Microbiome-metabolite linkages drive greenhouse gas dynamics over a permafrost thaw gradient.

Interactions between microbiomes and metabolites play crucial roles in the environment, yet how these interactions drive greenhouse gas emissions during ecosystem changes remains unclear. Here we analysed microbial and metabolite composition across a permafrost thaw gradient in Stordalen Mire, Sweden, using paired genome-resolved metagenomics and high-resolution Fourier transform ion cyclotron resonance mass spectrometry guided by principles from community assembly theory to test whether microorganisms and metabolites show concordant responses to changing drivers. Our analysis revealed divergence between the inferred microbial versus metabolite assembly processes, suggesting distinct responses to the same selective pressures. This contradicts common assumptions in trait-based microbial models and highlights the limitations of measuring microbial community-level data alone. Furthermore, feature-scale analysis revealed connections between microbial taxa, metabolites and observed CO2 and CH4 porewater variations. Our study showcases insights gained by using feature-level data and microorganism-metabolite interactions to better understand metabolic processes that drive greenhouse gas emissions during ecosystem changes.

Quantifying microbial DNA in metagenomes improves microbial trait estimation.

Shotgun metagenomics is a powerful tool for studying the genomic traits of microbial community members, such as genome size, gene content, etc. While such traits can be used to better understand the ecology and evolution of microbial communities, the accuracy of their estimations can be critically influenced by both known and unknown factors. One factor that can bias trait estimations is the proportion of eukaryotic and viral DNA in a metagenome, as some bioinformatic tools assume that all DNA reads in a metagenome are bacterial or archaeal. Here, we add to a recent debate about the influence of eukaryotic DNA in the estimation of average genome size from a global soil sample dataset using a new bioinformatic tool. Contrary to what was assumed, our reanalysis of this dataset revealed that soil samples can contain a substantial proportion of non-microbial DNA, which severely inflated the original estimates of average genome size. Correcting for this bias significantly improves the statistical support for the negative relationship between average bacterial genome size and soil pH. These results highlight that metagenomes can contain large quantities of non-microbial DNA and that new methods that correct for this can improve microbial trait estimation.

Phylogenetic reconciliation: making the most of genomes to understand microbial ecology and evolution.

In recent years, phylogenetic reconciliation has emerged as a promising approach for studying microbial ecology and evolution. The core idea is to model how gene trees evolve along a species tree and to explain differences between them via evolutionary events including gene duplications, transfers, and losses. Here, we describe how phylogenetic reconciliation provides a natural framework for studying genome evolution and highlight recent applications including ancestral gene content inference, the rooting of species trees, and the insights into metabolic evolution and ecological transitions they yield. Reconciliation analyses have elucidated the evolution of diverse microbial lineages, from Chlamydiae to Asgard archaea, shedding light on ecological adaptation, host-microbe interactions, and symbiotic relationships. However, there are many opportunities for broader application of the approach in microbiology. Continuing improvements to make reconciliation models more realistic and scalable, and integration of ecological metadata such as habitat, pH, temperature, and oxygen use offer enormous potential for understanding the rich tapestry of microbial life.

Borg extrachromosomal elements of methane-oxidizing archaea have conserved and expressed genetic repertoires.

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle.

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.

Methylotrophy in the Mire: direct and indirect routes for methane production in thawing permafrost.

While 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.

Taxonomic and carbon metabolic diversification of Bathyarchaeia during its coevolution history with early Earth surface environment.

Bathyarchaeia, as one of the most abundant microorganisms on Earth, play vital roles in the global carbon cycle. However, our understanding of their origin, evolution, and ecological functions remains poorly constrained. Here, we present the largest dataset of Bathyarchaeia metagenome assembled genome to date and reclassify Bathyarchaeia into eight order-level units corresponding to the former subgroup system. Highly diversified and versatile carbon metabolisms were found among different orders, particularly atypical C1 metabolic pathways, indicating that Bathyarchaeia represent overlooked important methylotrophs. Molecular dating results indicate that Bathyarchaeia diverged at ~3.3 billion years, followed by three major diversifications at ~3.0, ~2.5, and ~1.8 to 1.7 billion years, likely driven by continental emergence, growth, and intensive submarine volcanism, respectively. The lignin-degrading Bathyarchaeia clade emerged at ~300 million years perhaps contributed to the sharply decreased carbon sequestration rate during the Late Carboniferous period. The evolutionary history of Bathyarchaeia potentially has been shaped by geological forces, which, in turn, affected Earth's surface environment.

CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning.

Advances in sequencing technologies and bioinformatics tools have dramatically increased the recovery rate of microbial genomes from metagenomic data. Assessing the quality of metagenome-assembled genomes (MAGs) is a critical step before downstream analysis. Here, we present CheckM2, an improved method of predicting genome quality of MAGs using machine learning. Using synthetic and experimental data, we demonstrate that CheckM2 outperforms existing tools in both accuracy and computational speed. In addition, CheckM2's database can be rapidly updated with new high-quality reference genomes, including taxa represented only by a single genome. We also show that CheckM2 accurately predicts genome quality for MAGs from novel lineages, even for those with reduced genome size (for example, Patescibacteria and the DPANN superphylum). CheckM2 provides accurate genome quality predictions across bacterial and archaeal lineages, giving increased confidence when inferring biological conclusions from MAGs.

A functional microbiome catalog crowdsourced from North American rivers.

Predicting elemental cycles and maintaining water quality under increasing anthropogenic influence requires understanding the spatial drivers of river microbiomes. However, the unifying microbial processes governing river biogeochemistry are hindered by a lack of genome-resolved functional insights and sampling across multiple rivers. Here we employed a community science effort to accelerate the sampling, sequencing, and genome-resolved analyses of river microbiomes to create the Genome Resolved Open Watersheds database (GROWdb). This resource profiled the identity, distribution, function, and expression of thousands of microbial genomes across rivers covering 90% of United States watersheds. Specifically, GROWdb encompasses 1,469 microbial species from 27 phyla, including novel lineages from 10 families and 128 genera, and defines the core river microbiome for the first time at genome level. GROWdb analyses coupled to extensive geospatial information revealed local and regional drivers of microbial community structuring, while also presenting a myriad of foundational hypotheses about ecosystem function. Building upon the previously conceived River Continuum Concept 1 , we layer on microbial functional trait expression, which suggests the structure and function of river microbiomes is predictable. We make GROWdb available through various collaborative cyberinfrastructures 2, 3 so that it can be widely accessed across disciplines for watershed predictive modeling and microbiome-based management practices.

Anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens' has a pleomorphic life cycle.

'Candidatus Methanoperedens' are anaerobic methanotrophic (ANME) archaea with global importance to methane cycling. Here meta-omics and fluorescence in situ hybridization (FISH) were applied to characterize a bioreactor dominated by 'Candidatus Methanoperedens nitroreducens' performing anaerobic methane oxidation coupled to nitrate reduction. Unexpectedly, FISH revealed the stable co-existence of two 'Ca. M. nitroreducens' morphotypes: the archetypal coccobacilli microcolonies and previously unreported planktonic rods. Metagenomic analysis showed that the 'Ca. M. nitroreducens' morphotypes were genomically identical but had distinct gene expression profiles for proteins associated with carbon metabolism, motility and cell division. In addition, a third distinct phenotype was observed, with some coccobacilli 'Ca. M. nitroreducens' storing carbon as polyhydroxyalkanoates. The phenotypic variation of 'Ca. M. nitroreducens' probably aids their survival and dispersal in the face of sub-optimal environmental conditions. These findings further demonstrate the remarkable ability of members of the 'Ca. Methanoperedens' to adapt to their environment.

Diverse sediment microbiota shape methane emission temperature sensitivity in Arctic lakes.

Northern post-glacial lakes are significant, increasing sources of atmospheric carbon through ebullition (bubbling) of microbially-produced methane (CH4) from sediments. Ebullitive CH4 flux correlates strongly with temperature, reflecting that solar radiation drives emissions. However, here we show that the slope of the temperature-CH4 flux relationship differs spatially across two post-glacial lakes in Sweden. We compared these CH4 emission patterns with sediment microbial (metagenomic and amplicon), isotopic, and geochemical data. The temperature-associated increase in CH4 emissions was greater in lake middles-where methanogens were more abundant-than edges, and sediment communities were distinct between edges and middles. Microbial abundances, including those of CH4-cycling microorganisms and syntrophs, were predictive of porewater CH4 concentrations. Results suggest that deeper lake regions, which currently emit less CH4 than shallower edges, could add substantially to CH4 emissions in a warmer Arctic and that CH4 emission predictions may be improved by accounting for spatial variations in sediment microbiota.

Successional dynamics and alternative stable states in a saline activated sludge microbial community over 9 years.

BACKGROUND: Microbial communities in both natural and applied settings reliably carry out myriads of functions, yet how stable these taxonomically diverse assemblages can be and what causes them to transition between states remains poorly understood. We studied monthly activated sludge (AS) samples collected over 9 years from a full-scale wastewater treatment plant to answer how complex AS communities evolve in the long term and how the community functions change when there is a disturbance in operational parameters. RESULTS: Here, we show that a microbial community in activated sludge (AS) system fluctuated around a stable average for 3 years but was then abruptly pushed into an alternative stable state by a simple transient disturbance (bleaching). While the taxonomic composition rapidly turned into a new state following the disturbance, the metabolic profile of the community and system performance remained remarkably stable. A total of 920 metagenome-assembled genomes (MAGs), representing approximately 70% of the community in the studied AS ecosystem, were recovered from the 97 monthly AS metagenomes. Comparative genomic analysis revealed an increased ability to aggregate in the cohorts of MAGs with correlated dynamics that are dominant after the bleaching event. Fine-scale analysis of dynamics also revealed cohorts that dominated during different periods and showed successional dynamics on seasonal and longer time scales due to temperature fluctuation and gradual changes in mean residence time in the reactor, respectively. CONCLUSIONS: Our work highlights that communities can assume different stable states under highly similar environmental conditions and that a specific disturbance threshold may lead to a rapid shift in community composition. Video Abstract.

Recoding of stop codons expands the metabolic potential of two novel Asgardarchaeota lineages.

Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative heterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including the deep subsurface, brackish shallow lakes, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four additional lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Sifarchaeia class nov. and Ca. Jordarchaeia class nov., derived from the gods Sif and Jord in Norse mythology. Metabolic inference suggests that both classes represent hetero-organotrophic acetogens, which also have the ability to utilise methyl groups such as methylated amines, with acetate as the probable end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e., stop codon recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding, on the other hand, is restricted to Sifarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic archaeal lineage with an inferred complete Pyl machinery, likely providing members of this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of bacteria and eukaryotes, in both newly described classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.

Sequenceserver: A Modern Graphical User Interface for Custom BLAST Databases.

Comparing newly obtained and previously known nucleotide and amino-acid sequences underpins modern biological research. BLAST is a well-established tool for such comparisons but is challenging to use on new data sets. We combined a user-centric design philosophy with sustainable software development approaches to create Sequenceserver, a tool for running BLAST and visually inspecting BLAST results for biological interpretation. Sequenceserver uses simple algorithms to prevent potential analysis errors and provides flexible text-based and visual outputs to support researcher productivity. Our software can be rapidly installed for use by individuals or on shared servers.

An evolving view of methane metabolism in the Archaea.

Methane is a key compound in the global carbon cycle that influences both nutrient cycling and the Earth's climate. A limited number of microorganisms control the flux of biologically generated methane, including methane-metabolizing archaea that either produce or consume methane. Methanogenic and methanotrophic archaea belonging to the phylum Euryarchaeota share a genetically similar, interrelated pathway for methane metabolism. The key enzyme in this pathway, the methyl-coenzyme M reductase (Mcr) complex, catalyses the last step in methanogenesis and the first step in methanotrophy. The discovery of mcr and divergent mcr-like genes in new euryarchaeotal lineages and novel archaeal phyla challenges long-held views of the evolutionary origin of this metabolism within the Euryarchaeota. Divergent mcr-like genes have recently been shown to oxidize short-chain alkanes, indicating that these complexes have evolved to metabolize substrates other than methane. In this Review, we examine the diversity, metabolism and evolutionary history of mcr-containing archaea in light of these recent discoveries.

Divergent methyl-coenzyme M reductase genes in a deep-subseafloor Archaeoglobi.

The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for ß-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.

Discovery and ecogenomic context of a global Caldiserica-related phylum active in thawing permafrost, Candidatus Cryosericota phylum nov., Ca. Cryosericia class nov., Ca. Cryosericales ord. nov., Ca. Cryosericaceae fam. nov., comprising the four species Cryosericum septentrionale gen. nov. sp. nov., Ca. C. hinesii sp. nov., Ca. C. odellii sp. nov., Ca. C. terrychapinii sp. nov.

The phylum Caldiserica was identified from the hot spring 16S rRNA gene lineage 'OP5' and named for the sole isolate Caldisericum exile, a hot spring sulfur-reducing chemoheterotroph. Here we characterize 7 Caldiserica metagenome-assembled genomes (MAGs) from a thawing permafrost site in Stordalen Mire, Arctic Sweden. By 16S rRNA and marker gene phylogenies, and average nucleotide and amino acid identities, these Stordalen Mire Caldiserica (SMC) MAGs form part of a divergent clade from C. exile. Genome and meta-transcriptome and proteome analyses suggest that unlike Caldisericum, the SMCs (i) are carbohydrate- and possibly amino acid fermenters that can use labile plant compounds and peptides, and (ii) encode adaptations to low temperature. The SMC clade rose to community dominance within permafrost, with a peak metagenome-based relative abundance of ∼60%. It was also physiologically active in the upper seasonally-thawed soil. Beyond Stordalen Mire, analysis of 16S rRNA gene surveys indicated a global distribution of this clade, predominantly in anaerobic, carbon-rich and cold environments. These findings establish the SMCs as four novel phenotypically and ecologically distinct species within a single novel genus, distinct from C. exile clade at the phylum level. The SMCs are thus part of a novel cold-habitat phylum for an understudied, globally-distributed superphylum encompassing the Caldiserica. We propose the names Candidatus Cryosericota phylum nov., Ca. Cryosericia class nov., Ca. Cryosericales ord. nov., Ca. Cryosericaceae fam. nov., Ca. Cryosericum gen. nov., Ca. Cryosericum septentrionale sp. nov., Ca. C. hinesii sp. nov., Ca. C. odellii sp. nov., and Ca. C. terrychapinii sp. nov.

Soil Viruses Are Underexplored Players in Ecosystem Carbon Processing.

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.