Horizontal gene transfer (HGT) is a promising avenue for microbiome engineering that enables DNA delivery to microbes in their native environment. Integrative and conjugative elements (ICEs), a class of genomically-integrated mobile elements, are ideal vectors for this purpose. Developing targeted ICE transfer for controlled microbiome engineering requires a better understanding of ICE mobility in complex communities. However, current methods for tracking horizontal gene transfer are not high throughput. To improve upon current methods, we developed a sequencing-based strategy to track and quantify ICE transfer in complex mixtures that we refer to as ‘ICE-seq.’ This method was able to assess ICE transfer in a synthetic community of 150 recipients simultaneously and in combination with phenotypic assays demonstrated that restriction-modification (RM) systems in the donor and recipient alter ICE transfer rate with subspecies resolution by up to 10,000-fold. We propose that manipulating RM systems can be a strategy to engineer targeted ICE transfer for directed microbiome engineering.

Radiolaria are unicellular marine organisms (protists) that have been drifting in oceanic plankton for hundreds of millions of years. These mineral architects can build extraordinarily complex skelet...

Methanogenic archaea affect the climate through their production of the greenhouse gas, methane. However, it is unclear how a changing climate and other anthropogenic influences impact methanogen physiology and consequent methane flux. The Great Salt Lake (GSL) is an environment that has been heavily impacted by human activity; more than doubling its salt concentration since the last methanogen was cultured from it in 1985. In this study, we enriched a novel methanogen, for which we propose the name Candidatus Methanohalophilus hillemani, from the GSL at a time when its salinity reached a historical high. Interestingly, Ca. M. hillemani does not increase expression of energy-conservation or osmo-tolerance proteins when challenged with salinity or oxygen. In contrast, Ca. M. hillemani prioritizes trace metal uptake and immune functions in response to the presence of the sulfate-reducing bacterium Desulfovermiculus . 16S rRNA gene amplicon data from GSL shore soils with extremely high and variable methane flux indicated the presence of Ca. M. hillemani. Our results show that Ca. M. hillemani is active when challenged with environmental stressors and contributes to the methane flux emanating from the GSL.

The in situ relevance of biosynthetic gene clusters (BGCs) remains poorly understood. We applied meta-omics to characterize BGC diversity and activity along a peatland redox gradient. From seven metagenomes, we recovered 9,694 BGCs spanning diverse taxa, most lacking close relatives in reference databases, indicating extensive novelty. Only 9-27% of this potential was expressed in situ, with Acidobacteriota, despite moderate repertoires, accounting for over half of all BGC transcription. “Talented producers” with up to 24 clusters were largely silent, and expression was inversely related to BGCs per genome. Acidobacteriota and other oligotrophic taxa expressed most of their BGCs, whereas copiotrophic Pseudomonadota expressed a few, suggesting that life-history and stress-responsive regulation govern activation. PKS and terpene BGCs expression was enriched in genomes expressing markers of aerobic respiration and stress, whereas RiPPs were associated with antimicrobial resistance, stationary phase and stress. Thus, although BGCs are widespread, environmental constraints determine which are realized. ### Competing Interest Statement The authors have declared no competing interest.

Microorganisms colonize all environments on Earth, yet it remains unclear which taxa merely broadly persist and which consistently outperform others across environments. Here we introduce the Baas-Becking score (BB-score), a performance metric to rank taxa across communities that integrates occupancy, relative abundance, and penalized absences. Applying BB-scores to 576,531 microbial communities spanning 24 biomes (categorized as either host-associated or free-living), we found that most species-level operational taxonomic units (OTUs) were widespread, but their success was substantially more restricted. Although 64% of OTUs were detected in at least one host-associated and one free-living biome, only six taxa ranked within the top 1% of performers in >50% of biomes: Aerococcus viridans, Faucicola (previously: Moraxella) osloensis, Lawsonella clevelandensis, Methylorubrum populi, Sphingobium yanoikuyae, and Pseudomonas fluorescens complex. These globally successful taxa were present in a quarter of all airborne communities, consistent with the atmosphere acting as a dispersal corridor. Network analysis of shared top 5% performers identified the phyllosphere and freshwaters as hubs linking animal-associated, plant-associated, and soil biomes. BB-score provides a scalable framework to map microbial success across Earth's biomes and to put new focus on globally successful yet woefully understudied taxa. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, 10000877

Introns were traditionally thought to be rare in bacteria, yet their occurrence and diversity may have been underestimated. Here, we present the first comprehensive overview of group I and group II introns in Patescibacteria. While most introns are readily identified, group I introns inserted at position 35/36 within the anticodon loop often escape detection by standard annotation tools; through experimental verification, we demonstrate that these introns are accurately spliced despite their unusual insertion site. Notably, approximately 40% of genomes in both Patescibacteria and Cyanobacteriota harbor group I introns; however, while around 20% of Cyanobacteriota genomes also contain group II introns, none were detected in Patescibacteria. These results illustrate a previously overlooked phylogenetic distribution of group I and group II introns across the bacterial domain.

Background Processing of archaeal 16S and 23S rRNAs is believed to involve excision of individual rRNAs from polycistronic precursors, circularization of excised rRNAs, and re-linearization before the incorporation into ribosomes. However, all the knowledge is derived from several isolated species, leaving open the possibility that different processes may occur in other archaeal groups. Results Here, we investigate rRNAs from diverse and mostly uncultivated archaea. Sequencing of total cellular RNA from eight phylum-level lineages indicates that archaeal circular 23S rRNA transcript abundances vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggest that ribosomes contain circular 23S rRNAs. Thus, we directly sequence RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirm that the 23S rRNAs in the ribosomes are circular. Structural modeling places the 5′ and 3′ ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their existence as circular molecules. We also confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups, suggesting that certain archaea do not circularize 16S rRNA during processing. Conclusions Our findings uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

The rapid accumulation of viral genome sequences presents major challenges for downstream analysis tools, including tools for multiple sequence alignments, phylogeny, and genome/alignment visualization, due to computational constraints and sampling biases caused by outbreak-driven over- representation. Selecting representative genomes through clustering offers a principled alternative to random subsampling, yet choosing appropriate clustering strategies remains non-trivial and context dependent. Here, we present ViralClust, a modular Nextflow pipeline for bias-aware representative selection from large viral genome datasets. ViralClust integrates five distinct clustering algorithms (CD-HIT-EST, SUMACLUST, VSEARCH, MMSeqs2, and HDBSCAN) within a unified workflow, enabling direct comparison of clustering outcomes and flexible adaptation to diverse biological questions, considering a balanced phylogenic distribution of the selected sequences. We evaluated ViralClust on six RNA and DNA virus datasets ranging from 632 to 156,586 sequences and spanning genome lengths from 890 to 197,185 nucleotides. Across all datasets, clustering reduced dataset size by 95% or more while preserving genetic diversity across species, genera, and families, and effectively mitigating biases introduced by outbreaks, partial genomes, and sequence orientation artifacts. By supporting whole-genome clustering and scalable representative selection, ViralClust enables efficient and reproducible downstream analyses that would otherwise be computationally infeasible. Our framework provides a flexible foundation for large-scale viral genomics and supports future applications in comparative analysis and virus classification. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy, EXC 2051 - Project-ID 390713860 BMBF-funded project ADAPTI-M, Project-ID 031L0322H Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under NFDI4Microbiota, NFDI 28/1 - Project-ID 460129525

Tang et al. performed a large-scale metagenomic analysis that provided a deeper understanding of the microbial composition of the gut and skin of wild freshwater fish as well as the phylosymbiosis relationships between them. The assembled 705 MAGs have greatly enriched microbial genome resources for fish species.

The prokaryotic pangenome, the full complement of genes within a species, is strikingly large. To understand how ecological forces shape this diversity, it is useful to examine the variable gene pool within a single population, defined as cells of the same species coexisting in the same time and place. This single-population pangenome reflects the minimal flexible gene repertoire required in a specific environmental context. Recent long-read metagenomic studies of marine prokaryotes show that local population pangenomes remain large, often comprising thousands of genes. Specifically, cells belonging to the same species of the streamlined alfaproteobacterium Pelagibacter, coming from the same sampling site and even sample, contain more than a thousand genes. Many of these genes are related variants that collectively expand the population’s metabolic potential, akin to paralogs within a single large genome. We propose for them the name 'metaparalogs' together with the idea that these data reflect cooperative, population-level strategies, where the flexible genome operates as a public good (sensu Samuelson), enhancing both adaptability and ecological resilience. A role for extracellular vesicles in facilitating resource sharing is also suggested.

De novo gene evolution entails the birth of new genes from previously non-coding DNA. In this Review, Bornberg-Bauer and Eicholt overview how protein-coding de novo genes are identified, the mechanistic and evolutionary processes underlying their emergence and evolution, and the patterns in their encoded protein structures.

Protein structure is conserved beyond sequence, making multiple structural alignment (MSTA) essential for analyzing distantly related proteins. Computational prediction methods have vastly extended our repository of available protein structures, ...

AlphaGenome, a deep learning model that inputs 1-Mb DNA sequence to predict functional genomic tracks at single-base resolution across diverse modalities, outperforms existing models in variant effect prediction and enables comprehensive genomic analysis.

Microorganisms play essential roles in mediating biogeochemical cycling of carbon across Earth`s ecosystems. Understanding the processes and underlying mechanisms for microbially mediated carbon cycling is therefore critical for advancing global ecology and climate change research. To comprehensively depict these complex biogeochemical processes, we developed CCycDB, a knowledge-based functional gene database, to accurately fingerprint microbially-mediated carbon cycling pathways and gene families, particularly from shotgun metagenomes. The CCycDB database comprises 4,676 gene families classified into six major functional categories, further structured into 45 level-1 and 188 level-2 sub-categories, encompassing a total of 10,991,724 high-quality reference sequences. Validation using both synthetic and real-world datasets demonstrated that CCycDB outperforms existing orthology databases in terms of accuracy, coverage and specificity. By directly targeting carbon-cycling functional gene families, CCycDB provided promising routines to reconstruct both functional gene and taxonomic profiles associated with microbially mediated carbon cycling. Application of CCycDB to shotgun metagenomes from diverse and complex ecosystems revealed pronounced habitat-specific differences in carbon cycling processes and their associated microbial taxa. Collectively, CCycDB provides a powerful and reliable tool for profiling carbon cycling processes from both functional and taxonomic perspectives in complex ecosystems. CCycDB is accessible at https://ccycdb.github.io/. ### Competing Interest Statement The authors have declared no competing interest. Ocean Negative Carbon Emissions (ONCE) program National Key Research and Development Program of China, 2020YFA0607600 National Natural Science Foundation of China, 32371598, 42225708 Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), SML2024SP022, SML2024SP002 Taishan Young Scholarship of Shandong Province

The in situ relevance of biosynthetic gene clusters (BGCs) remains poorly understood. We applied meta-omics to characterize BGC diversity and activity along a peatland redox gradient. From seven metagenomes, we recovered 9,694 BGCs spanning diverse taxa, most lacking close relatives in reference databases, indicating extensive novelty. Only 9-27% of this potential was expressed in situ, with Acidobacteriota, despite moderate repertoires, accounting for over half of all BGC transcription. Talented producers with up to 24 clusters were largely silent, and expression was inversely related to BGCs per genome. Acidobacteriota and other oligotrophic taxa expressed most of their BGCs, whereas copiotrophic Pseudomonadota expressed a few, suggesting that life-history and stress-responsive regulation govern activation. PKS and terpene BGCs expression was enriched in genomes expressing markers of aerobic respiration and stress, whereas RiPPs were associated with antimicrobial resistance, stationary phase and stress. Thus, although BGCs are widespread, environmental constraints determine which are realized. ### Competing Interest Statement The authors have declared no competing interest.

Many bacterial defense systems restrict phage infection by breaking the molecule NAD+ to its constituents, adenosine diphosphate ribose (ADPR) and nicotinamide (Nam). To counter NAD+ depletion-mediated defense, phages evolved NAD+ reconstitution pathway 1 (NARP1), which uses ADPR and Nam to rebuild NAD+. Here we report a bacterial defense system called aRES, involving RES-domain proteins that degrade NAD+ into Nam and ADPR-1″-phosphate (ADPR-1P). This molecule cannot serve as a substrate for NARP1, so that NAD+ depletion by aRES defends against phages even if they encode NARP1. We further discover that some phages evolved an extended NARP1 pathway capable of overcoming aRES defense. In these phages, the NARP1 operon also includes a specialized phosphatase, which dephosphorylates ADPR-1P to form ADPR, a substrate from which NARP1 then reconstitutes NAD+. Other phages encode inhibitors that directly bind aRES proteins and physically block their active sites. Our study describes new layers in the NAD+-centric arms race between bacteria and phages and highlights the centrality of the NAD+ pool in cellular battles between viruses and their hosts. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, ERC-AdG GA 101018520 Israel Science Foundation, MAPATS grant 2720/22 Deutsche Forschungsgemeinschaft, SPP 2330, grant 464312965 Minerva Foundation with funding from the Federal German Ministry for Education and Research research grant from Magnus Konow in honor of his mother Olga Konow Rappaport Ministry of Aliyah and Immigrant Absorption, https://ror.org/05aycsg86 Clore Scholars Program

Biologics are delivered to the gut using phage that infects resident commensal bacteria.

Abstract. The genetic material of bacteria and archaea is organized into various structures and set-ups, attesting that genome architecture is dynamic in t

Plasmids are extrachromosomal DNA molecules that spread by horizontal transfer and shape bacterial evolution. Plasmids are typically present at mul...

We show how to localize and quantify the functional evolutionary constraints on natural proteins. Protein folding has been one of the strongest con...

A Gifsy-1 prophage–encoded higher eukaryotes and prokaryotes nucleotide-binding protein, HepS, senses Siphoviridae infection, activates abortive defence by cleaving host transfer RNAs, blocks rival phages and avoids self-targeting via tail-tip variation.