Diel study revealed fine scale coupling between microbial activity and DOM biogeochemistry in the oligotrophic ocean.

Abstract. Viruses are the most abundant biological entities on Earth, yet their global diversity remains largely unexplored. Here, we present VIRE, a compr

While viruses affect the flow of elements and energy at a planet-wide scale through lysis, gene transfer, and metabolic reprogramming, they are yet to be inc...

Abstract. Extracting and directly amplifying DNA from small-volume, low-biomass samples would enable rapid, ultra-high-throughput analyses, facilitating th

Chloroflexus aurantiacus succinate dehydrogenase at apo- and MK-bound forms reveal electron transfer pathways connecting succinate oxidation to menaquinone reduction at a canonical QP site and a newly identified QD site with atypical configuration.

Environmental metagenomic explorations show that Mirusviricota lineages lack essential replication and transcription genes and contain spliceosomal introns, suggesting nuclear reproduction.

Motivation: Protein-protein interactions are central for understanding biological processes. The ability to predict interaction partners is extremely valuable for avoiding costly, time-consuming experiments. It has been shown that AlphaFold has an unsurpassed ability to accurately evaluate interacting protein pairs. However, a protein can also form homomeric interactions, i.e. interact with itself. Results: We found that AlphaFold yielded a significantly higher false-positive rate for identifying homodimers than for heterodimers. True Positive Rate (TPR) at 1% False Positive Rate (FPR) drops from 63% for heterodimers to 18% for homodimers. When we investigated the high-scoring false positives, i.e., non-homodimers with high AlphaFold scores when predicted as such, we found that their homologs were enriched for homomultimeric proteins. Using a simple logistic regression model that combines AlphaFold scores with structural and homology information, we increased the TPR (at 1% FPR) to 42 +/- 8% (5-fold cross-validation) from 19%. If we excluded the homology information, we achieved a TPR of 28 +/- 7%, which is still better than using AlphaFold metrics. Availability and implementation: All data are available from Zenodo DOI:\10.5281/zenodo.17738668 and all code from https://github.com/SarahND97/alphafold-homodimers ### Competing Interest Statement The authors have declared no competing interest. Knut and Alice Wallenberg, 2022.0032 Vetenskaprådet, 2021-03979

Non-tailed phages remain an underexplored group in marine environments, as tailed phages have dominated sequence and culture collections. However, recent surveys indicate that non-tailed phages might be more abundant than tailed phages, and with different effects on microbial mortality and gene transfer. Here, we report the structural characterization of bacteriophage NO16, a non-tailed vibriophage, one of the simplest members of the Double Jelly Roll (DJR) lineage. Mass spectrometry analyses indicate that the virion is composed of at least nine different proteins. NO16 has a pseudoT = 21 capsid, similar to other phages in the lineage but differs in the organization of minor capsid proteins, mainly in terms of membrane-capsid contacts. The DJR major capsid protein GP19 is stabilized by a cation in the base as previously observed in corticovirus PM2, and by strong electrostatic interactions between monomers. Notably, localized reconstruction reveals a symmetry mismatch at the vertex, where two trimeric GP13 spikes are anchored to a pentamer of the penton base GP14. GP13 presents carbohydrate-binding modules and putative glycosylase activity, indicating implications for host entry. Finally, we use structural and functional predictions for NO16 non-structural proteins to propose a complete atlas of the NO16 infectious cycle. ### Competing Interest Statement The authors have declared no competing interest. Agencia Estatal de Investigación, https://ror.org/003x0zc53, PID2019-104098GB-I00/AEI/10.13039/501100011033, PID2022-136456NB-I00/AEI/10.13039/501100011033/, SEV-2017-0712/AEI/10.13039/501100011033/, CEX2023-001386-S/AEI/10.13039/501100011033/ Consejo Superior de Investigaciones Científicas, https://ror.org/02gfc7t72, JAE-SOMdM20-20 Ministerio de Ciencia, Innovación y Universidades, FPU2020-05148 European Commission, Horizon Europe Programme, Grant Agreement No. 101084204 (Cure4Aqua) Innovation Fund Denmark, https://ror.org/00daj4111, Project No. 2105-00014B (AQUAPHAGE) Danish National Research Foundation, HADAL, Grant No. DNRF145

Non-tailed phages remain an underexplored group in marine environments, as tailed phages have dominated sequence and culture collections. However, recent surveys indicate that non-tailed phages might be more abundant than tailed phages, and with different effects on microbial mortality and gene transfer. Here, we report the structural characterization of bacteriophage NO16, a non-tailed vibriophage, one of the simplest members of the Double Jelly Roll (DJR) lineage. Mass spectrometry analyses indicate that the virion is composed of at least nine different proteins. NO16 has a pseudoT = 21 capsid, similar to other phages in the lineage but differs in the organization of minor capsid proteins, mainly in terms of membrane-capsid contacts. The DJR major capsid protein GP19 is stabilized by a cation in the base as previously observed in corticovirus PM2, and by strong electrostatic interactions between monomers. Notably, localized reconstruction reveals a symmetry mismatch at the vertex, where two trimeric GP13 spikes are anchored to a pentamer of the penton base GP14. GP13 presents carbohydrate-binding modules and putative glycosylase activity, indicating implications for host entry. Finally, we use structural and functional predictions for NO16 non-structural proteins to propose a complete atlas of the NO16 infectious cycle. ### Competing Interest Statement The authors have declared no competing interest. Agencia Estatal de Investigación, https://ror.org/003x0zc53, PID2019-104098GB-I00/AEI/10.13039/501100011033, PID2022-136456NB-I00/AEI/10.13039/501100011033/, SEV-2017-0712/AEI/10.13039/501100011033/, CEX2023-001386-S/AEI/10.13039/501100011033/ Consejo Superior de Investigaciones Científicas, https://ror.org/02gfc7t72, JAE-SOMdM20-20 Ministerio de Ciencia, Innovación y Universidades, FPU2020-05148 European Commission, Horizon Europe Programme, Grant Agreement No. 101084204 (Cure4Aqua) Innovation Fund Denmark, https://ror.org/00daj4111, Project No. 2105-00014B (AQUAPHAGE) Danish National Research Foundation, HADAL, Grant No. DNRF145

Background The Roseobacter group is a major component of prokaryotic communities in the global oceans. Information on this group is based predominantly on isolates and their genomic features and on the 16S rRNA gene. Assessments of prokaryotic communities in the pelagic of the global oceans indicated an unveiled diversity of this group but studies of the diversity and global biogeography of the entire group are still missing. Hence, we aimed at a comprehensive assessment of the Roseobacter group in the global oceans on the basis of MAGs and SAGs. Results The obtained 610 MAGs and 43 SAGs of high quality were subjected to in-depth analyses of their phylogeny, genomic and functional features. The recruitment locations range from the tropics to polar regions, include all major ocean basins. The phylogenetic analysis delineated the known RCA cluster and five pelagic clusters, two of which were completely novel: TCR (Temperate and Cold Roseobacter), AAPR (Arctic-Atlantic-Pacific Roseobacter, novel), AAR (Arctic-Atlantic Roseobacter, novel), COR (Central Oceanic Roseobacter), LUX (Cand. Luxescamonaceae) cluster. These clusters account for ~ 70% of all Roseobacter MAGs and SAGs in the epipelagic. The TCR, AAPR, AAR, and LUX clusters are among the most deeply branching lineages of the Roseobacter group. These clusters and several sublineages of the RCA and COR clusters exhibit distinct features of genome streamlining, i.e. genome sizes of < 2.9 Mbp and G + C contents of < 40%. The clusters exhibit differences in their functional features and also compared to other lineages of the Roseobacter group. Proteorhodopsin is encoded in most species of the AAPR, AAR, TCR, and RCA clusters and in a few species of the COR cluster, whereas in most species of the latter, the LUX cluster and in a few species of the RCA cluster aerobic anoxygenic photosynthesis is encoded. Biogeographic assessments showed that the AAPR, AAR, TCR and RCA clusters constitute the Roseobacter group in the temperate to polar regions to great extent whereas the COR and LUX clusters in the tropics and subtropics. Conclusions Our comprehensive analyses shed new light on the diversification, genomic features, environmental adaptation, and global biogeography of a major lineage of pelagic bacteria. Video Abstract

Background The Roseobacter group is a major component of prokaryotic communities in the global oceans. Information on this group is based predominantly on isolates and their genomic features and on the 16S rRNA gene. Assessments of prokaryotic communities in the pelagic of the global oceans indicated an unveiled diversity of this group but studies of the diversity and global biogeography of the entire group are still missing. Hence, we aimed at a comprehensive assessment of the Roseobacter group in the global oceans on the basis of MAGs and SAGs. Results The obtained 610 MAGs and 43 SAGs of high quality were subjected to in-depth analyses of their phylogeny, genomic and functional features. The recruitment locations range from the tropics to polar regions, include all major ocean basins. The phylogenetic analysis delineated the known RCA cluster and five pelagic clusters, two of which were completely novel: TCR (Temperate and Cold Roseobacter), AAPR (Arctic-Atlantic-Pacific Roseobacter, novel), AAR (Arctic-Atlantic Roseobacter, novel), COR (Central Oceanic Roseobacter), LUX (Cand. Luxescamonaceae) cluster. These clusters account for ~ 70% of all Roseobacter MAGs and SAGs in the epipelagic. The TCR, AAPR, AAR, and LUX clusters are among the most deeply branching lineages of the Roseobacter group. These clusters and several sublineages of the RCA and COR clusters exhibit distinct features of genome streamlining, i.e. genome sizes of < 2.9 Mbp and G + C contents of < 40%. The clusters exhibit differences in their functional features and also compared to other lineages of the Roseobacter group. Proteorhodopsin is encoded in most species of the AAPR, AAR, TCR, and RCA clusters and in a few species of the COR cluster, whereas in most species of the latter, the LUX cluster and in a few species of the RCA cluster aerobic anoxygenic photosynthesis is encoded. Biogeographic assessments showed that the AAPR, AAR, TCR and RCA clusters constitute the Roseobacter group in the temperate to polar regions to great extent whereas the COR and LUX clusters in the tropics and subtropics. Conclusions Our comprehensive analyses shed new light on the diversification, genomic features, environmental adaptation, and global biogeography of a major lineage of pelagic bacteria. Video Abstract

Borgs are large extrachromosomal elements of anaerobic methane-oxidizing archaea. Here, via in silico protein structure prediction of ~10,000 Borg proteins, the authors reveal that Borgs share numerous features with giant eukaryotic viruses, suggesting that Borgs have a viral-like lifestyle and evolutionary convergence of large extrachromosomal elements across the Domains of Life.

Primer bias in 16S rRNA gene amplicon sequencing can distort microbial diversity estimates by underrepresenting key taxa. We introduce a modified primer pair (V4-EXT) targeting the hypervariable V4 region of bacterial and archaeal 16S rRNA genes, with improved in silico taxonomic inclusivity. To benchmark performance, we analyzed 938 samples from terrestrial, aquatic, and host-associated habitats, comparing microbial community profiles derived with V4-EXT and the currently most widely used V4-targeted primers. V4-EXT substantially improved the detection of Patescibacteria and other underrepresented lineages, such as Chloroflexota and Iainarchaeota, while enhancing recovery of novel amplicon sequence variants across sample types. Overall, V4-EXT provides broader taxonomic coverage and more inclusive microbial community profiles, particularly in high-diversity ecosystems such as groundwater and soils. We propose V4-EXT as a robust successor for comprehensive microbial community analysis across diverse habitats. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, Z383-B, 10.55776, COE 7, 10.55776, T 1218, ZK-74B Research Foundation - Flanders, G0A4821N, G000821N, 1174925N, BOF.MET.2021.0004.01

Vibrio natriegens is a Gram-negative, halophilic member of the Harveyi clade and Vibrio core group that inhabits marine and estuarine coastal waters and sediments. While generally considered nonpathogenic, this species shares this clade with serious human pathogens (e.g., Vibrio parahaemolyticus) and aquatic animal pathogens (e.g., Vibrio harveyi, Vibrio campbelllii, and Vibrio alginolyticus). Although it was discovered more than 65 years ago, most of our knowledge regarding this bacterium has been generated in the past decade.

Abstract. Bacterial flagella are known for facilitating motility to support nutrient acquisition and predator evasion, but can also serve as receptors for

Complex prophage integration dynamics, including low-level induction, cross-family host range and transposase-mediated mobilization, challenge existing paradigms and deepen our understanding of phage–bacterial interactions in the human gut microbiome.

Differences in genomic GC content skew species abundances in microbial communities. Here, the authors develop GuaCAMOLE, a computational method that quantifies this GC bias from metagenomic sequencing data and corrects abundances accordingly, showing its application increases abundances of some pathogenic bacteria up to twofold in human gut microbiomes.

Abstract. Microbes differ greatly in their organismal structure, physiology, and environmental adaptation, yet information about these phenotypic traits is

Abstract. Taxonomic classification underlies all biological science and is the basis for comparative analysis of biological organisms and therefore our und

This graphical abstract illustrates the integrated molecular dating workflow in PhyloSuite v2. The new Molecular Dating Suite unifies data configuration, analysis, and visualization into a single, st...

Daily dynamics in the composition and function of the human gut microbiota have been recognized since 2014, yet the molecular mechanisms underlying these rhythms and their impact on human health remain unclear. Disrupted microbial oscillations are increasingly linked to metabolic diseases such as obesity and type 2 diabetes, and to inflammatory conditions in the gut and beyond. We propose advancing from observational studies to experimentally targeting microbial rhythms and clocks to uncover causal relationships. In vivo and in vitro models offer opportunities to uncover how signaling cues and dietary patterns influence microbial oscillations and, in turn, host metabolic and immune functions. Manipulating microbial rhythmicity independent of host physiology represents a new frontier for microbiota-based strategies to promote health and prevent diseases.

Background: Organized collections of bacterial strains can help bridge the gap between studying model organisms and communities, through comparative experiments between genetically and phenotypically diverse strains. We describe the establishment and initial characterization of a library of 62 marine heterotrophic bacteria, selected to represent a significant fraction of the genome-encoded functional diversity and a wide range of known phytoplankton-bacteria interactions. We focus on important but often undiscussed aspects of collecting and maintaining such a library, verifying strain identity, and applying classical microbiological methods across diverse strains. Results: Cultured strains contain up to hundreds of mutations compared with the reference genomes, with non-synonymous mutations in rpoB and/or rpoC genes observed in ~15% of the cultures. Most strains grow well at 25C, but the dependence of growth rate on temperature and the width of the temperature niche vary between strains in a systematic manner. We describe steps towards designing a universal, defined, minimal media for marine bacteria, revealing that growth inhibition on amino acids and peptides by carbohydrates is widespread. Cell counts obtained from flow cytometry and colony plating differ systematically, as do different methods to assess motility. Finally, we discuss traits potentially related to microbial interactions such as hemolysis, biofilm formation, and antibiotic resistance. Gammaproteobacteria such as Alteromonas, Pseudoalteromonas, and Vibrio reveal consistently robust growth, and activity, perhaps explaining why these clades are well-explored. Conclusion: Explicitly discussing the insights and challenges of working with strain libraries will pave the way to robust, reproducible, and generalizable mapping of bacterial traits across diversity. ### Competing Interest Statement The authors have declared no competing interest. Human Frontiers Research Program, RGP0020/2016 National Science Foundation - United States-Israel Binational Science Foundation, NSFOCE-BSF 1635070, NSF-BSF 2246707 NSF Center for Chemical Currencies of a Microbial Planet, publication #81 Israel Science foundation, 1786/20

Oxygenic phototrophs fix CO2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) that shows relatively low CO2 affinity and specificity. To circumvent low and fluctuating CO2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence are tightly regulated at multiple layers. The control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. Here we report on the discovery of a so far non-annotated gene in the model Synechocystis sp. PCC 6803, which is located upstream of the sbtAB operon and encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting after fusing the sbtC-coding sequence to a Flag-tag. Similar to sbtAB, transcription of the sbtC locus is induced by low CO2 availability but controlled independently. Mutation of the sbtC locus in a wild-type background showed only a mild phenotype even under low CO2, but the diurnal growth was impaired as found before in the mutant ∆sbtB. Biochemical analysis provided evidence for a trimeric SbtABC complex in the membrane. Recombinant Synechocystis strains harboring only SbtA as single Ci uptake system with either deleted sbtB or sbtC genes showed that bicarbonate leakage from the cell was strongly elevated in both mutants. Our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes pointing towards an important role in the cyanobacterial CCM. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, HA2002/27-1, KL 3114/10-1, HE 2544/22-1 German