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Fusion Model for Metagenomics

This work highlights the use of an embeddings approach that can encode multiple features and create efficient contextualization of profiled metagenomes derived from microbiome samples using computer vision models and image representations of the abundance profiles. The model's embeddings can be used to cluster existing samples based on multiple conditions and interpretations, and new embeddings can be quickly created for new samples and fitted to existing clusters to characterize them. This has practical applications for unknown, unlabeled microbiome samples. The model's embeddings can be used to cluster existing samples based on multiple conditions and interpretations, and new embeddings can be quickly created for new samples and fitted to existing clusters to characterize them. This has practical applications for unknown, unlabeled microbiome samples.

Valdes, CamiloA [Lawrence Livermore National Labor

Potential applications of microbial genomics in nuclear non-proliferation

As nuclear technology evolves in response to increased demand for diversification and decarbonization of the energy sector, new and innovative approaches are needed to effectively identify and deter the proliferation of nuclear arms, while ensuring safe development of global nuclear energy resources. Preventing the use of nuclear material and technology for unsanctioned development of nuclear weapons has been a long-standing challenge for the International Atomic Energy Agency and signatories of the Treaty on the Non-Proliferation of Nuclear Weapons. Environmental swipe sampling has proven to be an effective technique for characterizing clandestine proliferation activities within and around known locations of nuclear facilities and sites. However, limited tools and techniques exist for detecting nuclear proliferation in unknown locations beyond the boundaries of declared nuclear fuel cycle facilities, representing a critical gap in non-proliferation safeguards. Microbiomes, defined as “characteristic communities of microorganisms” found in specific habitats with distinct physical and chemical properties, can provide valuable information about the conditions and activities occurring in the surrounding environment. Microorganisms are known to inhabit radionuclide-contaminated sites, spent nuclear fuel storage pools, and cooling systems of water-cooled nuclear reactors, where they can cause radionuclide migration and corrosion of critical structures. Microbial transformation of radionuclides is a well-established process that has been documented in numerous field and laboratory studies. These studies helped to identify key bacterial taxa and microbially-mediated processes that directly and indirectly control the transformation, mobility, and fate of radionuclides in the environment. Expanding on this work, other studies have used microbial genomics integrated with machine learning models to successfully monitor and predict the occurrence of heavy metals, radionuclides, and other process wastes in the environment, indicating the potential role of nuclear activities in shaping microbial community structure and function. Results of this previous body of work suggest fundamental geochemical-microbial interactions occurring at nuclear fuel cycle facilities could give rise to microbiomes that are characteristic of nuclear activities. These microbiomes could provide valuable information for monitoring nuclear fuel cycle facilities, planning environmental sampling campaigns, and developing biosensor technology for the detection of undisclosed fuel cycle activities and proliferation concerns.

59 BASIC BIOLOGICAL SCIENCES

A stable 15-member bacterial SynCom promotes Brachypodium growth under drought stress

Introduction: Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes mimicking the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging. Methods: Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla. Genomic analysis of the individual strains was performed to reveal encoded plant growth-promoting traits, including genes for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters, and some predicted traits were validated by laboratory phenotypic assays. Results: The SynCom demonstrates strong stability both in vitro and in planta. Most strains encoded multiple plant growth-promoting functions, and several of these were confirmed experimentally. The presence of osmoprotectant and ion transporter genes likely contributed to the observed resilience of Brachypodium to drought stress, where plants amended with the SynCom recovered better than those without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria. Discussion: Our results demonstrate that trait-informed construction of synthetic communities can yield stable, functionally diverse consortia that enhance plant resilience under drought. Preferential colonization near root tips points to active, localized plant-microbe signaling as a component of stress-responsive recruitment. This stable SynCom provides a scalable platform for probing mechanisms of plant-microbe interaction and for developing microbiome-based strategies to improve soil and crop performance in variable environments.

Yadav, Archana

CAZyme domain architectures suggest fine-scale functional differentiation among anaerobic fungi and bacteria during lignocellulose conversion to volatile fatty acids

Anaerobic fermentation with microbial communities (microbiomes) is an emerging platform for conversion of lignocellulosic biomass to biofuels and bioproducts. The process relies on diverse anaerobic microbes that interact to deconstruct and convert lignocellulosic biomass into a range of products, such as volatile fatty acids (VFAs), which can be achieved by arresting methanogenesis during fermentation. However, defining the distinct functional roles played by various fungi and bacteria during anaerobic biodegradation remains poorly understood. Here, we performed parallel enrichment experiments from cow faeces, goat faeces, and anaerobic digester sludge, selecting for fungal or bacterial dominated communities that convert sorghum biomass into VFAs. Subsequently we reconstructed metabolic networks across these enrichments based on recovered bacterial metagenome-assembled genomes (MAGs) and fungal isolate genomes and profiled their metabolic activity using metatranscriptomics to identify potential functional niches. Our findings implicate diverse bacteria affiliated with the Bacteroidales and Lachnospiraceae in the direct conversion of lignocellulosic biomass to propionate and butyrate, respectively, whereas Neocallimastix-dominated fungal enrichments converted lignocellulose to lactate, acetate and formate. Analysis of carbohydrate-active enzymes (CAZymes) revealed fine-scale differences between microbes that expressed unique multi-functional enzymes linking two or more CAZymes together with distinct carbohydrate binding motifs, implicating lignocellulose structure as a key driver of selection and niche differentiation. Most of these multi-functional enzymes localized complementary degradation functions together, likely conferring synergistic degradation effects within and between microbiome members. We anticipate that these findings will help inform efforts to develop synthetic microbiomes with tailored functionality for low-cost conversion of lignocellulosic biomass to fuels and bio-based chemicals.

Lawson, Christopher E [University of Toronto;]

The Integrated Impact of Diet on Human Immune Response, the Gut Microbiota, and Nutritional Status During Adaptation to a Spaceflight Analog

Spaceflight impacts human physiology, including well documented immune system dysregulation. Diet, immune function, and the microbiome are interlinked, but diet is the only one of these factors that we have the ability to easily, and significantly, alter on Earth or during flight. As we understand dietary impacts on physiology more thoroughly, we may then improve the spaceflight diet to improve crew health and potentially reduce spaceflight-associated physiological alterations. It is expected that increasing the consumption of fruits and vegetables and bioactive compounds (e.g., omega-3 fatty acids, lycopene, flavonoids) and therefore enhancing overall nutritional intake from the nominal shelf-stable, fully-processed space food system could serve as a countermeasure to improve human immunological profiles, the taxonomic profile of the gut microbiota, and nutritional status, especially where currently dysregulated during spaceflight. This interdisciplinary study will determine the effect of the current shelf-stable spaceflight diet compared to an "enhanced" shelf-stable spaceflight diet (25% more foods rich in omega-3 fatty acids, lycopene, flavonoids, and more fruits, and vegetables in general). The NASA Human Exploration Research Analog (HERA) 2017 missions, consisting of four 45-day missions with closed chamber confinement and realistic mission simulation in a high-fidelity mock space vehicle, will serve as a platform to replicate mission stressors and the effects on crew biochemistry, immunology, and the gut microbiome. Bio sampling of crewmembers is scheduled for selected intervals pre- and in-mission. Data collection also includes dietary intake recording. Outcome measures will include immune markers (e.g., peripheral leukocyte distribution, inflammatory cytokine profiles, T cell function), the taxonomic and metatranscriptomic profile of the gut microbiome, and nutritional status biomarkers and metabolites. Statistical evaluations will determine physiological and biochemical shifts in relation to nutrient intake and study phase. To date, sample collection has been completed for 2 crewmembers from the first mission, aka Campaign 4 Mission 1. Mission 2 was terminated after 22 days due to effects of Hurricane Harvey, and sample collection was not completed. Sample collection will continue for Campaign 4 Mission 3 and 4 prior to comprehensive sample analysis. Beneficial improvements will provide evidence of the impact of diet on crew health and adaptation to this spaceflight analog, and will aid in the design and development of more-efficient targeted dietary interventions for exploration missions.

Douglas, G. L.

New approaches to secondary metabolite discovery from anaerobic gut microbes

The animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilize in silico tools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism.

59 BASIC BIOLOGICAL SCIENCES

A roadmap to understanding and anticipating microbial gene transfer in soil communities

Engineered microbes are being programmed using synthetic DNA for applications in soil to overcome global challenges related to climate change, energy, food security, and pollution. However, we cannot yet predict gene transfer processes in soil to assess the frequency of unintentional transfer of engineered DNA to environmental microbes when applying synthetic biology technologies at scale. This challenge exists because of the complex and heterogeneous characteristics of soils, which contribute to the fitness and transport of cells and the exchange of genetic material within communities. Here, we describe knowledge gaps about gene transfer across soil microbiomes. Here, we propose strategies to improve our understanding of gene transfer across soil communities, highlight the need to benchmark the performance of biocontainment measures in situ, and discuss responsibly engaging community stakeholders. We highlight opportunities to address knowledge gaps, such as creating a set of soil standards for studying gene transfer across diverse soil types and measuring gene transfer host range across microbiomes using emerging technologies. By comparing gene transfer rates, host range, and persistence of engineered microbes across different soils, we posit that community-scale, environment-specific models can be built that anticipate biotechnology risks. Such studies will enable the design of safer biotechnologies that allow us to realize the benefits of synthetic biology and mitigate risks associated with the release of such technologies.

bioccontainment

Identification of carbohydrate gene clusters obtained from in vitro fermentations as predictive biomarkers of prebiotic responses

Prebiotic fibers are non-digestible substrates that modulate the gut microbiome by promoting expansion of microbes having the genetic and physiological potential to utilize those molecules. Although several prebiotic substrates have been consistently shown to provide health benefits in human clinical trials, responder and non-responder phenotypes are often reported. These observations had led to interest in identifying, a priori, prebiotic responders and non-responders as a basis for personalized nutrition. In this study, we conducted in vitro fecal enrichments and applied shotgun metagenomics and machine learning tools to identify microbial gene signatures from adult subjects that could be used to predict prebiotic responders and non-responders. Using short chain fatty acids as a targeted response, we identified genetic features, consisting of carbohydrate active enzymes, transcription factors and sugar transporters, from metagenomic sequencing of in vitro fermentations for three prebiotic substrates: xylooligosacharides, fructooligosacharides, and inulin. A machine learning approach was then used to select substrate-specific gene signatures as predictive features. These features were found to be predictive for XOS responders with respect to SCFA production in an in vivo trial. Our results confirm the bifidogenic effect of commonly used prebiotic substrates along with inter-individual microbial responses towards these substrates. We successfully trained classifiers for the prediction of prebiotic responders towards XOS and inulin with robust accuracy (≥ AUC 0.9) and demonstrated its utility in a human feeding trial. Overall, the findings from this study highlight the practical implementation of pre-intervention targeted profiling of individual microbiomes to stratify responders and non-responders.

59 BASIC BIOLOGICAL SCIENCES

Montane Conifer, Aspen, Meadow, and Sagebrush Metagenome Resolved Genomes and Traits in East River Watershed, Colorado, USA

Climate change is driving vegetation shifts in mountain watersheds, with unknown impacts on biogeochemical cycles. We hypothesize that these shifts will reshape soil microbiomes and associated biogeochemical processes. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed microbiome and microbial functional trait differences between soils under conifer, aspen, forby meadows, and sagebrush across the East River Watershed, CO, controlling for elevation and aspect.Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from soils 0-20cm in depth across three locations in the watershed—Headwaters, Upper Reaches, and Lower Reaches from August 3-11th 2016. Each location was further subdivided into two blocks, with one block on a west facing aspect, and two on the east aspect of the valley. Within blocks, two samples per vegetation type were taken (one at each depth). This resulted in 66 samples, which were sequenced at JGI and can be found under the Joint Genome Institute (JGI) Genomes Online Database (GOLD) sequencing project Gs0118068. Metagenomes were assembled through an inhouse pipeline (see methods), binned using four autobinners (concoct, maxbin2, metabat2, and vamb) and consolidated using dastool. The consolidated bins from all metagenomes were pooled, filtered by completeness (>75%) and contamination (<25%), and dereplicated at 95% ANI using drep. The dataset includes a zip file of 687 genomes (Vegtype_MAGS.zip), the accession numbers for the underlying metagenomes, a csv file with MAG quality metrics and taxonomy from Genome Taxonomy Database (GTDB) and National Center for Biotechnology Information (NCBI) taxonomic representative genome proteins (EastRiver_Vegtype_drep_genome_info.csv), and a file containing MAG quality metrics and taxonomy (gtdb_drep_bin_taxonomy.csv). The dataset additionally includes a sample metadata file (EastRiver_Vegtype_sample_metadata.csv), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a Google KML file for the sampled locations (sample_collection_sites.kml), a location metadata file (locations.csv), a file-level metadata file (flmd.csv), and a data dictionary (dd.csv) file.This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

54 ENVIRONMENTAL SCIENCES

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

59 BASIC BIOLOGICAL SCIENCES

IMAGINE BioSecurity: Mesocosm-Based Methods to Evaluate Biocontainment Strategies and Impact of Industrial Microbes Upon Native Ecosystems

Project Goals: The Integrative Modeling and Genome-scale Engineering for Biosystems Security (IMAGINE BioSecurity) SFA project seeks to establish an understanding of the behavior of engineered microbes in controlled versus environmental conditions to predictively devise new strategies for responding to biological escape. To this end, the IMAGINE Team has established a plant-soil mesocosm platform to track and quantify the fate of industrial microbes in environmental systems and assess the efficacy of biocontainment constraints upon genetically engineered microbe escape frequency and the impact of industrial microbes upon native ecological microbiomes. Abstract Text: Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees, the effect of associated bio-products, and the impact on native ecologies. To this end, we have developed an approach that utilizes soil mesocosms and integrated systems analyses to evaluate the efficacy of novel biocontainment strategies and to assess the impact of production systems upon terrestrial microbiome dynamics. We demonstrate the utility of this approach by modeling a contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from both strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, and stains of Escherichia coli that are contained via genomic recoding. The resultant data demonstrate that this system has broad utility across diverse microbial chassis and biocontainment strategies, enables us to track the fate of our contaminating microbe with high sensitivity in the soil, as well as monitor broader impacts of the perturbation on the underlying soil system. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

BASIC BIOLOGICAL SCIENCES,INORGANIC, ORGANIC, PHYS

Symbiotic bacteriophages exhibit multiple adaptive strategies in activated sludge flocs and contribute to floc stability

Despite the importance of phages for the dynamics and functions of microbial communities, it remains largely unexplored how symbiotic phages adapt in activated sludge systems and influence microbial aggregate stability, which is critical for clarification and reliable performance. Here, based on 12,127 phage contigs recovered from 12 activated sludge microbiomes, the symbiotic phages exhibited broad host ranges and infected dominant prokaryotes in activated sludge. Moreover, these phage communities exhibited high lysogenicity (49% to 66% lysogenic phages) and harbored diverse auxiliary metabolic genes (AMGs) that could enhance microbial aggregate stability, such as genes coding for lipopolysaccharide, peptidoglycan, and extracellular polysaccharide biosynthesis. Additionally, prokaryotic antiviral systems were widely distributed in dominant prokaryotes, particularly the Restriction-Modification (RM) and CRISPR-Cas systems, which could mitigate potential harmful phage infections. Overall, this study reveals the contemporary beneficial relationship between phages and prokaryotic hosts in activated sludge microbiomes and the potential benefits of symbiotic phages on floc stability.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH

Eco-evolutionary strategies for relieving carbon limitation under salt stress differ across microbial clades

With the continuous expansion of saline soils under climate change, understanding the eco-evolutionary tradeoff between the microbial mitigation of carbon limitation and the maintenance of functional traits in saline soils represents a significant knowledge gap in predicting future soil health and ecological function. Through shotgun metagenomic sequencing of coastal soils along a salinity gradient, we show contrasting eco-evolutionary directions of soil bacteria and archaea that manifest in changes to genome size and the functional potential of the soil microbiome. In salt environments with high carbon requirements, bacteria exhibit reduced genome sizes associated with a depletion of metabolic genes, while archaea display larger genomes and enrichment of salt-resistance, metabolic, and carbon-acquisition genes. This suggests that bacteria conserve energy through genome streamlining when facing salt stress, while archaea invest in carbon-acquisition pathways to broaden their resource usage. These findings suggest divergent directions in eco-evolutionary adaptations to soil saline stress amongst microbial clades and serve as a foundation for understanding the response of soil microbiomes to escalating climate change.

54 ENVIRONMENTAL SCIENCES

BiG-SCAPE 2.0 and BiG-SLiCE 2.0: scalable, accurate and interactive sequence clustering of metabolic gene clusters

Microbial metabolic gene clusters encode the biosynthesis or catabolism of metabolites that facilitate ecological specialization, mediate microbiome interactions and constitute a major source of medicines and crop protection agents. Here, we present BiG-SCAPE and BiG-SLiCE 2.0, next-generation methods that facilitate scalable, accurate and interactive gene cluster analyses. BiG-SCAPE 2.0 updates its classification, alignment methods, and visualizations, enabling more accurate analysis, up to 8x faster runtimes and halved memory requirements. BiG-SLiCE 2.0 updates its distance metric, pHMM database, and classification logic, resulting in increased sensitivity nearing that of BiG-SCAPE. Analysis of 260,630 biosynthetic gene clusters from publicly available genomes reveals that both tools generate concurring estimates of gene cluster diversity, thus providing significantly extended methodological support for recent evidence indicating that the vast majority of natural product diversity remains unexplored. Together, these updates will facilitate global genome mining efforts for natural product discovery and microbiome analyses scalable with current data sizes.

Draisma, Arjan [Wageningen University & Research (

Metabolic interactions shape emergent biofilm structures in a conceptual model of gut mucosal bacterial communities

Abstract The gut microbiome plays a major role in human health; however, little is known about the structural arrangement of microbes and factors governing their distribution. In this work, we present an in silico agent-based model (ABM) to conceptually simulate the dynamics of gut mucosal bacterial communities. We explored how various types of metabolic interactions, including competition, neutralism, commensalism, and mutualism, affect community structure, through nutrient consumption and metabolite exchange. Results showed that, across scenarios with different initial species abundances, cross-feeding promotes species coexistence. Morphologically, competition and neutralism resulted in segregation, while mutualism and commensalism fostered high intermixing. In addition, cooperative relations resulted in community properties with little sensitivity to the selective uptake of metabolites produced by the host. Moreover, metabolic interactions strongly influenced colonization success following the invasion of newcomer species. These results provide important insights into the utility of ABM in deciphering complex microbiome patterns.

Biotechnology & Applied Microbiology

Metabolic complexity drives divergence in microbial communities

Microbial communities are shaped by environmental metabolites, but the principles that govern whether different communities will converge or diverge in any given condition remain unknown, posing fundamental questions about the feasibility of microbiome engineering. Here, in this work, we studied the longitudinal assembly dynamics of a set of natural microbial communities grown in laboratory conditions of increasing metabolic complexity. We found that different microbial communities tend to become similar to each other when grown in metabolically simple conditions, but they diverge in composition as the metabolic complexity of the environment increases, a phenomenon we refer to as the divergence-complexity effect. A comparative analysis of these communities revealed that this divergence is driven by community diversity and by the assortment of specialist taxa capable of degrading complex metabolites. An ecological model of community dynamics indicates that the hierarchical structure of metabolism itself, where complex molecules are enzymatically degraded into progressively simpler ones that then participate in cross-feeding between community members, is necessary and sufficient to recapitulate our experimental observations. In addition to helping understand the role of the environment in community assembly, the divergence-complexity effect can provide insight into which environments support multiple community states, enabling the search for desired ecosystem functions towards microbiome engineering applications.

59 BASIC BIOLOGICAL SCIENCES

A global atlas of soil viruses reveals unexplored biodiversity and potential biogeochemical impacts

Historically neglected by microbial ecologists, soil viruses are now thought to be critical to global biogeochemical cycles. However, our understanding of their global distribution, activities and interactions with the soil microbiome remains limited. Here we present the Global Soil Virus Atlas, a comprehensive dataset compiled from 2,953 previously sequenced soil metagenomes and composed of 616,935 uncultivated viral genomes and 38,508 unique viral operational taxonomic units. Rarefaction curves from the Global Soil Virus Atlas indicate that most soil viral diversity remains unexplored, further underscored by high spatial turnover and low rates of shared viral operational taxonomic units across samples. By examining genes associated with biogeochemical functions, we also demonstrate the viral potential to impact soil carbon and nutrient cycling. This study represents an extensive characterization of soil viral diversity and provides a foundation for developing testable hypotheses regarding the role of the virosphere in the soil microbiome and global biogeochemistry.

59 BASIC BIOLOGICAL SCIENCES

Novel adaptive immune systems in pristine Antarctic soils

Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.

59 BASIC BIOLOGICAL SCIENCES