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.

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).

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.

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.

Habitat transitions are central to microbial ecology and evolution and have been extensively studied across vastly different environments, such as between saline and non-saline environments. However, microbial habitat transitions along other large-scale environmental gradients remain poorly studied. This is particularly true for transitions involving the cryosphere, despite building evidence suggesting the Cryogenian as important for evolutionary radiation. Here, we investigated ecosystem transitions and related genomic adaptations of the cosmopolitan cryospheric Polaromonas bacterium. We constructed a pangenome from 282 high-quality genomes, sourced from glaciers, glacier-fed streams, lakes, wetlands, groundwater, rivers, and soils. Phylogenetic reconciliation revealed that the ancestral Polaromonas genome radiated from glacier ecosystems into various downstream environments through multiple independent transitions. These transitions were marked by extensive horizontal gene transfer and gene loss, with mobile genetic elements, such as plasmids and prophages playing key roles in genomic diversification. Predicted ancestral genomes encoded versatile metabolic and stress-response capacities, supporting adaptation to fluctuating and extreme conditions in the various cryospheric habitats. Compared to the ancestral Polaromonas genome, distinct genomic signatures were associated with specific habitats: glacier-fed stream lineages possess expanded stress tolerance repertoires, glacier lineages gained chemolithotrophic and anaerobic pathways, lake and wetland genomes acquired phototrophic functions, and soil lineages expanded substrate transport and stress tolerance. Together, our findings highlight the role of genomic plasticity in the ecological success of Polaromonas, and also underscore the cryosphere as a potential evolutionary cradle from which lineages dispersed and adapted to downstream aquatic and terrestrial environments.

The type VI secretion system (T6SS) ia a widespread bacterial nanomachine that mediates interbacterial competition by delivering toxic effectors into neighboring cells. Among these, enzymes targeting nicotinamide adenine dinucleotide cofactors (NAD and NADP) are particularly potent because they rapidly disrupt redox homeostasis and central metabolism. Several families of T6SS-associated NAD(P)-consuming effectors (Tne1-Tne4) have been described. Here, we characterize a T6SS-associated Rhs toxin from Pluralibacter gergoviae. Competition assays show that P. gergoviae kills Escherichia coli in a T6SS-dependent manner. Heterologous production reveals that the Rhs C-terminal extension is toxic in the E. coli cytoplasm and that co-production with the protein encoded downstream neutralizes this activity. AlphaFold3 modeling predicts that the toxin adopts an ADP-ribosyltransferase (ART)-like /{beta} fold with a putative catalytic pocket accommodating NAD. By contrast to T6SS ART toxins described so far, the toxin does not inhibit transcription, translation or cell division, but instead depletes NAD and NADP. Phylogenetic analyses and structural modeling show that this effector defines a new ART-related family of NAD(P) glycohydrolases, which we propose to name Tne5, broadly distributed across antagonistic systems.

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.

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.

Antibiotic persistence allows a subpopulation of bacterial cells to survive antibiotic treatment without acquiring resistance mutations, often contributing to treatment failure. Although many genes have been linked to persistence, their deletion rarely abolishes the phenotype, highlighting redundancy in persistence mechanisms. Moreover, the transient and heterogeneous nature of persister formation has made it difficult to resolve its molecular basis using bulk analyses. Here, we use bacterial single-cell RNA sequencing and functional assays in Klebsiella pneumoniae to demonstrate how transcriptional heterogeneity and redundancy in stress responses shape persistence outcomes. Using growth phase as a biologically meaningful axis of transcriptional variation, we reveal that even within an isogenic population, distinct transcriptional responses can be induced and co-contribute to survival. These responses are shaped by the cells pre-treatment transcriptional state and the mechanism of antibiotic action. Genetic and environmental perturbations, such as rpoS deletion and nutrient supplementation, shift pre-treatment cell states and alter persistence frequencies. Our findings establish the biological significance of transcriptional heterogeneity shaped by pre-treatment cell states, providing a systems-level framework for understanding persistence and suggesting strategies to enhance antibiotic efficacy by modulating cell states.

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.

Metabolic adaptation to nutrient stress is a key but poorly understood driver of fungal virulence. Here, we show that a dominant East Asian lineage of Cryptococcus neoformans (VNIa-5), which disproportionately infects immunocompetent hosts, has undergone lineage-specific rewiring of glucose-responsive stress pathways. Integrating population genomics, transcriptomics, and experimental infection models, we demonstrate that VNIa-5s clinical dominance is not explained by environmental prevalence. Instead, selective activation of Snf1 signalling links glucose limitation to mitochondrial tubularisation, extracellular vesicle remodelling, and enhanced melanization. Under low-glucose conditions, VNIa-5 exhibits marked mitochondrial plasticity and extracellular vesicle compositional shifts resembling hypervirulent outbreak lineages of Cryptococcus gattii. Following experimental induction of dormancy, VNIa-5 shows significantly increased virulence in vivo compared with the closely related but clinically rare VNIa-31 subclade, with host survival tightly correlated with mitochondrial morphology. These findings identify metabolic stress integration as a central mechanism shaping cryptococcal virulence and disease in immunocompetent human hosts.

Type III secretion (T3S) systems assemble bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. In both systems, completion of an early assembly intermediate triggers an irreversible switch from early to late substrate secretion. In the flagellar system, this switch is controlled by the secreted molecular ruler FliK acting on the core T3S component FlhB, but the molecular mechanism governing this transition has remained unclear. Here we show that removal of two components, Fluke and the cleaved C-terminal domain of FlhB (FlhBCCD), locks the secretion apparatus in a constitutive late secretion state. In these mutants, secretion specificity no longer requires completion of the hook-basal body or the FliK ruler, indicating that Fluke and FlhBCCD function to maintain the apparatus in early secretion mode. Consistent with this model, synchronized flagellar gene expression experiments reveal that FlhBCCD is retained during early assembly and is lost coincident with hook-basal body completion and activation of {sigma}28-dependent late gene expression of flagellin and chemosensory genes. Structural modeling of the FliK C-terminal switch domain and FlhBCCD supports a mechanism in which secretion of FliK promotes destabilization and ejection of FlhBCCD from the secretion apparatus. Disruption of a folded region within FliK switch domain uncouples secretion from switching, indicating that the timing of FliK unfolding during secretion is critical for activation of the specificity switch. These findings show that secretion specificity switching is driven by FliK-dependent removal of inhibitory components, rather than passive sensing of assembly completion. Significance StatementType III secretion (T3S) systems build complex bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. How these systems switch secretion specificity during assembly has remained a long-standing question. We demonstrate that the flagellar T3S specificity switch requires removal of two inhibitory components that actively maintain the apparatus in an early secretion state. Their FliK-dependent ejection irreversibly triggers the transition to late substrate export, revealing that secretion switching is controlled by active inhibitory regulation rather than passive sensing of assembly completion. These results define a molecular mechanism for hierarchical control in bacterial secretion systems and provide insights likely relevant to other T3S nanomachines.

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.

Anaerobic protists across diverse lineages have independently evolved intimate spatial associations between their hydrogen-producing mitochondrion-related organelles and prokaryotic symbionts, yet the cellular structures mediating these syntrophic partnerships remain poorly characterized. Anaeramoebae -- a recently described phylum of anaerobic amoeboflagellates -- have evolved a particularly elaborate solution: the symbiosome, a membrane organelle that houses sulfate-reducing Desulfobacter sp. symbionts alongside host hydrogenosomes and maintains direct connections to the extracellular environment. Previous FIB-SEM work using aldehyde-based fixation established the symbiosome as a dynamic structure but left critical architectural questions unresolved, including whether symbionts occupy separate compartments and how extensive the connections to the cell exterior truly are. Here, we use high-pressure freezing with optimized cultivation to achieve markedly improved membrane preservation in Anaeramoeba flamelloides. We show that the symbiosome is a single, fully interconnected compartment enclosing all Desulfobacter sp. symbionts, spanning up to 15% of total cell volume with a membrane surface area matching that of the plasma membrane. The number of symbiosome-to-surface connections is an order of magnitude higher than previously documented -- 12 and 29 pores in two cells, compared with three in an earlier published volume -- likely reflecting the metabolic requirement for extracellular sulfate access by the symbionts. These findings establish the Anaeramoeba symbiosome as one of the largest known membrane organelles in a single-celled eukaryote, with an architecture shaped by the demands of syntrophic exchange.

Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin developed to overcome multidrug resistance in Gram-negative bacteria. Despite its unique iron-dependent entry mechanism, CFDC resistance has emerged in Klebsiella pneumoniae, primarily driven by alterations in siderophore transport and {beta}-lactamase evolution; however, the broader intrinsic resistome that supports CFDC tolerance remains incompletely defined. Here, we performed high-density transposon mutagenesis (Tn-Seq) in the epidemic ST258 K. pneumoniae to map the functional genetic landscape of CFDC susceptibility. Tn-Seq identified siderophore uptake components (tonB and cirA) as the dominant determinants of CFDC resistance. In contrast, disruption of genes involved in peptidoglycan recycling (ampG, ldcA), synthesis (mrcB, lpoB) and enterobacterial common antigen (ECA) biosynthesis (wzxE, wzyE), as well as deletion of plasmid-encoded blaKPC-3, increased CFDC susceptibility. In a CFDC-resistant {Delta}tonB strain, targeting these envelope homeostasis pathways yielded only limited resensitization relative to the siderophore-competent parental strain. Deletion of blaKPC-3 produced the greatest increase in susceptibility, reducing the CFDC MIC four-fold. This pattern is consistent with a model in which reduced CFDC influx in the {Delta}tonB background lowers intracellular drug exposure to levels at which the otherwise limited anti-CFDC activity of KPC {beta}-lactamase becomes sufficient to drive resistance. Together, these data define a hierarchical genetic architecture for CFDC resistance in ST258 K. pneumoniae, in which iron-dependent drug uptake is primary, {beta}-lactamase activity is secondary, and intrinsic envelope stress buffering shapes bacterial fitness once CFDC enters the cell.

Whether co-occurring microbial populations share similar evolutionary dynamics remains poorly understood, because strain-level resolution across many lineages simultaneously has been unachievable from metagenomic assembly. Here we apply 720 Gbp of Nanopore sequencing and the assembler myloasm to a South San Francisco Bay microbiome, recovering 488 high-quality single-contig genomes -- including 328 circular chromosomes -- without manual curation. Strain-level resolution revealed striking contrasts in population structure: Pelagibacter yielded 78 high-quality single contig genomes spanning 18 species with no two sharing >=99% average nucleotide identity (ANI) -- every genome a distinct strain, consistent with phage-driven frequency-dependent selection -- while HIMB114 was nearly monotypic, with 9 of 11 high-quality genomes in a single species, suggesting periodic selection or a recent sweep. Using unsupervised Markov clustering of variational autoencoder embeddings, we assigned 99.3% of contigs >=5 kbp to a taxonomic domain and resolved nearly 50% of the community to species level. The assembly also captured nearly 95,000 viral contigs (>=5 kbp) including 502 giant viruses, novel eukaryotic lineages, and mercury resistance genes reflecting the Bays contamination legacy. These contrasting patterns -- coexisting in a single 10-liter sample -- reveal that fundamentally different evolutionary strategies operate simultaneously within a complex microbiome, a level of resolution not previously demonstrated from de novo assembly. Wed like to note that the results of this study mark a milestone in a 10 year hunt for Torben to get as many complete Pelagibacter genomes from a metagenome as possible.

Plant leaves harbor diverse microbial communities influenced by environmental inputs and host traits, yet it remains unclear whether leaves act as passive substrates or active ecological filters that reorganize microbial functional capacity. Phylloplane pH regulation is one hostplant trait that has been traditionally underexplored. We used metatranscriptomics to examine microbial gene expression on the phylloplane and within whole leaves of five plant species spanning the extremes of baseline phylloplane pH, including hyperalkalinizing Gossypium species, weakly buffering Beta vulgaris, and hyperacidifying Nepenthes species. Young leaves were inoculated with a common soil-derived microbial community to quantify host-associated restructuring of taxonomic and functional profiles, and short-term pH perturbations were applied to test the effect of transient abiotic stress. Across both phylloplane and whole-leaf datasets, host species identity was the primary axis structuring microbial taxonomic composition and expressed functional repertoires. Leaf-associated communities diverged from the source inoculum, but retained a substantial shared functional backbone enriched for central biosynthetic and core metabolic pathways. Host-associated differentiation reflected selective retention and redistribution of reactions within this shared environmental pool rather than acquisition of novel metabolic capacity. Enriched pathway subsets were metabolically coherent and taxonomically distributed across multiple bacterial orders, consistent with functional redundancy and trait-based assembly. Among hosts, Gossypium exhibited the strongest restructuring relative to inoculum, suggesting comparatively stronger host-associated filtering. In contrast, short-term pH manipulation did not induce consistent community-wide functional reorganization. Microbial physiological responses to the phylloplane environment and external pH were observed at the organismal level. Together, these results position leaves as active ecological filters that reorganize microbial functional landscapes through host-specific trait regimes. This work begins to implicate some role of phylloplane pH regulation in microbial assembly and function.

Revegetation is a key strategy for restoring degraded lands globally. While this process can reshape belowground microbial communities, the extent to which such changes restore soil ecosystem functions, and whether different microbial traits recover synchronously or on distinct timescales remains less clear. Understanding this is essential for evaluating restoration success, as different microbial traits underpin distinct ecosystem services, from carbon storage to plant growth promotion, and the sequence in which these are restored can inform restoration targets and provide meaningful indicators of long-term ecological recovery. To address this, we applied deep metagenomic sequencing to characterise soil microbial responses following revegetation (spanning 1-31 years prior to sampling) on grazing agricultural lands. We find that revegetation following grazing cessation drove significant and sequential shifts in dominant microbial functions and life-history strategies. Functional changes occurred in distinct phases: an early, rapid restructuring of core soil health processes, detectable as early as three years, including enrichment of nutrient retention and carbon fixation pathways, followed by a more gradual development of plant growth-promoting traits as the plant community matures. Genome-resolved analyses of nearly 500 metagenome-assembled genomes revealed a fundamental shift in dominant microbial life history strategies: from a resource-scavenging and stress tolerance profile in grazing soils to strategies that prioritise biosynthesis and growth yields in revegetated soils. These lifestyle shifts have important implications for enhancing the microbial biomass and carbon sink potential of soils. Together, these findings show that microbial functional shifts following revegetation are temporally structured -- informing expectations for when key restoration targets may be achieved, and providing a practical framework for monitoring ecosystem recovery.