Saturation mutagenesis is a powerful tool for understanding and engineering the function of biological systems, and has been applied successfully to characterize the mutational landscape of individual proteins and genetic loci. However, it has not been applied at the whole-genome scale due to the challenges of both creating and quantifying a saturating set of mutations. Here we introduce Biobloom, a retron-based method for barcoded saturation mutagenesis at the scale of a whole bacterial genome. We constructed a barcoded Biobloom library with >99% projected sampling of saturating single-nucleotide polymorphism (SNP) mutations of the E. coli genome, and applied it to identify beneficial mutations under salt and antibiotic selection. Relative to other techniques like CRISPR-enabled mutagenesis or Adaptive Laboratory Evolution, Biobloom excels at identifying diverse causal SNPs quickly and at smaller working volumes. A barcoded, saturating mutation library is also a shared resource, and we are releasing the updated Biobloom-E.coli-2.0 library to the scientific community for broader adoption and application. Together, Biobloom makes barcoded saturation mutagenesis accessible at whole-genome scale, creating new opportunities for large-scale data collection and bacterial engineering. ### Competing Interest Statement The authors have declared no competing interest. Astera Institute, https://ror.org/00ydx1s47 Experiment Foundation Schmidt Sciences, https://ror.org/044fk6795 The Align Foundation, https://ror.org/056ncc580

Mixotrophy is emerging as a default nutritional strategy in phytoplankton but research seems so far isolated and mostly focussing on single phytoplankton groups or strains. Here we combined data from 24 oceanic and 22 freshwater strains - as well as results from other studies - to analyze phytoplanktons ability to utilize dissolved organic compounds and highlight potential influencing factors. The results emphasize that mixotrophy is ubiquitous in phytoplankton across functional groups and taxa isolated from various habitats, and not strictly dependent on light or nutrient deficiencies. Several factors such as taxonomic affiliation, temperature and growth phase can affect mixotrophic behavior but no consistent patterns have emerged regarding their effects. Hence, mixotrophic traits remain so far unpredictable. There is some indication that the strains origin - potentially through adaptation to habitat DOM availability - might predetermine phytoplanktons mixotrophic skills. For example, freshwater strains used overall more compounds than oceanic strains in our study and Ostreococcus exhibited a different use pattern depending on its origin. Nevertheless, many aspects of mixotrophy in phytoplankton - e.g. metabolic pathways - remain cryptic. By summarizing available knowledge and knowledge gaps, the present synthesis provides a guideline for upcoming research further exploring mixotrophy. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 407270017 (RTG2530)

Saturation mutagenesis is a powerful tool for understanding and engineering the function of biological systems, and has been applied successfully to characterize the mutational landscape of individual proteins and genetic loci. However, it has not been applied at the whole-genome scale due to the challenges of both creating and quantifying a saturating set of mutations. Here we introduce Biobloom, a retron-based method for barcoded saturation mutagenesis at the scale of a whole bacterial genome. We constructed a barcoded Biobloom library with >99% projected sampling of saturating single-nucleotide polymorphism (SNP) mutations of the E. coli genome, and applied it to identify beneficial mutations under salt and antibiotic selection. Relative to other techniques like CRISPR-enabled mutagenesis or Adaptive Laboratory Evolution, Biobloom excels at identifying diverse causal SNPs quickly and at smaller working volumes. A barcoded, saturating mutation library is also a shared resource, and we are releasing the updated Biobloom-E.coli-2.0 library to the scientific community for broader adoption and application. Together, Biobloom makes barcoded saturation mutagenesis accessible at whole-genome scale, creating new opportunities for large-scale data collection and bacterial engineering. ### Competing Interest Statement The authors have declared no competing interest. Astera Institute, https://ror.org/00ydx1s47 Experiment Foundation Schmidt Sciences, https://ror.org/044fk6795 The Align Foundation, https://ror.org/056ncc580

Scientific research relies on transparent dissemination of data and its associated interpretations. This task encompasses accessibility of raw data, its metadata, details concerning experimental design, along with parameters and tools employed for data interpretation. Production and handling of these data represents an ongoing challenge, extending beyond publication into individual facilities, institutes and research groups, often termed Research Data Management (RDM). It is foundational to scientific discovery and innovation, and can be paraphrased as Findability, Accessibility, Interoperability and Reusability (FAIR). Although the majority of peer-reviewed journals require the deposition of raw data in public repositories in alignment with FAIR principles, metadata frequently lacks full standardization. This critical gap in data management practices hinders effective utilization of research findings and complicates sharing of scientific knowledge. Here we present a flexible design of a machine-readable metadata format to store experimental metadata, along with an implementation of a generalized tool named FRED. It enables i) dialog based creation of metadata files, ii) structured semantic validation, iii) logical search, iv) an external programming interface (API), and v) a standalone web-front end. The tool is intended to be used by non-computational scientists as well as specialized facilities, and can be seamlessly integrated in existing RDM infrastructure. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, ExStra EXC2026 Translational Hub 2 Max Planck Society, https://ror.org/01hhn8329

Wu et al. reveal a mechanism regulating cell division via post-translational modification in Sulfolobales archaea. Phosphorylation of the proteasome α subunit by a cyclically expressed eukaryote-like protein kinase, ePK2, inhibits 20S proteasome assembly and the degradation of cell division proteins.

Prokaryote evolution is driven in large part by the incessant arms race with viruses. Genomic investments in antivirus defense can be coarsely classified into two categories, immune systems that abrogate virus reproduction resulting in clearance, and programmed cell death (PCD) systems. Prokaryotic defense systems are enormously diverse, as revealed by an avalanche of recent discoveries, but the basic ecological determinants of defense strategy remain poorly understood. Through mathematical modeling of defense against lytic virus infection, we identify two principal determinants of optimal defense strategy and, through comparative genomics, we test this model by measuring the genomic investment into immunity vs. PCD among diverse bacteria and archaea. First, as viral pressure grows, immunity becomes the preferred defense strategy. Second, as host population size grows, PCD becomes the preferred strategy. We additionally predict that, although optimal strategy typically involves investment in both PCD and immunity, investment in immunity can also result in antagonism, increasing the likelihood that a PCD-competent cell will lyse due to infection. Together, these findings indicate that, generally, PCD is preferred at low multiplicity of infection (MOI) and immunity is preferred at high MOI. Finally, we demonstrate that PCD, which is typically considered to be an altruistic trait, is in some cases neutral and can be maintained in an unstructured population over an evolutionary timescale. Our work shows that the landscape of prokaryotic antivirus defense is substantially more complex than previously suspected.

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthesis when CO2 is limited. Because this system consumes a substantial po...

An antiphage defence system has an activation mechanism that relies on the sensing of phage-encoded proteins that enforce geometry crucial to activation and are not typically present in non-infected cells.

Bacteria use diverse defence systems against phages, including a 164-residue prophage-encoded protein, Rip1, which senses conserved phage assembly rings to form membrane pores that block virion maturation and trigger premature host cell death.

PlasMAAG uses cross-sample information to improve plasmid reconstruction from metagenomic samples.

PlasMAAG uses cross-sample information to improve plasmid reconstruction from metagenomic samples.

Soils harbour immense biosynthetic gene cluster (BGC) diversity that can mediate microbial interactions, yet this potential is still mapped unevenly across the tree of life. Ktedonobacteria , a class of actinomycete-like bacteria within phylum Chloroflexota , are widespread in terrestrial environments and repeatedly dominate pioneer communities in extremely oligotrophic volcanic bare-ground soils; however, their secondary metabolism and genome architecture remain poorly characterised. Here, we integrate targeted cultivation using volcanic soils from Mount Zao with genome-resolved metagenomics and public genomes to analyse 183 ktedonobacterial genomes. Using antiSMASH and BiG-SLiCE, we identified 1,546 BGCs comprising 1,162 non-redundant gene-cluster families (GCFs). In our dataset, nearly one quarter of genomes encode >10 distinct GCFs, and several family-level clades show mean GCF counts comparable to those in genus Streptomyces. Most ktedonobacterial BGCs are highly divergent from reference collections and exhibit unusually low intra-genomic redundancy, suggesting broad, underexplored chemotypes. Long-read assemblies from ten strains reveal recurrent 1.6 - 3.5 Mb chromid-like secondary replicons with chromosome-like composition but distinct maintenance signatures. These replicons are consistently enriched in BGCs and mobility-associated genes, with mobility loci concentrated near BGC boundaries. Collectively, our results expand the current knowledge of the phylogenetic landscape of soil biosynthetic diversity and highlight chromid-like secondary replicons as major genomic reservoirs for specialised metabolism in Ktedonobacteria . ### Competing Interest Statement The authors have declared no competing interest. JSPS KAKENHI, JP25K22374, JP25K01112 The Institute for Fermentation, G-2024-1-019

Chmielowska et al. reveal how arbitrium phages coordinate host stress and viral communication to control induction. An SOS-regulated antirepressor disables an unusual phage repressor by DNA mimicry, while quorum sensing prevents prophage activation when related lysogens are abundant.

The authors leverage experimental and phylogenetic data to propose that anammox bacteria during the Archaean period could have harvested photoholes from cyanobacterial mats for use as an electron acceptor prior to the availability of nitrite.