Abstract Microbes that remain uncultivated occupy nearly every ecosystem on the planet; this is particularly true in soils, where despite their prevalence, the roles of rarely cultivated microbes in driving biogeochemical cycles and ecosystem function remain poorly explored. We combine metagenome-informed substrate selection with enrichment sub-communities to generate reduced-complexity communities that preserve co-occurrence and expand experimental access to underrepresented soil lineages without requiring prior isolation of each member. Carbohydrate-active enzyme (CAZyme) profiles from soil-derived genomes were used to select carbon compounds predicted to enrich difficult to culture taxa, including members of the phylum Acidobacteriota. Based on 16S rRNA amplicon sequencing, we reproducibly enriched Terriglobus (Acidobacteriota ) on multiple metagenome-guided substrates. Select communities with consistent presence and varying abundance of Terriglobus were passaged in a longitudinal design to generate 89 metagenomes; genus-level profiling revealed that community composition varied between biological replicates but remained consistent within replicates over time, providing diverse Acidobacteriota-containing configurations for downstream analysis. Association network inference identified a core set of co-occurring taxa that positively tracked with Terriglobus across the longitudinal series. In parallel, the substrate-guided approach led to isolation of a novel Terriglobus species, the first cultured representative of its GTDB species cluster. Together, these results establish a generalizable strategy for generating communities enriched with rarely cultivated taxa, yielding tractable systems for studying microbial interactions and community assembly in soil.

DNA modification is central to microbial defense against extrachromosomal genetic elements (ECEs), consequently ECEs tend to adopt their host's modification patterns. Shared ECE-host modification patterns enable linking ECEs to their hosts, but modification detection tools are designed for single genomes and are ineffective at metagenome scale. Here, we present MODIFI, software for detecting DNA modifications in metagenomes. MODIFI assumes that each k-mer in a metagenome is mostly unmodified and calculates background signal levels for that k-mer from PacBio HiFi reads, eliminating the need for matched control experiments. MODIFI ECE-host linkages were validated using >1,000 isolate and mock microbiome datasets. Illustrating the approach, we identified 315 strain-resolved, non-redundant ECE-host linkages in environmental and human metagenomes. In infant gut microbiomes, a chromosomal inversion in Enterococcus faecalis alters host and associated plasmid methylation motifs simultaneously. Overall, MODIFI solves a major bottleneck in DNA modification analysis and provides a foundational tool for understanding microbial epigenomics.

Abstract Bacteria drive crucial processes across ecosystems and impact human health, yet tools to rewrite microbiomes remain limited. Here, we show that bridge recombinase enables versatile and programmable genome editing across the bacterial tree of life. In Escherichia coli , we achieved 142 kb insertions at >90% efficiency, megabase-scale inversions (2.3 Mb), and pathway-scale 50 kb excisions. With a single ortholog and bridge RNA (bRNA), we edited bacterial isolates spanning five phyla and diverse members of two human gut communities. We overcame cross-reactivity between co-expressed bRNAs to establish search-and-replace Targetable Recombinase Assisted DNA Exchange (TRADE) editing and demonstrated capture and interphylum transfer of chromosomal pathways, enabling programmable horizontal gene transfer. These advances establish bridge recombinase as a foundation for reprogramming gene flow in complex microbial communities.

Cyanobacteria are integral to biogeochemical cycles, influence climate processes, and hold promise for commercial applications. In natural habitats, they form complex consortia with other microorganisms, where interspecies interactions shape their ecological roles. Although in vitro studies of these consortia have significantly advanced our understanding, they often lack the biological replication needed for robust statistical analysis of shared microbiome features and functions. Moreover, the microbiomes of many model cyanobacterial strains, which are central to our understanding of cyanobacterial biology, remain poorly characterized. Here, we expanded on existing in vitro approaches by coculturing five well-characterized model cyanobacterial strains with microorganisms filtered from three distinct freshwater sources, generating 108 stable consortia. Metagenomic analyses revealed that, despite host and inoculum diversity, these consortia converged on a similar set of noncyanobacterial taxa, forming a 25-species core microbiome. The large number of stable consortia in this study enabled statistical validation of both previously observed and newly identified core microbiome functionalities in micronutrient biosynthesis, metabolite transport, and anoxygenic photosynthesis. Furthermore, core species showed significant enrichment of plasmids, and functions encoded on plasmids suggested plasmid-mediated roles in symbiotic interactions. Overall, our findings uncover the potential microbiomes recruited by key model cyanobacteria, demonstrate that laboratory-enriched consortia retain many taxonomic and functional traits observed more broadly in phototroph-heterotroph assemblages, and show that model cyanobacteria can serve as robust hosts for uncovering functional roles underlying cyanobacterial community dynamics.

Testing microbial interaction hypotheses remains a major challenge in microbiome research. Bacteriophages have the potential to probe organism-organism interactions within complex microbiomes, yet are rarely used for this purpose. Here, we isolated nine narrow host range phages that replicate in several ecologically important Variovorax species. As Variovorax CL14 degrades the plant root-stunting hormone auxin, we used phages to eliminate V. CL14 from a rhizosphere consortium and reestablished the stunted root phenotype. We used three of the phages to deplete another Variovorax , SCN45, from complex communities to test correlation network-based hypotheses of thiamine interdependencies. Genome-resolved metagenomics revealed that specific taxa decreased in relative abundance following Variovorax depletion and could be rescued by thiamine supplementation. Thus, we confirmed thiamine production as the mechanistic basis for interdependence. These experiments lay the foundation for research that employs wildtype and engineered phages to test interaction hypotheses and for targeted microbiome manipulation. ### Competing Interest Statement JFB is a co-founder of Metagenomi. U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, DE-AC02-05CH11231

BackgroundMacroalgae belonging to the genus Asparagopsis have shown to reduce methane (CH4) production during rumen fermentation, while increasing feed efficiency when added to the feed of cattle. However, little is known about how the rumen microbiome responds to Asparagopsis supplementation, and how changes in the microbiome may contribute to changes in rumen function and host phenotype. Here, we generated and analyzed metagenomic and metatranscriptomic data from the rumen microbiome from cows receiving (treatment) and not receiving (control) an Asparagopsis armata supplemented diet.ResultsUsing a combination of metatranscriptome and metagenome analysis, we found that reduction of CH4 emission from animals receiving A. armata was coupled to a significant reduction in the transcription of methanogenesis pathways. Additionally, a significant decrease in the transcription of genes for carbon catabolism and a reorganization of carbon catabolic gene expression occurred at the species level within the rumen microbiome of animals that received red seaweed with their diet. Increased H2 production, a consequence of methanogenesis suppression, was coupled to a significant increase in the transcription of hydrogenases that mediate hydrogenotrophic metabolism in the treatment group. Metatranscriptome analysis identified a single metagenome assembled genome (MAG) of a Duodenibacillus sp., a hitherto uncultured hydrogenotrophic bacterial species, as the dominant driver of this transcriptional change.ConclusionsComparative genomic analysis between the Duodenibacillus sp. and other hydrogenotrophic rumen organisms revealed metabolic traits that may provide Duodenibacillus sp. with a competitive advantage in H2 scavenging. Our findings provide an initial understanding of how the rumen microbiome responds to a promising CH4 reducing feed additive and serve as a model for alternative stable rumen microbiome states that produce less methane and increase animal productivity. Ultimately, insights from the work presented here might enable the development of advanced microbiome-based strategies to reduce enteric methane production.

Underground research laboratories (URLs) provide a window on the deep biosphere and enable investigation of potential microbial impacts on nuclear waste, CO2 and H2 stored in the subsurface. We carried out the first multi-year study of groundwater microbiomes sampled from defined intervals between 140 and 400 m below the surface of the Horonobe and Mizunami URLs, Japan. We reconstructed draft genomes for >90% of all organisms detected over a four year period. The Horonobe and Mizunami microbiomes are dissimilar, likely because the Mizunami URL is hosted in granitic rock and the Horonobe URL in sedimentary rock. Despite this, hydrogen metabolism, rubisco-based CO2 fixation, reduction of nitrogen compounds and sulfate reduction are well represented functions in microbiomes from both URLs, although methane metabolism is more prevalent at the organic- and CO2-rich Horonobe URL. High fluid flow zones and proximity to subsurface tunnels select for candidate phyla radiation bacteria in the Mizunami URL. We detected near-identical genotypes for approximately one third of all genomically defined organisms at multiple depths within the Horonobe URL. This cannot be explained by inactivity, as in situ growth was detected for some bacteria, albeit at slow rates. Given the current low hydraulic conductivity and groundwater compositional heterogeneity, ongoing inter-site strain dispersal seems unlikely. Alternatively, the Horonobe URL microbiome homogeneity may be explained by higher groundwater mobility during the last glacial period. Genotypically-defined species closely related to those detected in the URLs were identified in three other subsurface environments in the USA. Thus, dispersal rates between widely separated underground sites may be fast enough relative to mutation rates to have precluded substantial divergence in species composition. Species overlaps between subsurface locations on different continents constrain expectations regarding the scale of global subsurface biodiversity. Overall, microbiome and geochemical stability over the study period has important implications for underground storage applications. ### Competing Interest Statement The authors have declared no competing interest.

Underground research laboratories (URLs) provide a window on the deep biosphere and enable investigation of potential microbial impacts on nuclear waste, CO2 and H2 stored in the subsurface. We carried out the first multi-year study of groundwater microbiomes sampled from defined intervals between 140 and 400 m below the surface of the Horonobe and Mizunami URLs, Japan. We reconstructed draft genomes for > 90

Effective transport of biological systems as cargo during space travel is a critical requirement to use synthetic biology and biomanufacturing in outer space. Bioproduction using microbes will drive the extent to which many human needs can be met in environments with limited resources. Vast repositories of biological parts and strains are available to meet this need, but their on-site availability requires effective transport. Here, we explore an approach that allows DNA plasmids, ubiquitous synthetic biology parts, to be safely transported to the International Space Station and back to the Kennedy Space Center without low-temperature or cryogenic stowage. Our approach relied on the cyanobacterium Nostoc punctiforme PC73102, which is naturally tolerant to prolonged desiccation. Desiccated N. punctiforme was able to carry the non-native pSCR119 plasmid as intracellular cargo safely to space and back. Upon return to the laboratory, the extracted plasmid showed no DNA damage or additional mutations and could be used as intended to transform the model synbio host Escherichia coli to bestow kanamycin resistance. This proof-of-concept study provides the foundation for a ruggedized transport host for DNA to environments where there is a need to reduce equipment and infrastructure for biological parts stowage and storage.

For a deeper and comprehensive understanding of the composition and function of rhizosphere microbiomes, we need to focus at the scale of individual roots in standardized growth containers. Root exudation patterns are known to vary along distinct parts of the root even in juvenile plants giving rise to spatially distinct microbial niches. To address this, we analyzed the microbial community from two spatially distinct zones of the developing primary root (tip and base) in young Brachypodium distachyon grown in natural soil using standardized fabricated ecosystems known as EcoFABs as well as in more conventional pot and tubes. 16S rRNA based community analysis showed a strong rhizosphere effect resulting in significant enrichment of several OTUs belonging to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. However, microbial community composition did not differ between root tips and root base or across different growth containers. Functional analysis of bulk metagenomics revealed significant differences between root tips and bulk soil. The genes associated with different metabolic pathways and root colonization were enriched in root tips. On the other hand, genes associated with nutrient-limitation and environmental stress were prominent in the bulk soil compared to root tips, implying the absence of easily available, labile carbon and nutrients in bulk soil relative to roots. Such insights into the relationships between developing root and microbial communities are critical for judicious understanding of plant-microbe interactions in early developmental stages of plants.

Metagenomic or metabarcoding data are often used to predict microbial interactions in complex communities, but these predictions are rarely explored experimentally. Here, we use an organism abundance correlation network to investigate factors that control community organization in mine tailings-derived laboratory microbial consortia grown under dozens of conditions. The network is overlaid with metagenomic information about functional capacities to generate testable hypotheses. We develop a metric to predict the importance of each node within its local network environments relative to correlated vitamin auxotrophs, and predict that a Variovorax species is a hub as an important source of thiamine. Quantification of thiamine during the growth of Variovorax in minimal media show high levels of thiamine production, up to 100 mg/L. A few of the correlated thiamine auxotrophs are predicted to produce pantothenate, which we show is required for growth of Variovorax , supporting that a subset of vitamin-dependent interactions are mutualistic. A Cryptococcus yeast produces the B-vitamin pantothenate, and co-culturing with Variovorax leads to a 90-130-fold fitness increase for both organisms. Our study demonstrates the predictive power of metagenome-informed, microbial consortia-based network analyses for identifying microbial interactions that underpin the structure and functioning of microbial communities.

Metagenomics analyses can be negatively impacted by DNA contamination. While external sources of contamination such as DNA extraction kits have been widely reported and investigated, contamination originating within the study itself remains underreported. Here we applied high-resolution strain-resolved analyses to identify contamination in two large-scale clinical metagenomics datasets. By mapping strain sharing to DNA extraction plates, we identified well-to-well contamination in both negative controls and biological samples in one dataset. Such contamination is more likely to occur among samples that are on the same or adjacent columns or rows of the extraction plate than samples that are far apart. Our strain-resolved workflow also reveals the presence of externally derived contamination, primarily in the other dataset. Overall in both datasets, contamination is more significant in samples with lower biomass. Our work demonstrates that genome-resolved strain tracking, with its essentially genome-wide nucleotide-level resolution, can be used to detect contamination in sequencing-based microbiome studies. Our results underscore the value of strain-specific methods to detect contamination and the critical importance of looking for contamination beyond negative and positive controls.

Microbial communities are fundamental components of most ecosystems but little is known about the interactions that structure them. Metagenomic data have revolutionized our understanding of complex consortia, yet predicted interactions are rarely explored experimentally. We developed an organism abundance correlation network to investigate factors that control community organization in soil-derived laboratory microbial consortia grown under dozens of conditions. The network was overlaid with metagenomic information about functional capacities to generate testable hypotheses. We developed a metric to predict the importance of each node within its local network environments relative to correlated vitamin auxotrophs and predicted that a Variovorax species is a hub because it is a highly important source of thiamine. Quantification of thiamine during the growth of Variovorax in minimal media conditions showed extraordinarily high levels of production of thiamine, up to 100 mg/L. This over-production of thiamine explains why the auxotrophs correlated with Variovorax are reliant on this organism for thiamine, despite the presence of other predicted thiamine producers in the community. A few of these thiamine auxotrophs are predicted to produce pantothenate that we show that Variovorax requires to grow, implying that a subset of vitamin-dependent interactions are mutualistic. We found that Cryptococcus produces the B-vitamin pantothenate, and co-cultures led to a 90-130-fold fitness increase for both Cryptococcus and Variovorax. This study demonstrates the predictive power of metagenome-informed, microbial consortia-based network analyses for identifying microbial interactions that underpin the structure and functioning of microbial communities.

Cyanobacteria are central to biogeochemical cycling, climate change, and eutrophication. While they readily develop associations with environmental microorganisms, the question of whether they consistently recruit specific microbiomes remains unresolved. Here, we established in vitro cyanobacterial consortia by inoculating five different cyanobacterial strains with microbiomes from three freshwater environments to determine if similar non-cyanobacterial organisms were recruited into stable cyanobacteria-based communities. Using amplicon and genome-resolved metagenomic methods we determined that stable cyanobacterial communities largely share the same taxa, regardless of the host cyanobacterial species or inoculation source. Specifically, we identified a 25 species core microbiome, which was significantly enriched in functions for micronutrient biosynthesis, metabolite transport/exchange, and anoxygenic photosynthesis. Furthermore, core species were enriched in putative mobile genetic elements, including two large (> 100 kb) circularized and curated plasmids, encoding functions that may support cyanobacteria-heterotroph symbioses. The consistency observed across these systems indicate that cyanobacterial consortia maintained under similar conditions converge to a predictable structure, and support a core microbiome with taxonomic and functional similarity to core microbiomes of other phototroph-heterotroph symbiotic assemblages.### Competing Interest StatementThe authors have declared no competing interest.

Copper membrane monooxygenases (CuMMOs) play critical roles in the global carbon and nitrogen cycles. Organisms harboring these enzymes perform the first, and rate limiting, step in aerobic oxidation of ammonia, methane, or other simple hydrocarbons. Within archaea, only organisms in the order Nitrososphaerales (Thaumarchaeota) encode CuMMOs, which function exclusively as ammonia monooxygenases. From grassland and hillslope soils and aquifer sediments, we identified 20 genomes from distinct archaeal species encoding divergent CuMMO sequences. These archaea are phylogenetically clustered in a previously unnamed Thermoplasmatota order, herein named the Ca. Angelarchaeales. The CuMMO proteins in Ca. Angelarchaeales are more similar in structure to those in Nitrososphaerales than those of bacteria, and contain all functional residues required for general monooxygenase activity. Ca. Angelarchaeales genomes are significantly enriched in blue copper proteins (BCPs) relative to sibling lineages, including plastocyanin-like electron carriers and divergent nitrite reductase-like (nirK) 2-domain cupredoxin proteins co-located with electron transport machinery. Ca. Angelarchaeales also encode significant capacity for peptide/amino acid uptake and degradation and share numerous electron transport mechanisms with the Nitrososphaerales. Ca. Angelarchaeales are detected at high relative abundance in some of the environments where their genomes originated from. While the exact substrate specificities of the novel CuMMOs identified here have yet to be determined, activity on ammonia is possible given their metabolic and ecological context. The identification of an archaeal CuMMO outside of the Nitrososphaerales significantly expands the known diversity of CuMMO enzymes in archaea and suggests previously unaccounted organisms contribute to critical global nitrogen and/or carbon cycling functions.

Bacteria of the phylum Acidobacteria are one of the most abundant groups across soil ecosystems, yet they are represented by comparatively few sequenced genomes, leaving gaps in our understanding of their metabolic diversity. Recently, genomes of Acidobacteria species with unusually large repertoires of biosynthetic gene clusters (BGCs) were reconstructed from grassland soil metagenomes, but the degree to which species with this trait are widespread is still unknown. To investigate this, we assembled 46 metagenome-assembled genomes recovered from permanently saturated organic-rich soils of a vernal (spring) pool ecosystem in Northern California. We obtained high and medium-quality draft genomes for three novel species from Candidatus Angelobacter (a proposed subdivision 1 Acidobacterial genus), a genus that is genomically enriched in genes for specialized metabolite biosynthesis. Acidobacteria were particularly abundant in the vernal pool sediments, and a Ca . Angelobacter species was the most abundant bacterial species detected in some samples. We identified numerous diverse biosynthetic gene clusters in these genomes, and also in five additional genomes from other publicly available soil metagenomes for other related Ca . Angelobacter species. Metabolic analysis indicates that Ca . Angelobacter likely are aerobes that ferment organic carbon, with potential to contribute to carbon compound turnover in soils. Using metatranscriptomics, we identified in situ metabolic activity and expression of specialized metabolic traits for two species from this genus. In conclusion, we expand genomic sampling of the uncultivated Ca . Angelobacter, and show that they represent common and sometimes highly abundant members of dry and saturated soil communities, with a high degree of capacity for synthesis of diverse specialized metabolites.

Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control. A suite of methods enables programmable species- and locus-specific editing of bacteria in communities.

Gut microbiome succession affects infant development. However, it remains unclear what factors promote persistence of initial bacterial colonizers in the developing gut. Here, we perform strain-resolved analyses to compare gut colonization of preterm and full-term infants throughout the first year of life and evaluate associations between strain persistence and strain origin as well as genetic potential. Analysis of fecal metagenomes collected from 13 full-term and 9 preterm infants reveals that infants' initially distinct microbiomes converge by age 1 year. Approximately 11% of early colonizers, primarily Bacteroides and Bifidobacterium, persist during the first year of life, and those are more prevalent in full-term, compared with preterm infants. Examination of 17 mother-infant pairs reveals maternal gut strains are significantly more likely to persist in the infant gut than other strains. Enrichment in genes for surface adhesion, iron acquisition, and carbohydrate degradation may explain persistence of some strains through the first year of life.