Archaea play essential roles in global biogeochemical cycles, yet their diversity in environmental samples remains understudied. Limited coverage of universal primers towards archaea, their very low abundance relative to bacteria in the studied samples, inefficient cell lysis and the incompleteness of reference databases complicate archaeal 16S rRNA gene sequencing. Furthermore, standardization lacks due to the absence of commercially available mock communities. Here, we developed a mock community composed of mixtures of DNA from six archaeal and four bacterial strains with known genome sizes and 16S rRNA gene copy numbers. Five mock compositions with different archaeal–bacterial abundance ratios were used to evaluate two universal primer pairs and eight archaea-specific methods. The universal primer pairs detected all taxa but consistently overestimated bacterial abundance, indicating to be less suitable for environments where archaea are more dominant. Archaeal specific primer pairs showed variable specificity, with some exhibiting substantial bacterial co-amplification under certain conditions. The Arch349F–A806R primer pair demonstrated the highest archaeal specificity, the least technical variability, the most consistent agreement with expected mock compositions, and minimal artefactual amplification when applied to a complex bacterial mock community. To show its applicability on environmental samples, this method was applied on Boom Clay pore water samples, which indicated a much higher archaeal diversity than previously shown with a standard commercial method. Overall, this study showed the necessity of primer validation through mock communities and identified Arch349F–A806R as a promising primer pair for archaeal 16S rRNA gene sequencing, especially in samples containing methanogenic archaea.

Methane emissions from rice paddies are a major source of greenhouse gases, and silicate fertilizer (SF) derived from blast furnace slag has emerged as an environmentally friendly amendment that reduces methane release. SF modifies the soil redox environment by supplying electron acceptors such as iron, but the microbial mechanisms underlying methane mitigation remain unclear. Here, we combined metagenomic profiling, microbial isolation, and greenhouse experiments to show that SF consistently enriched Burkholderia vietnamiensis together with the methanotrophs in the rice rhizosphere. Inoculation of B. vietnamiensis strain HU01 alone reproduced the methane-suppressing effect of SF and increased methanotroph abundance. Cell-free culture supernatants of HU01 stimulated the growth of Methylococcus geothermalis and Methylocystis parvus through secreted, low-molecular-weight, heat-stable metabolites. Although metabolic modeling predicted primary metabolites such as L-glutamate and cobalamin as candidate cross-feeding factors, LC–MS/MS analysis of the highly active fractions indicated that the robust bioactivity was primarily driven by a chemically complex metabolite mixture rather than these ubiquitous single compounds. These results provide direct evidence that enrichment of a single heterotrophic species can enhance methane oxidation by stimulating methanotroph activity through metabolite exchange. B. vietnamiensis HU01 represents a promising microbial resource for methane mitigation and illustrates how targeted heterotroph–methanotroph interactions can be leveraged for climate-smart rice cultivation.

Genome stability is essential for cellular viability yet constantly threatened by DNA-damaging agents. Among these, DNA double-strand breaks (DSBs) are particularly harmful and in S/G2 phases are faithfully repaired through homologous recombination (HR). The RAD51 recombinase forms nucleoprotein filaments on single-stranded DNA (ssDNA) that performs critical functions in HR. The efficiency of HR depends on precise regulation of RAD51 filament dynamics by accessory factors, including RAD54B, a double-strand DNA-dependent ATPase. Here, using cryogenic electron microscopy, mutagenesis, and biochemical assays, we show that RAD54B stabilizes RAD51–DNA filaments, inhibits RAD51 ATPase activity, and promotes D-loop formation and strand exchange. RAD54B contains two main RAD51-interacting sites and a β-domain that bridges RAD51 protomers, is positioned to contact donor dsDNA, and regulates RAD54B’s ATPase activity. Cellular assays reveal that the RAD51-interacting sites are required for repairing DSBs by HR in human cells. Our findings uncover a modular architecture and mechanistic framework for RAD54B’s critical role in genome maintenance. RAD54B contains two main RAD51-interacting sites and a β domain that bridges RAD51 protomers, is positioned to contact donor dsDNA, and regulates RAD54B’s ATPase activity. Cellular assays reveal that the RAD51-interacting sites are required for repairing DSBs by HR in human cells

Single-molecule techniques provide exceptional resolution of biomolecular dynamics but are limited by low throughput. Here, we present a DNA barcoding strategy that enables parallel kinetic profiling of multiple protein variants in a single experiment. Each protein is covalently linked to a unique DNA barcode, decoded through transient hybridisation of fluorescent probes distinguished by color and binding duration. Applied to a library of 16 ClpS variants, this approach revealed ~5-fold variation in dissociation kinetics and uncovered previously unrecognised bimodal binding behavior arising from distinct structural subpopulations. By enabling scalable, multiplexed functional characterization at the single-molecule level, this platform provides a general framework for protein engineering, directed evolution and the development of next-generation single-molecule technologies. Single-molecule methods typically analyse only a few protein variants at a time, limiting high-throughput screening. Here, authors develop a DNA barcoding strategy that enables parallel identification and kinetic profiling of protein variants in a single experiment.

Nanopore protein sequencing has advanced from a theoretical ambition to a practical technology, overcoming major challenges in protein unfolding, transport and signal detection to achieve single-amino-acid and post-translational modification sensitivity. With both full-length and peptide-based approaches progressing towards commercialization, the field is now focused on scaling throughput and refining analysis tools to unlock a new generation of single-molecule proteomics.

Curiosity-driven studies of lipid organization and lipid asymmetry in biological membranes led to scalable methods for making liposomes, efficient loading of drugs and ionizable cationic lipids. In turn, these advances led to lipid nanoparticles enabling the RNA medicine Onpattro and the COVID-19 mRNA vaccines, and they are now facilitating gene therapies that can potentially treat most human diseases. lnp

Using near-infrared photoswitchable photochromic genetically encoded probes, differential photoacoustic tomography (PAT) enables deep-tissue imaging with high resolution and sensitivity; however, current bacteriophytochrome-based probes are bulky multidomain proteins exceeding 55 kDa, which constrains their use for designing PAT molecular reporters. Here we engineer rsmiRFPnano, a compact cyanobacteriochrome-derived photoswitchable PAT probe. With a molecular weight below 19 kDa, rsmiRFPnano efficiently incorporates biliverdin, exhibits high extinction coefficients in both photochromic states and demonstrates robust performance in live cells. We established rsmiRFPnano-based reporters for NF-κB and AP-1 pathways, which display low basal activity and strong activation-dependent responses. These reporters effectively visualized acute inflammation using noninvasive PAT in vivo, further validated through cellular-level spatial omics mapping. We then created photoswitchable nanobody probes, rsNIR-Fbs, stabilized upon antigen binding and rapidly degraded otherwise, enabling selective photoacoustic immuno-imaging of intracellular antigens in vivo. Together, these results establish rsmiRFPnano as an ultracompact and versatile platform for molecular PAT. A small, photoswitchable and genetically encodable protein operating in the near-infrared window is reported for photoacoustic tomography applications.

Soils are Earth’s largest carbon reservoir, yet microbial mechanisms controlling decomposition, and their warming response, remain poorly resolved, especially in subsoils. Using genome-resolved proteogenomics within a replicated whole-soil warming experiment (0–90 cm, +4 °C, 7.5 years) we map microbial traits, their depth stratification, and warming responses. Depth is the dominant driver of microbial community and trait structure. Depth-wise, metabolism converge on low-molecular-weight and C1 compounds, challenging glucose-centric models, with surface soils enriched in methanol oxidation and deep soils in alcohol fermentation, CO oxidation and N₂ fixation. Abundant sialidases in deep soils implicate glycoproteins as a key carbon source. Warming effects are smaller and decrease over time but are depth-specific: surface soils show enhanced amino sugar and sugar alcohol transport, phosphorus mobilization, and stress tolerance, whereas mid- and deep soils exhibit stimulated C1 metabolism, CO oxidation, and altered nitrogen turnover. These depth-specific microbial trait shifts may critically influence soil carbon–climate feedbacks. In this study, the authors use proteogenomics of a whole-soil warming experiment to show that soil depth dictates microbial community and trait structure more than soil warming. Deep-soil microbes rely on alternative survival strategies and actively produce enzymes that break down complex glycoproteins.

Aminoacyl-tRNA synthetases are the guardians of translational fidelity. Their complex function is mirrored by an elaborate structure, which includes multiple nested domains. While the evolutionary pressures that promoted the emergence of some domains, such as the editing domain, are clear, the pressures acting on other domains, particularly those at the C-terminus, are not. Here, we combine kinetic analysis, X-ray crystallography, and bioinformatics to unveil the history and evolutionary forces that have shaped isoleucyl-tRNA synthetase (IleRS) domain structure. We find that the traditional classification of IleRSs based on the C-terminal tRNA-recognition domains is incomplete, as it fails to capture features of the synthetic domain. Guided by a crystal structure of the Priestia megaterium IleRS2:tRNAIle complex, we removed key interactions between IleRS2 and its cognate tRNA and characterised their impact on enzyme activity. We found that D-loop interactions with the IleRS2 C-terminal domains are non-essential in prokaryotes, and their loss can even increase catalytic turnover. Further, we show that the zinc-binding domain of IleRS1 recognises the anticodon less stringently than the canonical C-terminal domain of IleRS2. Our data suggest that C-terminal evolutionary remodelling of IleRSs is an ongoing process with a historical precedent and is consistent with selection for faster aminoacylation rate. Aminoacyl-tRNA synthetases decipher the genetic code with remarkable speed and accuracy. Here, the authors show how evolution can increase aminoacylation speed by repeatedly reshaping the interactions between an enzyme and its tRNA substrate.