ISME Communications

Myxococcota are globally distributed bacteria renowned for their remarkable ecological and biotechnological significance due to their complex lifestyles, social behaviour, and secondary metabolite production. Despite their ubiquity in diverse environments, including soil, marine, and extreme habitats, their diversity and ecological roles remain underexplored. Here, we utilized the Microflora Danica dataset, encompassing >10,000 metagenomes and >400 rRNA gene datasets from various environments in Denmark, to investigate the distribution, diversity, and metabolic potential of Myxococcota. We show that Myxococcota are ubiquitous but strongly structured by environment, with soil-associated lineages enriched in predatory and multicellular development traits, whereas aquatic-associated taxa exhibit alternative lifestyles, including anaerobic metabolism and phototrophy. Comparative genomic analysis reveals widespread potential for secondary metabolite production, hydrocarbon degradation, and organohalide transformation, alongside diverse contribution to carbon and nutrient cycling. Together, these findings redefine Myxococcota as a functionally diverse and ecologically differentiated phylum, extending beyond canonical predation and multicellularity, and underscore their promise as large reservoir of unexplored functional potential for biotechnological applications in drug discovery and environmental remediation.

Microbial cultivation remains essential for understanding the physiology, ecology, and biotechnological potential of environmental microbes, yet conventional plate-based methods (CPM) recover only a minute fraction of the environmental microbiome. Polar regions, particularly Arctic soils, represent unique reservoirs of "microbial dark matter" that remain challenging to cultivate, owing to oligotrophic conditions, low temperatures and freeze-thaw cycles that impose severe physiological constraints on microbial growth. Here, we report the first systematic application of microfluidic droplet technology (MDT) to Arctic active-layer soil microbiota and benchmark its performance against CPM using identical starting cell numbers, R2A medium, and incubation at 15{degrees}C. MDT achieved 6.5- to 8.1-fold higher recovery rates than CPM and improved isolation throughput by >180-fold. Near-full-length 16S rRNA gene sequencing (PacBio) revealed that MDT recovered significantly higher taxonomic richness across all taxonomic levels, with 256 genera detected in the high-cell-input group (DropAS_H) versus 211 in the corresponding plate group (PlateAS_H). Notably, MDT yielded a more even community distribution, significantly reducing the dominance of fast-growing copiotrophs such as Pseudomonas and Flavobacterium. Moreover, approximately 50% of sequences from MDT were affiliated with potential novel species (<98.46% identity to type strains), and 27% with potential novel genera (<95% identity). Strain verification by Sanger sequencing confirmed 12 of 17 isolates as candidate novel species, among which one strain represented a potential novel genus within Devosiaceae. This study demonstrates that MDT is a powerful platform for accessing the uncultured majority of polar soil microbiota and establishes a pipeline for high-throughput isolation of novel cold-adapted bacteria. IMPORTANCEArctic soils harbor a vast reservoir of microbial diversity that remains largely inaccessible due to the extreme oligotrophic conditions and low temperatures characteristic of polar environments, leading to slow growth rates and extended lag phases in most microbes. Conventional plate-based methods (CPM) inherently favor fast-growing copiotrophs while suppressing rare or slow-growing lineages. Here we demonstrate that microfluidic droplet technology (MDT) overcomes these fundamental constraints, representing its first systematic application to polar microbiology. By physically isolating individual cells into nanoliter-scale bioreactors, MDT mitigates interspecific competition, thereby releasing slow-growing and oligotrophic taxa that are otherwise outcompeted in bulk cultures. The water-in-oil emulsion format further enables extended low-temperature incubation without evaporative loss or airborne fungal contamination, issues that frequently compromise long-term plate-based cultivation of Arctic samples. Relative to CPM, MDT increased recovery rates by >6-fold and isolation throughput by >180-fold, while markedly enhanced both taxonomic richness and evenness. Exclusively recovered by MDT, the oligotrophic genus Caulobacter and numerous cold-adapted genera underscore that MDT accesses physiologically distinct fractions of the cryospheric microbiome. Furthermore, the integration of near-full-length 16S rRNA gene sequencing with MDT cultivation assessment provided substantially improved phylogenetic resolution for species-level identification and novel taxon delineation. Collectively, these findings establish MDT as a transformative platform for cryospheric culturomics, accelerating the construction of comprehensive polar strain collections essential for understanding cold-adaptation mechanisms and exploiting the biotechnological potential of Earths frozen microbiomes.

Chytrids are unicellular fungi that infect and degrade phytoplankton as parasites or saprotrophs. They impact not only food availability and quality in surface waters but also carbon cycling and sequestration. So far, their ecological significance has mostly been investigated for freshwater environments, whereas observations for marine environments are scarce -- even though chytrids can be highly abundant there, too (as shown for the Arctic Ocean). To test the chytrids' potential to control phytoplankton dynamics in the Arctic Ocean, we analysed metabarcoding and photosynthetic pigment data from two expeditions, Tara Polar Circle and MOSAiC; the latter providing a dense sampling transect across one year from the under-ice water column and sea ice samples. The phytoplankton communities of both environments were dominated by diatoms, with strong seasonal effects indicating blooms in the water column. Chytrids dominated fungal communities in both environments and revealed a strong cryo-pelagic coupling. They were especially abundant during the sea ice melt in water samples and in ice-associated (sympagic) samples, where they represented >2% and up to 61%, respectively, of all combined reads assigned to chytrids or phytoplankton. Co-occurrences of the two most abundant chytrid taxa with some of the most abundant diatom taxa and niche differentiation from other potential diatom parasites are consistent with the chytrids' critical role in controlling diatom blooms, especially in sympagic habitats.

BackgroundCloud water harbors diverse microbial communities despite its extreme oligotrophic conditions. However, the ecological and evolutionary dynamics of viruses in these transient atmospheric habitats remain poorly understood. Clouds have traditionally been regarded primarily as passive carriers of microorganisms rather than as active ecological environments supporting microbial interactions. In this study, cloud water was sampled at Mount Verde, Cape Verde Islands (744 m a.s.l.). We performed metagenomic analyses of iron-flocculated cloud water alongside genome analyses of a bacterial isolate and metagenome-assembled genomes using established bioinformatic approaches. Viral diversity, virus-host interactions, metabolic functions, genetic adaptations, and viral population dynamics across cloud events were investigated. In addition, UV-B resistance experiments were conducted for a novel cloud-water isolate. ResultsWe isolated 24 cloud water bacteria, including four novel species lineages, and recovered 62 high-quality metagenome-assembled genomes, including 10 novel species lineages. We identified 458 viral operational taxonomic units and 237 virus-host linkages across diverse prokaryotic hosts, revealing active viral predation across diverse bacterial taxa. In addition, CRISPR spacer matches from isolates of novel bacterial lineages such as Deinococcus nubigenus MPC36 were found. Viruses carried genes involved in host adaptation to environmental stressors, including cold-shock response, UV radiation resistance, and osmotic stress. In addition, viral populations exhibited SNP-level microdiversity and shifts in single-nucleotide variant composition across temporally proximate cloud events, indicating rapid population turnover. Experimental characterization of the cloud isolate Curtobacterium nubigenum MPC39 further revealed pronounced resistance to UV-B radiation and the presence of an inducible prophage, Curtobacterium phage vB_CnuS_Cirrus1 assigned to the new viral family Nebulaviridae, which could be validated in transmission electron microscopy. Reconstructed genomes from cloud-associated bacteria encoded carbon monoxide dehydrogenase genes and UV resistance genes, suggesting trace gas metabolism and enhanced UV protection as survival strategies in oligotrophic cloud droplets. In silico replication rates estimated using iRep were consistent with active bacterial replication at the time of sampling. ConclusionsTogether, these findings demonstrate that clouds are not merely passive carriers of microorganisms, but dynamic atmospheric ecosystems in which virus-host interactions shape microbial diversity and contribute to microbial turnover, atmospheric dispersal, and cloud-water biogeochemistry.

Marine microbiology has long considered ultrasmall (<0.2 {micro}m) size fractions as virus-dominated, yet emerging evidence suggests potential prokaryotic activity with implications for global biogeochemical cycles. Here, we present the first comprehensive genome-resolved survey of this fraction, built on 4,058 metagenome-assembled genomes (MAGs) representing 1,152 species across Bacteria (90.7%) and Archaea (9.3%), dominated by Pseudomonadota, Bacteroidota, and Nanoarchaeota. Over 490 non-redundant MAGs occurred exclusively in the ultrasmall fraction, with 66.8% representing novel taxa, particularly abundant in Arctic surface waters. Beyond known ultrasmall organisms (e.g., DPANN archaea and Patescibacteria), which show expected genomic and metabolic reduction, functional annotation revealed diverse metabolic potential across nitrogen, sulfur, and carbon cycling. Focusing on nitrogen fixation, we identified 13 non-cyanobacterial diazotroph species with complete nitrogenase operons spanning four bacterial classes. These resolved into two ecotypes: cosmopolitan low-abundance taxa, and Arctic ultrasmall-restricted strains reaching up to 16.7% relative abundance. Two lineages (50-400-T64 and Novosphingobium) showed ultrasmall exclusive bipolar distributions across Arctic and Southern Oceans. Beyond nitrogen fixation, these diazotrophs encoded pathways for denitrification, DNRA, sulfur oxidation, and carbon fixation. Our findings reveal an overlooked reservoir of microbial diversity including diazotrophs in the ultrasmall ocean microbiome, with significant implications for polar nitrogen budgets and global biogeochemical models.

Root exudates influence soil microbial community assembly and function, yet plant species-specific effects on microbially-driven metabolic transformations remain insufficiently characterized under controlled conditions. Here, we present a multi-omic dataset of exudate-microbe interactions across six plant species, including tallgrass prairie species (Helianthus pauciflorus, Carex brevior, Eragrostis spectabilis, Astragalus canadensis, and Panicum virgatum) and a model grass (Brachypodium distachyon), generated using an in vitro incubation experiment. Sterile root exudates were used to amend a soil-mimicking medium inoculated with a native tall-grass prairie soil microbiome. The dataset includes full-length 16S rRNA gene sequencing for microbial community profiling, untargeted LC-MS/MS metabolomics for exometabolomic profiling, and optical density measurements of microbial growth. We describe the axenic plant growth protocols, experimental design, data acquisition, processing workflows, and technical validation. This dataset provides a resource for investigating microbially mediated exometabolite transformations across diverse plant species to help understand rhizosphere processes, microbiome assembly, and ecosystem function.

Agriculture is a major source of anthropogenic greenhouse-gas emissions, being the largest source of nitrous oxide (N2O), an extremely potent greenhouse gas and ozone-depleting agent. Soil N2O emissions are largely driven by microbial nitrification, in which ammonia-oxidizing microorganisms catalyze the rate-limiting oxidation of ammonia to nitrite. Nitrification not only mediates N2O fluxes but also reduces fertilization efficiency and contributes to eutrophication through nitrate leaching. Bacteriophage (phage)-based control of microbial communities is rapidly garnering interest in a number of fields; however, phages infecting ammonia-oxidizers are largely uncharacterized, with only one lytic phage having been described, limiting the potential for phage-mediated nitrification inhibition. Here, we show the largest set of phages infecting ammonia-oxidizing bacteria (AOB) to date: 45 dsDNA phages identified from urban wastewater, infecting four AOB species, with 16 demonstrating cross-genus host ranges and capable of eliminating nitrification activity in liquid cultures. Phylogenetic and taxonomic analyses revealed six proposed families of Caudoviricetes and numerous monophyletic clades, likely representing higher-level lineages. Structure-guided genome annotation revealed these phages to carry diverse and seldom-seen auxiliary metabolic genes, ranging from a complete ABC transporter cassette to a large antimicrobial resistance gene cluster. These results unveil the previously unrecognized diversity of AOB phages and their potential to alter host physiology. Our data demonstrates a broad taxonomic and functional repertoire of cultured AOB phages, greatly expanding the panel of known AOB phages, suggesting that viruses play a more significant and complex role in nitrification than previously understood. Moreover, we outline an effective methodological framework for isolating AOB phages from environmental samples. These results will help reframe our understanding of environmental nitrification and enable intensified selection and use of phages for its control.

Microbial communities are foundational to marine ecosystem function, yet their diversity is often obscured by broad taxonomic groupings and relative-abundance surveys that mask the dynamics of individual populations. This limitation is especially important across estuarine-coastal gradients, where microbial standing stocks, environmental conditions, and community composition vary sharply over space and time. Here, we used quantitative, genome-resolved metagenomics to examine microbial population dynamics across a one-year estuary-to-ocean transect spanning the Neuse River Estuary, Pamlico Sound, and adjacent North Atlantic shelf waters. Internal standard normalization enabled absolute abundance estimates for single-copy genes and metagenome-assembled genomes (MAGs), allowing individual populations to be tracked as genome equivalents per liter. Bacterial standing stocks were higher in estuarine waters, and communities varied primarily with salinity and season. We recovered 415 MAGs, including 386 bacterial genomes that represented, on average, 52% of bacterial genome equivalents, along with archaeal and eukaryotic representatives. Many abundant MAGs lacked close reference genomes, demonstrating that numerically important coastal populations remain poorly characterized. Genome-resolved abundances revealed pronounced niche partitioning among closely related taxa, including seasonal and spatial turnover of Synechococcus, Cyanobium, and Vulcanococcus populations associated with distinct pigment-defined cytometric groups. Rhodobacteraceae MAGs also showed population-specific correlations with picoeukaryotic MAGs, including a winter offshore Planktomarina population that reached 18% of total bacterial genome equivalents during a Micromonas-associated bloom. By providing absolute population abundances, this study transformed coastal microbiome surveys into numerical frameworks for resolving microbial population structure, ecological interactions, and biogeochemical relevance across dynamic environmental gradients. IMPORTANCEEstuarine and coastal waters contain diverse microbial communities that help regulate food webs and the cycling of carbon and nutrients, but many of the individual microbial populations responsible for these processes remain poorly understood. In this study, we examined bacteria, archaea, and small algae across the Neuse River Estuary, Pamlico Sound, and nearby coastal ocean waters over one year. By measuring the absolute abundance of individual microbial genomes, rather than only their relative proportions, we showed that closely related populations can have very different seasonal and spatial patterns. This was especially clear for cyanobacteria related to Synechococcus and heterotrophic bacteria in the family Rhodobacteraceae, which showed distinct population dynamics and associations with small algae. These results demonstrate how quantitative genome-resolved measurements can reveal hidden microbial population structure and improve our understanding of how microorganisms shape coastal ecosystem function.

Soils are recognized as reservoirs of antibiotic resistance genes (ARGs) with the potential to transfer to clinical pathogens, creating antimicrobial resistance (AMR) that poses a threat to human health. While large-scale AMR surveys have profiled how diverse biomes shape soil resistomes, less is known about the influence of specific soil properties. Here, we combined metagenomics and 16S rRNA amplicon sequencing with isolate-based approaches to investigate drivers of soil AMR across a tropical watershed from beach to mountaintop in Waimea Valley, Oahu, Hawai{square}i. We leveraged functional- and taxonomic-classification of resistances to unravel how soil properties interact with bacterial taxa to structure resistomes. Metagenomic- and isolate-resistomes showed remarkable consistency, including a general gradient of increasing AMR from ridge to beach. Resistome functional composition was significantly correlated with total bacterial community structure. The relationship between resistances and soil properties was primarily dictated by taxonomic composition of each resistance. Rifampin- and Vancomycin-ARGs associated with Actinomycetes negatively correlated with soil physical properties, while resistant genes and isolates from Gammaproteobacteria positively correlated with enzymatic activity metrics. These findings indicate that soil properties structure the resistome indirectly through taxonomic filtering of microbial hosts and challenge the notion that AMR is decoupled from phylogenetic relatedness.

Polinton-like viruses (PLVs) are an emerging group of double-stranded DNA viruses of microbial eukaryotes whose ecological roles in marine ecosystems remain poorly understood. Using a five-year monthly viromic time series from the San Pedro Ocean Time-series (SPOT), we investigated the diversity, temporal dynamics, and functional potential of PLVs in a coastal marine ecosystem. We identified 2,355 distinct PLV populations, revealing PLVs to be a highly abundant and diverse component of the marine virosphere. Phylogenetic analyses resolved multiple major PLV clades, including abundant Group X/Trimcap PLVs characterized by triplicate major capsid proteins, supporting the widespread occurrence of this unusual viral architecture in marine PLVs. Approximately half of PLV populations exhibited significant repeatable seasonal dynamics, partitioning into numerous chronotypes that reflect highly modular temporal niches. PLV abundance correlated positively with multiple productivity-linked environmental variables, including nitrate, particulate organic carbon, and primary productivity, suggesting close coupling between PLVs and seasonal ecosystem productivity. Functional analyses further identified diverse auxiliary metabolic genes in many PLV genomes involved in carbohydrate, lipid, redox, and nucleotide metabolism, with strong phylogenetic structuring across PLV clades. Together, these findings demonstrate that PLVs are abundant, functionally diverse, and ecologically dynamic members of marine viral communities, and suggest they are important yet underappreciated regulators of protist ecology and evolution in marine ecosystems.

Coastal marine environments are increasingly recognised as reservoirs of antimicrobial-resistant (AMR) pathogens. However, it remains challenging to recover high-quality genomes of clinically relevant bacteria present at low abundance from complex natural systems. Here, we applied culture-enriched metagenomics to systematically track the diversity and dynamics of major AMR pathogens within the coastal marine system of St. Johns Island, Singapore, as a model ecosystem for pathogen surveillance. Selective media-based enrichment recovered 773 metagenome-assembled genomes (MAGs) from 92 multi-matrix environmental samples, which includes coastal water, sediment, and seaweed, capturing diverse AMR ESKAPE and Vibrio species. Distinct bacterial signatures and dispersal patterns were observed in each niche, for example, microbes that signal human impact was detected at the beach, while fish-associated pathogens were present at the aquaculture facility outlet. Notably, the high-quality MAGs enabled subspecies-level identification and supported the AMR gene detection across six distinct coastal habitats. Detailed differences in the recovery of specific pathogens across enrichment media were also identified, demonstrating the methods efficacy in finding media suitable for surveillance of specific organisms, such as deciding between liquid or solid formulations. MAGs recovered from culture-enriched metagenomics were highly similar to genomes obtained from pure isolates, as demonstrated for Klebsiella pneumoniae. The preserved culture-enriched stocks were capable of recovering organisms of interest when individual isolates were required for further study. Overall, our findings highlight the utility of culture-enriched metagenomics as a cost-effective, sensitive approach to uncovering the genomic landscape of pathogens with environmental reservoirs, with implications for AMR surveillance and ecological risk assessment.

Fungal hyphae form spatially confined interfaces in soil that mediate close associations with bacteria, collectively referred to as the hyphosphere. Despite its recognized ecological importance, experimental access to hyphosphere-associated microbial communities under realistic soil and plant-associated conditions has remained limited. Here, we present a soil-mimetic microcosm that enables controlled reconstruction and recovery of hyphosphere bacterial communities embedded within plant-associated soil. The system integrates field-derived soil, a native soil microbial inoculum, living cotton seedlings, and a spatially constrained fungal inoculum housed within sterile cell-strainer assemblies, permitting hyphal extension into soil while preserving a recoverable fungal-soil boundary. Using the soil-borne plant pathogen Fusarium oxysporum f. sp. vasinfectum as a model filamentous fungus, we show that the microcosm enables reproducible recovery of hypha-associated soil microaggregates containing physically attached bacterial cells. Full-length 16S rRNA profiling revealed pronounced reductions in bacterial richness and evenness in hyphosphere samples relative to bulk and rhizosphere soils, consistent with recruitment of a restricted subset of the surrounding microbiota. Ordination analyses demonstrated clear compositional separation between soil and hyphosphere compartments, with convergence of hypha-associated communities across bulk and rhizosphere contexts. Phylogenetic turnover analyses indicated phylogenetic structuring, whereas taxonomic analyses identified a conserved set of bacterial genera consistently associated with hyphae alongside compartment-specific taxa influenced by soil and plant context. Together, these findings establish the novel hyphal release-and-capture microcosm as a reproducible, ecologically grounded platform for studying hyphosphere-associated bacterial communities in plant-associated soils.

Nitrous oxide (N2O) is a potent greenhouse gas and is largely produced by incomplete denitrification. Although we know many of the microbial species that denitrify, we are still unable to reliably predict N2O production from soils. Recent work in microbial ecology has shown that when key microbes are considered as members of functional ensembles rather than isolated, the predictive power linking their activity to emergent properties increases dramatically. We hypothesized that the active microbial community during high N2O production would be taxonomically distinct from the inactive portion and increases in N2O production rates would correlate more strongly with increased abundance across multiple active taxa than with dominance by a single active species. We conducted a microcosm experiment where agricultural soil was incubated in anaerobic vials for up to 15 hours while tracking N2O production. Using bioorthogonal non-canonical amino acid tagging paired with fluorescence-activated cell sorting and 16S rRNA amplicon sequencing (BONCAT-FACS-Seq), we probed the active subset of the microbial community throughout the incubation period. Analysis of 16S rRNA gene amplicons revealed that the active and inactive fractions contained distinct taxa, and the taxonomic composition of the active fraction shifted over time. We found that less than 1% of the microbial community was responsible for N2O flux rates as high as 3.84 {micro}g N2O-N g dry soil-1 hr-1. The level of activity (median fluorescent intensity of active cells) correlated well with N2O production rates. The Ensemble Quotient Optimization for Microbiomes (mEQO) tool was used to identify an ensemble of eight organisms whose combined abundance best correlated with N2O fluxes. Overall, our results reveal that N2O fluxes are driven not by changes in a single taxon but by shifting ensembles of active microorganisms whose combined functional potential supports consistent emissions. This study applied a novel conceptual and methodological framework with a distinct focus on the active microbial community, rather than the entire community; if our observation that N2O flux rates are correlated with an ensemble of organisms is broadly confirmed, then framing denitrification as a community trait may increase predictability of this key process.

Protistan predators are key regulators of microbial food webs, yet most are considered to occupy relatively narrow trophic niches. Here, we demonstrate that Mayorella spp. (Amoebozoa), isolated from marine and freshwater environments, exhibits exceptional trophic breadth spanning multiple trophic levels. Live-cell imaging revealed predation on bacteria, algae, dinoflagellates, diatoms, flagellates, ciliates, and multicellular prey including rotifers. Large or filamentous prey were engulfed whole or mechanically fragmented during ingestion. Notably, Mayorella consumed both trophozoites and cysts of free-living amoebae (Naegleria and Acanthamoeba), with clear digestion of cyst contents. Dense cultures showed aggregation around large prey and facultative cannibalism. Ingestion of microplastic-like particles occurred without evidence of digestion. Predator cell size and population density increased markedly when feeding on protist or mixed prey relative to bacterial diets, indicating pronounced trophic plasticity. These findings establish Mayorella as a broad-spectrum, cross-trophic predator with the capacity to exert top-down effects across microbial food webs and suggest a previously underappreciated role in the suppression of pathogenic free-living amoebae.

Competition for a single limiting resource is expected to lead to competitive exclusion, yet diverse microbial communities persist even in nutrient-poor environments. Cross-feeding of essential metabolites is one mechanism that can promote coexistence between species, but its contribution is difficult to pinpoint experimentally. Here, we studied a prototroph-auxotroph pair growing on a single carbon source in chemostats. In minimal medium, the prototroph Comamonas testosteroni (Ct) supplied thiamine to the thiamine-auxotroph Ochrobactrum anthropi (Oa), allowing stable coexistence in agreement with consumer-resource theory. Contrary to our expectation that supplying thiamine would remove the dependency and lead to exclusion of Ct, coexistence persisted even when thiamine was supplemented. Our theoretical anlaysis showed that coexistence between competitors can be maintained by trace concentrations of an additional metabolite if it is taken up at sufficiently high affinity by the weaker competitor. Consistent with this prediction, targeted metabolomics and spent-medium assays identified growth-enhancing compounds at micromolar concentrations in Oa spent medium and as residues in fresh medium. Model analysis further showed that such weak positive effects can qualitatively change coexistence outcomes in chemostats while remaining undetected in standard batch interaction assays. Together, our results show that trace metabolites and subtle positive effects can reshape coexistence outcomes and should be incorporated into ecological models and interaction measurements.

Virus-host interactions are fundamental drivers of microbial community structure, yet whether viral ecological niches are confined within individual host niches (nested host niche scenario) or span multiple hosts and exceed any single host niche (expanded host niche scenario) remains poorly understood. To explore these patterns, we characterized prokaryotic and viral distributions and predicted virus-host interactions along a salinity gradient at Bras del Port salterns (Spain), ranging from seawater (3.6% salinity) to salt saturation (39.0%). We analyzed metagenomes and viromes from six ponds supplemented by 27 additional published viromes from the same hypersaline system, recovering 170 metagenome-assembled genomes (MAGs) dereplicated at the genomospecies level (MAGs clustered at 95 % average nucleotide identity), approximately 55,000 viral operational taxonomic units (vOTUs), and nearly 4,000 predicted virus-host pairs. Viruses exhibited broader niches than their putative hosts at the highest salinities, while at lower salinities the pattern was reversed or inconsistent depending on the site, and niche breadths of both viruses and hosts increased steadily toward higher salinities. Host taxonomy at the class level and below was the primary driver of viral genomic clustering, explaining more variance than salinity provenance (approximately 30% vs. approximately 19%), while the contribution of salinity to viral genomic composition appeared indirect, mediated through the salinity-driven distribution of distinct host classes rather than direct environmental filtering of viral sequences. Together, these findings support the expanded host niche scenario as the predominant virus-host interaction strategy, with evolutionary and ecological dynamics jointly shaped by salinity and host identity.

Amplicon sequencing remains the benchmark technique for characterizing microbial communities, but the limitations of PCR bias and single-locus targets conspire to limit conclusions. Metagenomics, the primary alternative, shares with amplicon sequencing challenges of economic scaling at adequate sequencing depth. Targeted enrichment strategies can improve the data resolution and economics of sequencing efforts while reducing methodological bias. Here, we develop a target capture strategy for metagenomic characterization of eukaryotic ribosomal DNA and root nodule symbiosis genes and test it in the metagenomes of plant root nodules. We utilize biotinylated RNA probes to selectively capture genomic regions of interest from complex environmental DNA samples, avoiding forms of PCR bias that can undermine community characterization and overcoming the need for conserved priming sites often lacking in functional genes. We designed custom probe sets targeting conserved flanking regions of eukaryotic ITS and known root nodule symbiosis (RNS)-related genes and tested them on diverse root nodule metagenomes and a mock community. We observed high recovery of target loci from samples, very high on-target read proportions, and enhanced detection of low-abundance taxa compared to amplicon sequencing, including stronger alignment with known mock community compositions. This approach will enable deeper insights into the phylogenetic diversity of eukaryotic symbionts, their genomic adaptations, and the functional potential of symbiotic interactions in a cost-effective manner suitable for large-scale projects. This strategy advances our understanding of microbial community dynamics and symbiotic relationships in natural and anthropogenic ecosystems.

Viruses of the phylum Nucleocytoviricota are paradigm-shifting entities due to their exceptionally large genomes and complex gene repertoires, which blur the lines between viral and cellular life. Previous research has leveraged computational approaches to map their extensive diversity, while experimental work has started to elucidate the intricate networks they form with hosts, bacterial and other symbionts, co-infecting virophages and other mobile genetic elements. Here, we analyzed deeply sequenced metagenomes sampled from wastewater treatment plants in Denmark, an environment with rapid abiotic changes and known to be a hotbed of dense microbial communities. We discovered 61 novel nucleocytoviruses, 15 virophages and 14 polinton-like viruses. By integrating them with microbial contigs into a multilayered interaction network, we explore the role of these entities on a mesocosm scale. We demonstrate the centrality of nucleocytoviruses, positioning them as important players shaping microbial community structure and evolution in wastewater treatment plants.

Microbial communities occur in all habitats, yet how individual growth on available nutrients scales to community assembly remains poorly understood. This gap stems largely from the unknown effects of species interactions. These interactions arise because individual populations both consume and transform primary substrates into metabolites exploitable by others, and because parasitic and predatory mechanisms can release cellular building blocks that enable nutrient reuse. Here, we present a mathematical framework that predicts community growth and compositional succession from monoculture growth kinetics, resource availability, and species interaction parameters. To parametrize species interactions, we use a simulated-annealing optimization algorithm to search parameter space for sets that minimize the difference between modeled community growth and experimental time series from soil microcosms inoculated with defined communities of 20 or 21 soil isolates, with or without an opportunistic bacteriovorous member. The optimized interaction parameter sets were then used to predict growth dynamics in an independent 21-member community and in species drop-out communities. We find that community development is biphasic: an initial phase dominated by competition for primary resources driven by inherent strain growth kinetics, followed by a phase governed by cross-feeding and biomass formation on released byproducts. Paired metatranscriptomic analysis corroborated predicted shifts in individual growth states and revealed metabolic repurposing associated with the sudden renewed availability of metabolites and cellular building blocks. Model simulations that excluded species interactions reproduced only one-fifth of the observed community biomass, highlighting the importance of cross-feeding for soil community growth. Overall, models that integrate monoculture growth kinetics with inferred species interactions can predict the dynamics of medium-complexity communities from starting inocula even when environmental nutrient composition is largely unknown.

Identifying discrete microbial assemblages and their environmental drivers across multiple biological fractions simultaneously remains a central challenge in aquatic microbial ecology. We applied an integrated analytical pipeline built around Latent Dirichlet Allocation (LDA) to an eight-year 16S rRNA amplicon time series from the Laurentian Great Lakes, spanning four size-fractionated biological blocks -- free-living prokaryotes, particle-associated prokaryotes, and small and large chloroplast-containing eukaryotes. LDA resolved ecologically coherent subcommunities whose taxonomic identity was consistently defined at the order and class level, with fingerprint taxa confirmed by discriminant analysis. Shannon entropy differences between blocks reflected fundamental differences in dispersal capacity and environmental filtering -- free-living prokaryotes and large eukaryotes showed higher mixing than particle-associated prokaryotes and small eukaryotes. Temperature dominated environmental structuring across all blocks, assessed through Limma and random forest with SHAP, with secondary drivers differing by size fraction. Group Compositional Analysis jointly integrating all four blocks revealed that thermal stratification and lake chemistry organize microbial communities coherently across all size fractions simultaneously. Warm stratified and cold inversely-stratified waters harbored largely non-overlapping assemblages across all four blocks, with cold water specialists -- including chemolithotrophic deep-branching lineages and silica-dependent diatoms -- having no warm water equivalents. ImportanceThe Laurentian Great Lakes are among the fastest warming lakes in the world, yet the microbial communities that drive their biogeochemical cycles remain poorly characterized across size fractions and thermal habitats. Using eight years of samples spanning all five Great Lakes, we show that free-living bacteria, particle-associated bacteria, small phytoplankton, and large phytoplankton all respond coherently to the same master environmental gradients -- thermal stratification and lake chemistry -- despite fundamental differences in organism size, trophic role, and sequencing protocol. Warm stratified and cold water habitats support distinct microbial communities, and the cold water specialists identified here have no warm water equivalents. As the Great Lakes warm and cold water habitats shrink, the microbial communities that depend on them will not simply become less abundant they will be replaced by fundamentally different assemblages.