Nature Communications

Borgs are giant linear extrachromosomal elements (ECEs) of methane-oxidizing Methanoperedens archaea whose evolutionary origin and ecosystem distribution remain unknown. Here we detected 240 Borgs in diverse saline, soil, and freshwater ecosystems. 27 encode methyl-coenzyme M reductases central to methane metabolism, eight have full rRNA operons related to those of Methanoperedens, and some contain up to four ribosomal proteins. We also identified 323 mini-Borgs whose proteome content and gene phylogenies classify as a distinct ECE type. Based on 105 complete and near-complete genomes, Borgs and mini-Borgs share core genes in conserved order, indicating common ancestry. Phylogenetic and diversity analyses suggest that Borgs evolved via gene acquisition into a backbone inherited from mini-Borgs. This evolutionary trajectory mirrors those proposed for giant viruses of both eukaryotes and bacteria.

Agricultural intensification has reduced biodiversity and weakened ecosystem functions essential for sustainable crop production. Agroforestry has been proposed as a regenerative strategy to restore these functions through vegetation diversification, yet its ecological effects at the farm scale remain insufficiently documented, particularly in Mediterranean perennial systems. Here, adaptive field monitoring and soil shotgun metagenomics were combined to investigate ecological responses across a 10-year agroforestry transition gradient in a Mediterranean citrus and olive farm. Mature agroforestry plots supported higher insect richness and functional diversity while maintaining stable pollinator communities. Plant-insect interaction analyses suggested that potential pest activity was largely associated with spontaneous and supportive vegetation rather than crops. In contrast, soil communities showed limited changes in overall richness but substantial compositional and functional restructuring, including enrichment of the arbuscular mycorrhizal fungus Rhizophagus and proteins with nutrient-cycling functions in advanced plots. Together, these findings suggest that agroforestry transition can promote functional diversification above and below ground and highlight the value of farmer-led regenerative transitions coupled with integrative ecological monitoring for the development of more resilient agricultural systems.

Gram-negative bacteria maintain an asymmetric outer membrane that protects cells from environmental stresses and antibiotics. The maintenance of lipid asymmetry (Mla) pathway contributes to outer membrane lipid homeostasis through phospholipid transport between the outer and inner membranes. Although the periplasmic lipid carrier MlaC is thought to transfer phospholipids to the inner membrane MlaFEDB transporter via the hexameric protein MlaD, the molecular mechanism underlying this process remains unclear. Here we show crystal structures of two distinct MlaC-MlaD complexes from Pseudomonas aeruginosa that reveal distinct conformational states of MlaC. In these structures, an ordered conformation of the C-terminal 8 helix of MlaC positions MlaC distally from the central pore of the MlaD hexamer and limits accessibility of the lipid-binding cavity, whereas partial disordering of the 8 helix allows closer association with the MlaD hexamer and increased exposure of the cavity. Structure-based biochemical analyses further demonstrate that the C-terminal region negatively regulates MlaC- MlaD interaction while stabilizing phospholipid binding. These findings identify the C-terminal 8 helix as a conformational switch that couples MlaC positioning with lipid cavity accessibility, providing structural insight into phospholipid transfer at the MlaC-MlaD interface.

Phloem-feeding insects execute complex behavioral decisions to secure essential nutrients from a diet characterized by nitrogen scarcity and severe osmotic pressure due to high sucrose concentrations. We investigated the sensory mechanisms underlying these decisions in the phloem-feeding whitefly Bemisia tabaci. We demonstrate that the sweet taste receptor BtabGR1, expressed in mouthpart and gut tissues, integrates three environmental chemical cues: sucrose concentration, the presence of the essential amino acid arginine, and pH values. Arginine is a pH-dependent positive modulator of sucrose sensing, increasing receptor responses nearly fourfold under apoplast-like conditions and more than doubling the receptor responses in the gut luminal environment. Insects show a strong feeding preference for arginine-containing diets in dual-choice bioassays, with markedly higher intake when arginine is present. RNAi-mediated silencing of BtabGR1 disrupt intake regulation, leading to increased honeydew excretion. These findings suggest a putative link between arginine and the BtabGR1 receptor in regulating both feeding-site evaluation and diet ingestion. Furthermore, the ability to integrate three distinct environmental cues makes BtabGR1 one of the most complex interdependent sensory systems described for a single insect chemoreceptor.

Northern peatlands store approximately one-third of global soil organic carbon, yet the anaerobic respiratory pathways governing carbon turnover remain unclear. In ombrotrophic bogs, the scarcity of inorganic terminal electron acceptors (TEAs) and high CO2:CH4 ratios indicate that methanogenesis alone cannot account for the observed CO2 production. Peat particulate organic matter (POM) has been proposed as an alternative TEA, but whether resident microorganisms encode and express extracellular electron transfer (EET) machinery required to use such extracellular TEAs remains unknown. Using depth-resolved metagenomics and metatranscriptomics across peat profiles from four ombrotrophic Swedish bogs, we identified conserved EET machinery in dominant yet uncultured Acidobacteriota and Verrucomicrobiota, comprising multiheme cytochromes and outer-membrane porins arranged in syntenic gene clusters. This machinery was transcriptionally active up to several meters depth, alongside broader anaerobic respiratory pathways, while methane-cycling processes were more prominent in the upper layers. These results provide systematic genomic and transcriptomic evidence for EET capacity in peatland microorganisms, establishing a molecular foundation for EET-based respiration and its potential role in suppressing methane formation and emissions.

The multidrug efflux pump EmrE is one of the smallest known active transporters and has become a model system for studying multidrug recognition and transport. While recent high-resolution structures have illuminated its dynamic substrate binding pocket, the conformations of its interhelical loops and C-terminal tail, regions critical for controlling proton coupling and gating, remain poorly characterized. Here, we report the high-resolution structure of protonated S64V EmrE determined using solution and solid-state NMR data. This new structural model shows the C-terminal tail occluding the open face of the transport pore, providing a structural basis for how EmrE minimizes proton leak in the absence of substrate. These findings support growing evidence that relatively simple model transporters must leverage an occluded state during alternating access to avoid physiologically unfavorable proton leak.

Durum wheat (Triticum turgidum subsp. durum) is a Mediterranean dietary staple threatened by accelerating climate change, yet the genomic basis of adaptation in North African landraces remains poorly characterised. We present the first integrated whole-genome sequencing (WGS) and RNA-seq study of two contrasting Tunisian landraces: humid-adapted Chili and arid-adapted Mahmoudi. From 27,777 high-confidence SNPs, permutation-based FST outlier analysis (1,000 shuffles) identified 46 selection hotspots across six chromosomes, with a peak signal on chromosome 6B (FST = 0.833; p = 0.013). Constitutive transcriptome profiling (38,159 expressed genes) revealed 406 expression-divergent observations (|log2FC|{square}>{square}1) between landraces. Physical co-localisation analysis confirmed that 99.5% of expression-divergent observations are independent of selection hotspots, implicating trans-regulatory rewiring as the dominant adaptive mechanism. Trans-regulated genes are significantly enriched for disease-resistance (NBS-LRR, RLK, PR; FDR = 1.4 x 10-9) and ubiquitin-proteasome components (FDR = 0.049). Mahmoudi constitutively upregulates ROS-scavenging and dehydrin networks ("store-and-protect"), while Chili elevates aquaporins and transcription factors ("acquire-and-distribute"). These findings identify six chromosomal breeding targets, establish chromosome 6B as a priority fine-mapping locus, and demonstrate that arid-zone adaptation is orchestrated primarily through trans-regulatory stress-network rewiring.

Amyloid deposition in the central nervous system is increasingly recognized in transthyretin (ATTR) amyloidosis, particularly in patients with prolonged survival following liver transplantation or disease-modifying therapies. However, the structural basis of transthyretin aggregation in the brain remains unknown. Here we determine cryo-electron microscopy (cryo-EM) structures of ex vivo brain-derived ATTR fibrils from patients carrying the ATTRv-V30M and ATTRv-V30G variants. Both fibrils adopt folds distinct from those previously reported in peripheral tissues and the vitreous humor. V30M fibrils exhibit a continuous ordered core spanning residues Pro11-Asn124, whereas V30G fibrils consist of a substantially reduced ordered core, revealing pronounced structural divergence even within the same tissue environment. Despite this diversity, comparative analyses identify conserved regions across ATTR fibrils, including a segment implicated in transthyretin aggregation and targeted for diagnostic and therapeutic development. These results provide direct evidence that local tissue context can shape amyloid fibril architecture in human disease.

The structural states of proteins in cells and tissues provide important insight into their functional states, but studying protein structures in situ remains challenging. Furthermore, a single protein can adopt multiple conformations in cells, which typically cannot be assessed by most structural approaches. Here we developed a novel approach, based on structural proteomics fingerprints, for the quantitative analysis of the distribution of structural states of a protein in vitro and in situ. We applied it to the Parkinsons disease hallmark protein alpha-synuclein (aSyn), for which various structural states (disordered, helical, oligomeric and amyloid fibrillar, among others) have been characterized in vitro, but for which the in vivo structural states remain hotly debated. We measured structure-specific proteolytic fingerprints from well-characterized aSyn in vitro conformations and used them to quantitatively determine the aSyn conformational composition in samples of interest. We first benchmarked our approach using ground truth datasets of known composition and showed that, during in vitro amyloid fibril formation, we could simultaneously detect a time-dependent decrease in disordered monomeric aSyn, an increase in {beta}-sheet-rich oligomers, and a delayed rise in amyloid fibrils. We then applied the method to complex, biologically relevant samples. In a S. cerevisiae aSyn overex-pression model, aSyn was predominantly helical, with an increased helical fraction accompanying its relocalization from the plasma membrane to cytosolic lipid droplets. This shift was linked to proteome-wide changes in lipid droplet homeostasis and fatty acid and ergosterol metabolism, underscoring the role of lipid metabolism and droplet formation in aSyn biology. Importantly, we also detected helical aSyn in human iPSC-derived cortical neurons, supporting the physiological relevance of this conformation. Finally, neurons differentiated from PD patient-derived iPSCs showed elevated levels of {beta}-sheet-rich aSyn compared to wild-type cells. Our approach allowed the in situ identification and quantification of different structural states of aSyn directly in cell lysates. Since several proteins can adopt multiple, functionally-relevant conformations in cells, our approach should be broadly applicable to in situ, quantitative structural and functional studies of proteins.

Culex pipiens is a major vector of West Nile Virus (WNV) in Europe and has a complex evolutionary history that has been linked to the probability of WNV spillover to humans. Here, we present a population genomic analysis of Cx. pipiens from a WNV hotspot in southwestern Spain. Whole-genome sequencing of 217 individuals from 24 localities revealed that population structure is mainly shaped by the coexistence of pipiens and molestus ecotypes. Despite clustering, genome-wide divergence between ecotypes was low, consistent with shared ancestral variation. Furthermore, Wolbachia infection was heterogeneous across the study area, where some Culex pipiens pipiens remain uninfected. We also identified three new polymorphic chromosomal inversions on chromosomes 1 and 3 that segregate independently. Although inversions did not underlie ecotypic differentiation, they were enriched for genes involved in olfaction and insecticide resistance pathways, suggesting potential adaptive and epidemiological relevance. Distance-based redundancy analyses showed that ecotype identity explained the largest fraction of genetic variance, with additional contributions from Wolbachia infection, habitat type (urban versus rural) and geographic distance. These results reveal that partial ecotypic differentiation, symbiont infection, and habitat interact to shape population structure in Cx. pipiens, with implications for vector ecology and disease transmission.

Bacterial elongation factor G (EF-G) facilitates mRNA and tRNA translocation during peptide elongation as well as promotes splitting of ribosomal subunits during ribosome recycling in coordination with ribosome recycling factor (RRF). Two homologs of EF-G (EF-G1 and EF-G2) have been identified in some bacterial species, including mycobacteria and in mitochondria. We have previously reported that mycobacterial EF-G2 functions as a stress-specific factor. Here, we demonstrate that EF-G1 functions as the canonical elongation factor responsible for ribosome recycling in mycobacteria. In addition, our structural analyses explain why mycobacterial EF-G2 lacks recycling activity, in contrast to its mitochondrial counterpart. We report cryo-EM structures of the M. smegmatis 70S ribosome and 50S subunit in complex with RRF and/or EF-G1 at 3-4 [A] resolution, elucidating the molecular architecture of intermediate structures and mechanistic basis of ribosome recycling. Notably, GTP hydrolysis on EF-G1 is not required for ribosomal disassembly in mycobacteria, unlike in E. coli.

Some melanoma-prone families linked to the 9p21 locus, harboring the established susceptibility gene CDKN2A, lack pathogenic protein-coding variants. Using whole-exome and targeted sequencing, we identified three rare single-nucleotide variants in two melanoma-prone families and one sporadic melanoma case. Variants map to a conserved CTCF-bound region within the first intron of CDKN2B that physically interacts with CDKN2A. Analysis of UK Biobank showed significant enrichment of variants in this region in melanoma cases. Variants result in diminished CTCF binding in vitro. CTCF ChIP-seq in fibroblasts from the carriers of the largest family demonstrated loss of CTCF binding, accompanied by weakened promoter interactions and allele-specific reduction of CDKN2A p16 transcript expression from the variant haplotype. CRISPR-based perturbation of this region and editing of the large family variant into melanocytes resulted in reduced expression of p14 and p16 CDKN2A transcripts. These findings suggest that non-coding regulatory variants function as high-penetrance susceptibility alleles in melanoma families by altering CDKN2A function.

Refractile bodies (R-bodies) are protein assemblies that form tightly rolled morphologies in cells and undergo large-scale extension into long spirals in response to environmental stimuli. The type 51 R-body, the focus of this study, is assembled from four Reb proteins and undergoes a rapid repeatable [~]50-fold extension. Although R-bodies were first described more than 70 years ago, it has remained unclear how RebC and RebD contribute to the formation and function of this four-protein machinery even though RebA and RebB have been proposed as its major components. We characterized the wild-type R-body (Rb_WT) and a series of reb gene knockout mutants by combining in-cell, biochemical analyses, small-angle X-ray scattering (SAXS) and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. We found that RebD is incorporated into Rb_WT as a minor component, while RebC is not detectably incorporated into the final assembly. Mutants lacking either RebA or RebB still form roll-like assemblies but lack pH-dependent extension. In contrast, mutants lacking RebC and/or RebD exhibit a substantially lower propensity for roll formation and instead undergo off-pathway aggregation with increased {beta}-sheet content. SAXS analyses further indicate that roll-like morphology does not ensure formation of the ordered lamellar architecture characteristic of functional Rb_WT. Thus, pH-responsive R-body actuation is dictated not simply by the major proteins that constitute the final architecture, but by a regulated assembly pathway that builds the ordered lamellar architecture required for actuation. These findings establish assembly-pathway regulation as a key principle for constructing dynamic protein architectures capable of stimuli-responsive mechanical actuation.

Vitellogenins are essential transport lipoproteins and precursors to egg-yolk proteins in oviparous species. Several molecular structure studies have elucidated most of their multi-domain organization, yet the structure and function of their C-terminal cysteine knot (CTCK) domain have remained largely elusive. In this study, we present the 1.2 [A] resolution crystal structure of the recombinant CTCK domain from a vitellogenin isoform of the mosquito Aedes albopictus (Vg-CTCK). The molecular architecture reveals a CTCK fold defined by two antiparallel beta sheets stabilized by three intramolecular disulfide bonds, featuring a unique 12-amino acid insertion shaping an alpha helix positioned between the two main beta sheets. Analysis of the crystal packing and biophysical characterization in solution consistently confirms that recombinant Vg-CTCK is monomeric. To validate these findings in a native context, we employed ex vivo cross-linking mass spectrometry (XL-MS) on intact mosquito ovaries, which corroborated the molecular architecture of the Vg-CTCK observed in the crystal structure and highlighted the absence of inter-molecular cross-links. Collectively, our data highlight an evolutionarily divergent, monomeric assembly for the mosquito Vg-CTCK domain, challenging previous hypotheses that suggested this domain might facilitate vitellogenin oligomerization.

H3K27ac and H3K4me3 are enriched at transcriptional start sites and have been implicated in transcription. However, how these marks concertedly regulate transcription is not fully understood. Here, we developed a dual chemically inducible CRISPR/dCas9-based epigenome editing system that enables independent, temporal and transcription stage-specific modulation of H3K27ac and H3K4me3 at a specific gene locus. Stage-specific removal of H3K4me3 impaired RNA polymerase II recruitment, increased promoter-proximal pausing, reduced productive elongation, and accelerates mRNA decay via increased m6A deposition. Losing both H3K27ac and H3K4me3 rapidly abolished transcriptional activity, while preserving H3K4me3 without H3K27ac can partially sustain transcription. These findings revealed a functional hierarchy and interdependence between H3K27ac and H3K4me3 in different transcription stages and the established versatile tool will contribute to the functional dissection of the temporal dynamics of chromatin modifications in gene regulation.

Spatial multi-omics can provide a unique understanding in molecular organisation and heterogeneity of organs including the heart. Heart function is dependent on the abundance and the spatial arrangement of proteins and lipids. Yet, an integrated multi-omics landscape of the heart at subcellular scale remains unknown. Here, we used a sequential lipid-proteomic analytical pipeline applied to the mouse heart to identify, map, and integrate lipid and protein features, revealing coordinated distinct molecular networks within defined subcellular niches. Here, we benchmark this dual extraction workflow for integrated global proteome-lipidome analyses to demonstrate its performance. We developed conserved subcellular proteome of mouse heart across 14 niches and applied the knowledge of subcellular proteome to spatially resolve the heart lipidome, identifying unique lipid features from different subcellular niches, including mitochondria (e.g., cardiolipin, LPC) and plasma membrane-enriched lipids (e.g., plasmalogens). We identified sex-dependent molecular differences in heart subcellular proteome between male and female, including RNA-protein complexes, mitochondrial calcium handling and immune regulatory pathways. We demonstrate that lipid-protein integrated multi-omics analysis of the heart by dual extraction workflow and mass spectrometry could enable previously unidentified discoveries in heart molecular composition and organisation and spatial cardiac biology. HighlightsO_LIResolved tissue-wide subcellular architecture of cardiac tissue through simultaneous high-resolution proteomics and lipidomics profiling. C_LIO_LISystematically evaluated MS-compatible dual extraction strategies for integrated proteome-lipidome recovery and performance. C_LIO_LIProteomics revealed major sex-dependent differences in mouse left ventricles, primarily involving cellular metabolism, immune regulation, detoxification pathways, and signalling responses. C_LIO_LILipidomics identified strong sex-specific lipid signatures, including differences in omega-3 vs omega-6 fatty-acid containing lipid species, triacylglycerides, and acylcarnitines. C_LIO_LIDeveloped supervised machine-learning models to classify 14 subcellular niches from tissue-wide fractionation proteomics and identified a conserved core subcellular proteome. C_LIO_LIApplied the tissue-wide subcellular proteome model to infer lipid subcellular localisation, revealing mitochondria-enriched lipids (e.g., cardiolipin, LPC) and plasma membrane-enriched lipids (e.g., plasmalogens) in the mouse left ventricle. C_LIO_LIMapped sex-specific subcellular distribution in the heart, highlighting pathways linked to RNA-protein complexes, mitochondrial calcium handling, and immune regulatory networks. C_LI

PIEZO proteins (PIEZO1 and PIEZO2) are essential mechanosensitive channels. PIEZO1 is thought to be selectively activated by Yoda molecules (Yoda1 and Yoda2). Although a structural framework for PIEZO1 activation by Yoda1 exists, a molecular mechanism underlying this selective activation is lacking. Here, using electrophysiology and calcium imaging, we show that Yoda1 increases PIEZO2 open probability and stretch sensitivity as efficaciously as PIEZO1 but elicits weaker PIEZO2-dependent calcium entry, rationalizing why its effect on PIEZO2 has been overlooked. Both Yoda1 and its more potent Yoda2 analog slow down inactivation of PIEZO2 currents with potency similar to PIEZO1 but with lower efficacy. Using mutagenesis and molecular dynamics simulations, we further show that Yoda2s benzoic acid group forms a transient salt bridge with a conserved arginine in the Yoda binding site, providing a molecular basis for Yoda2s increased potency. Our study cautions a reevaluation of studies using these molecules to untangle biological functions mediated by PIEZO channels.

Archaellum-associated motility has been viewed as solely archaeal, yet new findings in Chloroflexota prompt a broader perspective. By analysing a curated [~]22,000 NCBI reference genomes alongside 2,397 archaeal and 226 archaellum-encoding Chloroflexota genomes, this study systematically characterises the co-distribution of archaellum loci with chemosensory system (CSS) classes. Maximum-likelihood phylogeny of 3,727 F1-type CheA proteins reveals three major clades, with Clade 1 comprising [~]80% monoderm representation, uniting archaeal and monoderm bacterial lineages in a shared evolutionary grouping. Overall, this work shows that not only archaeal-type motility, but also F1-CSS based sensing system, might have been gained from Archaea to Chloroflexota via horizontal gene transfer and both systems shared an evolutionary trajectory altogether.

Membrane transporters of the major facilitator superfamily (MFS) mediate uptake of diverse metabolites, yet the molecular basis of substrate recognition within many bacterial families remains unclear. The anion:cation symporter (ACS) family is conserved from bacteria to humans and includes medically relevant solute carrier transporters, but only few bacterial members have been functionally characterized. Here, we combine genetics, biochemistry, transport assays, and structural biology to define the substrate specificity of five ACS transporters from Escherichia coli. Using systematic growth complementation assays in deletion strains, we assign physiological substrates to each transporter, identifying DgoT, LgoT, and ExuT as specific uptake systems for D-galactonate, L-galactonate, and galacturonate/glucuronate, respectively, while revealing overlapping roles for GarP and GudP in C6 sugar acid uptake. NanoDSF ligand binding assays show highly selective recognition of sugar acids but do not predict transport activity. Proton-coupled uptake was directly demonstrated using reconstituted proteoliposomes, defining strict stereoselectivity of LgoT and DgoT. We determined the 2.2 [A] X-ray structure of LgoT in an inward-open conformation, revealing a canonical MFS fold with a conserved but differentially tuned substrate-binding cavity. Comparative structural and sequence analyses across ACS members identify a conserved core for coordination of sugar acid carboxylate and hydroxyl groups, while localized substitutions modulate steric and electrostatic properties to enable discrimination of substrate size and stereochemistry. These results provide a framework for substrate recognition in bacterial ACS transporters and establish LgoT as a structural model for stereo-selective proton-coupled organic anion transport.

The red seaweed genus Ahnfeltia is an ancient lineage that has persisted for over 500 million years with remarkably limited diversification despite a global distribution in cold-temperate intertidal habitats. Compared to the highly diverse sister lineage, Rhodymeniophycidae, Ahnfeltia provides a unique system for investigating long-term evolutionary persistence in marine macroalgae. Here, we generated chromosome-scale genomes from five populations across three species and combined population genomics with paleogeographic niche modelling. Our results reveal remarkable genomic conservation, strong geographic isolation with limited gene flow, high homozygosity, and evidence of purifying selection. Niche projections indicate long-term stability and spatial connectivity of suitable cold-temperate habitats. These findings suggest that Ahnfeltias persistence and limited diversification are linked to genomic constraints and stable ecological niches over geological timescales. This study provides new insights into the genomic basis of evolutionary stasis in ancient marine lineages and highlights potential vulnerability to ongoing climate change affecting cold-water coastal ecosystems.