Nature Microbiology

Conventional genomic risk classification of Listeria monocytogenes assigns clonal complexes to hypervirulent (CC1, CC2, CC4, CC6) or hypovirulent (CC9, CC121) categories based on population-level frequency ratios, leaving all remaining diversity in an undifferentiated "intermediate" category that carries no defined risk assessment. We analysed 436 genomes from confirmed invasive listeriosis across 19 countries using multi-dimensional genomic profiling of virulence and persistence determinants and demonstrate that this approach systematically misclassifies a major fraction of clinically relevant L. monocytogenes. Amphitrophic lineages -- carrying simultaneous genomic competence for clinical virulence (functional inlA, mean virulence score 52.7 +/- 6.6) and industrial persistence (SSI-1 in 94.1%, mean persistence score 66.8 +/- 11.6) -- constitute 31.0% of invasive disease, within 3.6 percentage points of the established hypervirulent category (34.6%). Of these 135 amphitrophic clinical isolates, 91.1% were classified as "intermediate" under conventional taxonomy. The five principal amphitrophic CCs (CC8, CC7, CC3, CC5, CC88) appear with indistinguishable dual-fitness genotypes in both clinical and food-chain datasets, establishing that the same organisms persist in processing facilities and cause invasive human disease. Decomposition of the species-level virulence-persistence trade-off (rho = -0.523) by trophic strategy reveals it to be a Simpsons paradox: no within-strategy correlation is significantly negative, and the only significant signal is a positive amphitrophic correlation (rho = +0.221, p = 0.010) indicating synergy rather than trade-off. Multi-dimensional profiling increases risk-stratified detection from 32.3% (conventional) to 65.6% of clinical isolates -- a 103% improvement. These findings demonstrate that clonal complex identity alone leaves one-third of clinically significant L. monocytogenes uncharacterised, and that effective One Health genomic surveillance requires simultaneous assessment of virulence and persistence at the isolate level.

The archaeal phylum Nanobdellota (formerly Nanoarchaeota) was previously represented by four complete genomes. We present 208 complete Nanobdellota genomes from Oxford Nanopore metagenomes of the Baltic Sea water column and Fennoscandian groundwater (69-201 m below sea level), rotated to the ORC1/Cdc6 replication origin -- a 52-fold expansion of complete-genome representation. Across the ar53 supermatrix and a Nanobdellota-tuned 71-marker supermatrix on 1,239 taxa, the named GTDB orders within Nanobdellota are recovered as monophyletic clades, including the three orders that dominate our environmental sampling: Woesearchaeales, Pacearchaeales, and the GTDB placeholder order SCGC-AAA011-G17. This is consistent with the existing GTDB R232 order-level circumscription. We retire the SCGC-AAA011-G17 placeholder name, replacing it with a complete-genome-anchored SeqCode nomenclatural chain (Maxwellarchaeales ord. nov., Maxwellarchaeaceae fam. nov., Maxwellarchaeum gen. nov., and Maxwellarchaeum balticum sp. nov.) without altering the order-level circumscription. Pacearchaeales and Maxwellarchaeales retain no central or energy metabolism beyond Form III RuBisCO, PEP synthase, and ferredoxin; Woesearchaeales retains partial glycolysis and a V/A-type ATPase. A 4,262-tip phylogeny of rbcL (the RuBisCO large-subunit gene) identifies nine candidate archaea-to-Patescibacteriota Form III RuBisCO transfer events -- including one to a Baltic Minisyncoccia -- versus two reciprocal candidates, consistent with archaea-to-CPR being the more frequently identified direction in our data. All 256 Nanobdellota genomes (208 complete + 48 high-quality non-circular), the ar71 marker set with its 1,239-taxon ML tree, 154 Nanobdellota-trained HMMs for KEGG-ortholog detection in DPANN proteomes (94 ROBUST), and the 4,262-tip rbcL reference tree are released as a community resource, alongside the full analysis archive -- alignments, intermediate trees, structural predictions, and per-step scripts -- at Zenodo (DOI 10.5281/zenodo.20174424; see Using the resource).

Pelagibacter, the largest genus within the SAR11 clade, is the most abundant bacterium in the ocean, yet the vast majority of its species-level diversity remains uncharacterized at the genomic level. Here we present 135 complete Pelagibacter genomes -- the largest such collection assembled to date -- comprising 75 from Oxford Nanopore metagenomes of the San Francisco Estuary (SFE), 31 from a deeply sequenced station within the same transect, and 29 from public databases. These genomes define 52 species at 95% ANI, of which 44 (85%) are taxonomically novel. An expanded phylogeny incorporating 89 additional high-quality NCBI genomes confirms that our collection captures the phylogenetic backbone of the genus, with genomes from Hawaii, Namibia, and the Sargasso Sea nesting within SFE clades. The pangenome is open (14,862 singletons, 62%), driven by two distinct mechanisms. First, a universal hypervariable region (HVR) at a conserved chromosomal position (7-15% from dnaA) is present in all 135 genomes, anchored by tRNA genes at both boundaries (Phe/His and Arg). The HVR carries genome-specific surface polysaccharide biosynthesis genes with a GC age gradient -- highest GC at the tRNA boundaries, lowest in the center -- consistent with a two-ended phage insertion model. Only this HVR is positionally conserved across the genus; the three other hypervariable regions previously described in a single reference genome are not. Second, scattered genomic islands throughout the chromosome contribute the remaining singleton content, including chimeric islands with genes from four bacterial phyla. Biosynthetic pathway reconstruction reveals auxotrophies that are phylogenetically structured, not uniform: biotin, reduced sulfur, and glycine are genus-wide dependencies, while isoleucine, pantothenate, histidine, and glyoxylate cycle capacity vary across lineages with significant phylogenetic clustering. Structural annotation with ESMFold and Foldseek resolved 3,125 hypothetical proteins; 1,222 remain uncharacterized by any method, including a 47-amino-acid protein conserved in two-thirds of all genomes within a fixed operonic context -- independently predicted by two gene callers yet matching nothing in any database. A controlled depth comparison at one station demonstrates that standard metagenome sequencing systematically underestimates Pelagibacter diversity, with three species recovered only at elevated depth and the species count at that station more than doubling (9 vs 4).

Halogenated one-carbon (C1) compounds, such as dichloromethane (DCM), drive critical fluxes in global carbon and halogen cycles. While the genus Dehalobacter is canonically defined by obligate organohalide respiration, its physiological and ecological roles in anaerobic C1 metabolism have remained fundamentally ambiguous. Here, we document a paradigm-shifting metabolic capacity within a sediment-derived microbial consortium: the autonomous fermentation of DCM by a novel population, Candidatus Dehalobacter formatiformans strain J1. Over successive transfers, strain J1 outcompeted co-existing Dehalobacterium formicoaceticum to become the overwhelmingly dominant population (>80% relative abundance), converting DCM stoichiometrically to acetate and formate (4:1) without auxiliary substrates. Genome-resolved metagenomics revealed that strain J1 couples a distinct mec gene cassette--mediating methyl-transfer reactions during DCM activation--to a complete Wood-Ljungdahl pathway for efficient C1 assimilation. Crucially, strain J1 lacks the complete genetic repertoire for de novo cobamide biosynthesis. Physiological validation confirmed that this fermentative pathway is strictly dependent on exogenous cobamides, exposing a profound reliance on community cross-feeding. These findings reveal an unexpected acetogenic lifestyle within Dehalobacter, a lineage historically viewed as comprising obligate organohalide-respiring bacteria. More broadly, this work identifies cobamide-dependent methyl-transfer metabolism as an ecological control on anaerobic DCM fermentation and expands the known roles of Dehalobacter in carbon-halogen cycling in anoxic environments.

In a single Oxford Nanopore long-read metagenome from a Fennoscandian deep-groundwater borehole (KR0015B, Aspo Hard Rock Laboratory, Sweden), 791 protein clusters span at least one chromosomal contig and at least one co-sampled circular mobile element -- the cross-replicon LGT-candidate cohort. The 199 participating chromosomes are dominated by three small-genome / symbiont-associated lineages -- Patescibacteriota, Omnitrophota, and Nanobdellota -- but the per-chromosome participation rate tells a different story: Omnitrophota chromosomes participate at an order-of-magnitude higher rate (mean 56 cross-replicon clusters per genome), while Patescibacteriota and Nanobdellota dominate by compositional abundance only. Two large divergent circular mobile elements (233-kbp u20424375 and 123-kbp u29249220) -- each lineage-restricted within a single Omnitrophota genus, with sparse cross-phylum reach (only 12 of their combined 289 cross-replicon clusters involve a non-Omnitrophota partner) -- together account for 37% of the cohort and lack canonical plasmid or phage signatures. The 233-kbp element carries a Mu-class DDE transposase, is found integrated in one host chromosome at 99.3% nucleotide identity over 87% of element length, and carries an essentially complete bacterial big-operon r-protein cluster (31 r-protein KOs) as cargo with no rRNA genes -- a cargo profile with no published precedent in the mobile-element literature. Seven cross-replicon clusters span both domains; per-cluster phylogenies confirm gene-tree topologies that violate the species-tree expectation in 6 of 10 callable smoking-gun trees. We release the cross-replicon cluster table, integrated mobile-partner classifications, and chromosome taxonomy as a community resource. A parallel cross-chromosome catalog without the mobile-partner requirement contains 957 clusters, 95% of which carry no co-sampled circular plasmid or virus partner -- a chromosome-only LGT footprint that bounds the MGE-coupled cohort and is consistent with vehicle-free / direct-contact transfer in lineages whose close-contact symbiotic biology is well-documented.

The biosynthetic locus encoding the exopolysaccharide poly-N-acetyl-glucosamine (PNAG) is widely conserved across bacteria, including the WHO critical-priority pathogen Klebsiella pneumoniae (Kp). In Kp, PNAG synthesis is mediated by the pgaABCD operon, yet its lineage-specific regulation remains incompletely defined. Using a comparative genomics approach to interrogate the pgaABCD locus across the high-risk clonal Kp complex 258 (CC258) lineage, we identified a previously uncharacterised positive transcriptional regulator located immediately upstream of pgaA, which we designate pgaR. Phylogenetic analysis revealed recurrent evolutionary events affecting this regulatory region, including repeated deletion or truncation of pgaR and a G>A substitution upstream of the pgaR start codon. Functional characterisation demonstrated that loss of pgaR abolishes pgaABCD expression and PNAG production, whereas the upstream G>A substitution drives PNAG hyper-production. In vitro, Kp produce extensive extracellular PNAG networks under static growth conditions, consistent with a role in biofilm architecture. Despite this, PNAG expression was dispensable in murine pneumonia and peritonitis models, while PNAG hyper-production significantly attenuated virulence and disease severity, indicating a fitness cost associated with sustained overexpression. Collectively, we discovered PgaR as a novel gene regulator of the pgaABCD operon. We show a previously unrecognised lineage-specific layer of PNAG regulation in Kp and demonstrate that opposing PNAG phenotypes: loss and hyper-production, have independently and repeatedly emerged among clinical CC258 isolates, highlighting dynamic selection acting on biofilm-associated traits in this high-risk pathogen. ImportanceThe exopolysaccharide poly-N-acetyl-glucosamine (PNAG) is widely conserved in bacteria, including the WHO critical-priority pathogen Klebsiella pneumoniae. However, how PNAG production is regulated in high-risk lineages has remained unclear. Here, we identify PgaR as a previously unrecognised positive regulator of the pgaABCD operon in clonal complex 258, a globally disseminated and drug-resistant lineage. We show that natural genetic variation within this regulatory region leads to strikingly different PNAG phenotypes: complete loss of production or hyper-production. While PNAG contributes to extracellular matrix formation in vitro, it is dispensable for virulence in murine infection models, and sustained overproduction imposes a fitness cost. The repeated and independent emergence of both loss- and gain-of-function variants among clinical isolates reveals dynamic evolutionary pressures acting on biofilm-associated traits. These findings uncover a lineage-specific layer of PNAG regulation and highlight how modulation of surface polysaccharide expression shapes pathogen fitness and adaptation.

Streptococcus pneumoniae is a leading cause of childhood meningitis, sepsis and pneumonia despite widespread implementation of pneumococcal conjugate vaccines (PCVs). Serotype 2, once a major invasive serotype that nearly disappeared in the mid-20th century, is not included in current vaccine formulations. Recent reports from multiple countries suggest potential re-emergence of serotype 2. Here, we present 30 years of hospital-based surveillance from Bangladesh (1993-2022), where serotype 2 accounted for 7.8% of invasive pneumococcal disease cases. Infections occurred predominantly in very young infants (median age, 3 months) and were largely associated with meningitis (91.3%), with nearly 90% of isolates recovered from cerebrospinal fluid. Comparative analysis of otitis media and nasopharyngeal carriage isolates demonstrated high invasive propensity relative to other serotypes. Whole genome sequencing of 170 serotype 2 isolates from 21 countries revealed that all modern isolates belong to the globally disseminated lineage GPSC96, which is distinct from the prototypical laboratory strain D39 (GPSC622). Phylodynamic reconstruction dated the emergence of GPSC96 to the late 19th century, with continued global circulation and largely preserved antibiotic susceptibility. These findings highlight serotype 2 as a potential invasive pneumococcal threat in countries such as Bangladesh and supports consideration of its inclusion in the next-generation conjugate vaccines.

Bacteriophages of environmental bacteria remain underrepresented, lending paucity to phage-biofilm research beyond clinical and model species domains. Here, we present the Alpine Lotic Phage (ALP) collection, curated through an isolation campaign from biofilm-forming bacteria of alpine streams. We obtained 57 phage isolates, which were dereplicated to 28 unique genomes following sequencing. The collection consists of tailed phages infecting 14 bacterial host species with genomes spanning 37 to 363 kb while exhibiting diverse plaque morphologies, depolymerase activity, and distinct impacts on host biofilm architecture. Comparative analyses against public viral genomes and a curated planetary-scale contig database revealed limited sequence similarity, underscoring the novelty of ALP phages. Functional annotation resolved 9 - 54% of predicted genes which encoded viral structural components, nucleotide metabolism functions, anti-defence mechanisms, and auxiliary genes that facilitate viral infection and replication. Together, the ALP collection represents a foundational resource for investigating phage evolution and ecology in natural bacterial communities.

Across the biosphere, microbiomes play essential roles in shaping the health of their host. One notable example of such a microbiome-associated phenotype is disease-suppressive soils, where susceptible plant hosts enrich and activate specific rhizosphere microbial consortia for protection against fungal root pathogens. However, identifying and reconstructing microbial consortia responsible for host protection remains challenging, given the inherent taxonomic and functional complexity of microbiomes. Here, we integrated metagenomic profiling of disease-suppressive microbiomes perturbed by dilution-to-extinction (DTE) with comprehensive culturing and synthetic ecology to identify the key bacterial taxa conferring suppressiveness to the fungal wheat pathogen Fusarium culmorum. Metagenomics of wheat rhizosphere samples along the DTE trajectory revealed bacterial taxa and functions associated with the disease-suppressive phenotype. Crosslinking these DTE metagenome data with a genome-sequenced collection of 336 rhizobacterial isolates from the suppressive soil allowed the reconstruction of synthetic communities (SynComs) of 11 de-replicated strains negatively associated with disease severity. Upon re-introduction in sterilized suppressive soils, this SynCom consistently reproduced the disease-suppressive phenotype. Paired time-series metagenomics and metatranscriptomics of the SynComs pinpointed candidate biosynthetic gene clusters, including a novel non-alpha poly-amino-acid (NAPAA) gene cluster from Arthrobacter, upregulated in presence of F. culmorum. Chemically synthesized NAPAA variants {varepsilon}-poly-L-lysine and {delta}-poly-L-ornithine significantly inhibited F. culmorum hyphal growth. Collectively, our work establishes a transformative strategy for reconstructing microbial consortia that recapitulates beneficial microbiome-associated phenotypes in plant and animal kingdoms.

Fungal hyphae form spatially confined interfaces in soil that mediate close associations with bacteria, collectively referred to as the hyphosphere. Despite its recognized ecological importance, experimental access to hyphosphere-associated microbial communities under realistic soil and plant-associated conditions has remained limited. Here, we present a soil-mimetic microcosm that enables controlled reconstruction and recovery of hyphosphere bacterial communities embedded within plant-associated soil. The system integrates field-derived soil, a native soil microbial inoculum, living cotton seedlings, and a spatially constrained fungal inoculum housed within sterile cell-strainer assemblies, permitting hyphal extension into soil while preserving a recoverable fungal-soil boundary. Using the soil-borne plant pathogen Fusarium oxysporum f. sp. vasinfectum as a model filamentous fungus, we show that the microcosm enables reproducible recovery of hypha-associated soil microaggregates containing physically attached bacterial cells. Full-length 16S rRNA profiling revealed pronounced reductions in bacterial richness and evenness in hyphosphere samples relative to bulk and rhizosphere soils, consistent with recruitment of a restricted subset of the surrounding microbiota. Ordination analyses demonstrated clear compositional separation between soil and hyphosphere compartments, with convergence of hypha-associated communities across bulk and rhizosphere contexts. Phylogenetic turnover analyses indicated phylogenetic structuring, whereas taxonomic analyses identified a conserved set of bacterial genera consistently associated with hyphae alongside compartment-specific taxa influenced by soil and plant context. Together, these findings establish the novel hyphal release-and-capture microcosm as a reproducible, ecologically grounded platform for studying hyphosphere-associated bacterial communities in plant-associated soils.

Bacteria encounter structurally complex extracellular polysaccharides in natural environments, yet the regulatory and evolutionary basis of their utilization remains poorly understood. Here, we isolated a soil-derived Microbacterium strain, named Microbacterium xanthanicum UB-LE1, that grows on xanthan as the sole carbon source. We dissected the genetic and regulatory architecture underlying this capability. Genome sequencing combined with transcriptomic and proteomic profiling uncovered a discrete, strongly inducible regulon associated with xanthan utilization, encoding 23 proteins with five secreted proteins and three candidate transcriptional regulators. DNA-affinity purification sequencing confirmed two regulators binding to operons within the xanthan utilization locus. Comparative genomics across the Microbacteriaceae revealed conserved and lineage-specific features of this system and supports recent acquisition and modular integration of the locus, with at least two predominant architectural variants possibly shaped by substrate availability and ecological specialization. Coordinated induction at both the transcript and protein levels, together with two experimentally validated regulators, points to tight regulatory control of complex polysaccharide degradation in Microbacterium xanthanicum UB-LE1. Together, these findings provide mechanistic and evolutionary insight into how bacteria adapt to complex extracellular carbohydrates, expand current knowledge of xanthan turnover in microbial ecosystems, and establish a framework for exploring the emergence and diversification of specialized polysaccharide utilization pathways across bacterial taxa. IMPORTANCEMicroorganisms are central drivers of carbon turnover in soils and other terrestrial ecosystems, determining the availability of nutrients and shaping microbial community structure. A significant portion of soil carbon is contained in extracellular polysaccharides, yet the pathways by which microorganisms degrade these complex polymers remain poorly understood. Xanthan, a structurally complex and widely produced microbial exopolysaccharide, represents a persistent and largely overlooked carbon pool. By dissecting the genetic, regulatory, and evolutionary basis of xanthan utilization in Microbacterium xanthanicum UB-LE1, this study advances our understanding of how soil bacteria adapt to complex extracellular carbohydrates and how substrate availability shapes the emergence and diversification of specialized metabolic pathways. Importantly, the identification of additional xanthan-active enzymes and regulatory components in M. xanthanicum UB-LE1 opens opportunities for targeted modification of xanthan structure and properties, paving the way for new biotechnological applications in food, materials, and industrial biotechnology, while linking microbial ecology to functional innovation.

Microbes frequently encounter fluctuating environments, requiring dynamic energy management strategies for survival. While carbon storage polymers like polyhydroxybutyrate (PHB) are ubiquitous across bacterial taxa, their precise ecological advantage remains poorly understood.1 Here we show that carbon storage drives conditional fitness during environmental transitions. Using a high-throughput single-cell microfluidic platform, we tracked tens of thousands of Cupriavidus necator cells under precisely controlled carbon and nitrogen fluctuations. We found that PHB provides no advantage under nutrient abundance but becomes decisive at starvation boundaries: during carbon starvation, it enables [~]30% more progeny before arrest; during recovery from nitrogen starvation, it shortens lag and accelerates regrowth. Strikingly, at the single-cell level, PHB granules are inherited in an asymmetric, all-or-nothing fashion, concentrating resources into specific lineages to overcome the discrete energetic threshold required for cell division. Despite this single-cell variance, at the population level, PHB fractions robustly return to a common setpoint after nutrient shifts--a homeostatic behavior consistent with integral feedback control. These findings reveal that while PHB does not increase the basal exponential growth rate, it confers a distinct fitness advantage by prolonging the proliferative phase during nutrient depletion and facilitating successful recovery from starvation, explaining the evolutionary persistence of carbon storage in environments with pulsed resource availability.

Anaeramoeba pumila is a free-living anaerobic amoeba and the smallest known member of the Anaeramoebae, a phylum characterized by elaborate membrane-bound symbiosomes housing sulfate-reducing bacterial symbionts. Here, we report a draft nuclear genome assembly of A. pumila LANTAAN and describe the discovery, genomic characterization, and metabolic reconstruction of Candidatus Centrionella anaeramoebae gen. nov., sp. nov., an obligate intracellular symbiont of A. pumila belonging to the order Legionellales. Ca. Centrionella is a rare anaerobic member of Legionellales, a lineage otherwise comprising aerobic intracellular pathogens. Its genome (1.52 Mbp, 1,249 genes) is highly reduced and encodes an entirely anaerobic metabolism centered on substrate-level phosphorylation, arginine fermentation, and hydrogen oxidation via a bidirectional [NiFe]-hydrogenase -- metabolic strategies that parallel those independently evolved in the distantly related Anoxychlamydiales. The complete Dot/Icm type IVB secretion system is retained and likely mediates ongoing host manipulation, including via a large repertoire of predicted effector proteins. Strikingly, Ca. Centrionella has acquired eukaryotic Rac1-like GTPase genes from its host through horizontal gene transfer, with subsequent domain shuffling and duplication, that it may use to manipulate the cytoskeleton of its host. Unlike other Anaeramoeba symbionts, Centrionella localizes to the host microtubule-organizing center rather than a symbiosome, a localization consistent with cytoskeletal anchoring strategies described in other endosymbionts. The symbiosome, present in other Anaeramoeba species, appears to have been secondarily lost in A. pumila. A co-occurring Desulfobacter sp. LANTAAN, related to symbionts of other Anaeramoebidae, likely forms a tripartite syntrophic consortium by consuming hydrogenosomal fermentation end-products and supplying vitamin B12. Together, these findings illuminate the evolutionary transition in Legionellales from aerobic pathogenesis to anaerobic mutualism, providing a new model for the origins of intracellular symbiosis.

Identifying antibiotic resistance genes (ARGs) from metagenomic data is critical for studying antimicrobial resistance across microbial communities and pathogens. However, there is no standardized methodology for ARG annotation. Here, we compare ten commonly used ARG detection pipelines by analysing over 270 million prokaryotic genes from the Global Microbial Gene Catalogue across 13 distinct habitats. We observed up to a 45-fold difference in the number of reported ARGs, with a mean Jaccard index of only 16% between pipelines. Pipeline selection profoundly impacted downstream biological interpretations, with drastic changes to estimates of ARG relative abundance and richness, to the characterization of pan- and core-resistomes, and to the class-level composition of the inferred resistome. ARG detection pipelines make different, defensible trade-offs, and no single approach should be treated as authoritative. Therefore, users should justify and communicate choices carefully, as our analyses show that, taken uncritically, the same data can support conflicting biological and ecological interpretations.

Dryland ecosystems rely on infrequent rainfall pulses to activate soil microbial communities, yet the fraction and identity of microbes resuscitating after hydration remain unclear. We applied bioorthogonal non-canonical amino acid tagging coupled with fluorescence-activated cell sorting (BONCAT-FACS) and 16S rRNA gene sequencing to identify translationally active bacteria in early (L-BSC) and late (D-BSC) successional cyanobacteria-dominated biocrusts subjected to simulated rainfall under light and dark conditions. Our results reveal that only a small subset of the microbial community resumes activity within six hours, with higher active cell abundances in mature crusts. Microbial activity patterns were largely independent of light exposure and showed partial decoupling from total community composition, indicating that presence does not predict short-term function. These findings suggest that biocrust maturity shapes microbial activation dynamics and that functional responses to precipitation pulses are governed by a conserved pool of fast responders, informing predictions of dryland soil microbiome resilience under changing precipitation regimes.

The restoration of species-rich calcareous grasslands is a critical conservation objective, yet the recovery of the invisible below-ground microbiome remains poorly quantified compared to above-ground vegetation. Using a unique 143-year land-use chronosequence on Salisbury Plain, UK, we investigated the trajectory of ecosystem reassembly across arable, regenerating (23 and 67 years), and ancient grasslands. By integrating vegetation surveys with soil physiochemistry, microbial profiling, and shotgun metagenomics, we identified a profound functional decoupling between floral and edaphic recovery. While vegetation diversity recovered relatively rapidly, approaching saturation within 23-67 years, soil properties exhibited persistent legacy effects and slow convergence. Bacterial richness decreased with restoration age, reflecting a transition from disturbance-adapted copiotrophs in arable soils to a specialised, oligotrophic community in ancient sites. This taxonomic contraction was conversely matched by an expansion in functional potential, driven by the emergence of specific taxa (e.g., Microthrixaceae, Aquihabitans sp.) and metabolic pathways associated with complex carbon cycling and stress tolerance. Crucially, the soil ecosystem did not reach equilibrium even after 67 years, characterised by persistent legacy phosphorus and a slow accumulation of soil organic matter. These findings suggest that passive regeneration alone may be insufficient for full soil functional recovery, and that strategies targeting microbial assembly and long-term carbon dynamics warrant further evaluation.

Background Bacterial fitness is shaped by interactions between genome variation and environmental context, yet how these interactions determine its predictability and heritability remains unclear. In the clinically important pathogens of Klebsiella pneumoniae, a leading cause of hospital-acquired infections, this question is particularly pressing. Despite extensive genomic characterization, we still lack a systematic understanding of how genome-wide variation translates into fitness across diverse environments in K. pneumoniae. Methods We filled this gap by profiling a systematic collection of 1,462 clinical K. pneumoniae isolates across 214 diverse environmental and pharmacological stress conditions using high-throughput chemical genomics. Fitness was quantified from colony growth and integrated with whole-genome sequencing data. Genome-wide association analyses identified genetic determinants of fitness, and machine learning models incorporating genomic features were used to predict fitness.Results Fitness exhibited a strongly environment-dependent genetic architecture, with modest but significant concordance between genetic background and phenotypic variation. Under antibiotic and stress-combination conditions, fitness was driven by discrete, high-effect determinants, including known resistance genes, resulting in stronger signals and improved predictability. In contrast, non-antibiotic environments showed more polygenic and distributed architectures with weaker associations. Genome-wide analyses identified both established and previously uncharacterized genes linked with fitness across conditions. Resistance and virulence determinants exhibited clear context-dependent trade-offs, conferring fitness advantages under selection but imposing costs in non-selective environments. Consistent with this, plasmid carriage showed environment- and genotype-dependent fitness effects, with benefits under antibiotic pressure and measurable costs otherwise. Genomic variant-based models for fitness prediction achieved moderate performance (Mean Spearman correlation ({rho}) = 0.36 (95% CI: 0.18-0.67) for predicted versus observed values in unseen data) across conditions, with improved accuracy under strong antibiotic selective pressures, and produced well-calibrated prediction intervals with high coverage. Despite strong population structure effect on predictions, models captured predictive gene and SNP biomarkers for fitness. Conclusion These findings highlight that bacterial fitness is an emergent property of genome-environment interactions rather than a fixed attribute of genotype. This work establishes a unified high-dimensional genotype-phenotype framework linking genomic variation to fitness across diverse conditions in a major pathogen, with broader implications for other pathogenic bacterial species.

Bacteria can outcompete rivals by using specialized secretion systems to deliver toxic effectors into prey cells. How these systems distinguish prey from kin remains a fundamental unanswered question. In many Xanthomonadales (Lysobacterales) species, the antibacterial type IV secretion system (X-T4SS) mediates cell-cell contact-dependent killing of competing bacteria. Here, we identified XAC2611, a chromosomally encoded, cysteine-rich DUF4189 protein, as essential for preventing X-T4SS-mediated fratricide among sibling cells in Xanthomonas citri. XAC2611 homologs are found within or proximal to the genomic loci coding almost all identified X-T4SS. Live-cell microscopy and biochemical data reveal that XAC2611 is abundantly produced and widely distributed throughout the periplasm of recipient cells and is required to prevent X. citri cells from intoxicating each other in a contact-dependent manner. We also show that XAC2611 homologs from Stenotrophomonas maltophilia protect X. citri cells from attack by S. maltophilia. Protein-protein interaction assays show that XAC2611 interacts directly with the VirB5 subunit. Since VirB5 is predicted to be localized at the tip of the X-T4SS pilus, its interaction with XAC2611 in a neighboring sister cell could block X-T4SS-mediated effector delivery. We therefore name this family of proteins trans-intoxication protection factors (Tpfs).

Most bacteria are enclosed by a peptidoglycan (PG) cell wall that must be expanded for growth. In rod-shaped species, PG elongation is spatially organized in a species-specific manner, occurring either at the cell poles or along the lateral wall. MreB filaments organize the Rod machinery and are typically required for dispersed, nonpolar PG elongation but are dispensable for polar growth. Whether elongation modes are inherently fixed or can be reprogrammed remains unclear. Escherichia coli and Myxococcus xanthus both elongate PG in a dispersed, nonpolar fashion. Here, we show that heterologous expression of M. xanthus MreB in E. coli relocalizes native MreB to the cell poles, thereby redirecting the Rod machinery and PG elongation to polar sites. Moreover, direct targeting of the Rod synthase PBP2 to the poles is sufficient to drive polar PG elongation in E. coli while preserving rod shape. This reprogrammed growth mode bypasses the requirement for MreB filaments, highlighting a plasticity of the Rod system that suggests polar elongation may have emerged through the evolutionary loss of MreB.

Bacterial signals control the development of marine algae, yet the molecular basis of these cross-kingdom interactions remains largely unknown. Thallusin is the paradigmatic case: isolated in 2005, it induces rhizoid and cell wall formation in the green seaweed Ulva at picomolar concentrations, but its biosynthesis has remained elusive for two decades. Comparative genomics across five bacterial phyla identifies a conserved set of genes - the eustigmatophyte bacterial operon (ebo) - as determinants of thallusin biosynthesis. Isotope labeling, heterologous expression, and gene deletion in Stieleria maiorica show that the aromatic scaffold derives from a cyclitol precursor and L-aspartate, with subsequent prenylation and cyclization. Searching 124,295 prokaryotic genomes identifies producers in eleven bacterial lineages, including soil cyanobacteria, establishing thallusin as a widespread cross-kingdom signal reaching beyond the ocean.