census tract data as a measure of population density (r=0.003, p>0.01), riparian zone width (r=0.107, p>0.01) and creek width (r=0.001, p>0.01). Ohio EPA biometric data were compared to percent plant cover data: f-IBI (r=0.185, p>0.01), MIWB (r=0.004, p>0.01), ICI (r=0.028, p>0.01) and QHEI (r=0.265, p>0.01). No significant correlations were found between urbanization and integrity, suggesting that land use does not directly affect the lower section of Big Walnut Creek. Therefore, it is possible (pending future research) that the mussel communities could recover in time, similar to the fish and invertebrate communities in the creek.