Nature Microbiology

Klebsiella pneumoniae is a WHO critical-priority pathogen for which strain-specific bacteriophages are being explored as precision antimicrobials, yet rapid phage-host matching remains a major barrier to therapeutic deployment. We constructed a comprehensive interaction atlas comprising 84 taxonomically diverse phages and 101 globally sourced, clinically representative K. pneumoniae strains, including multidrug-resistant isolates. Systematic pairwise profiling produced 8,484 interaction measurements, of which 2,656 (31.3%) scored positive for bacterial clearance. Genus was the dominant phage-side determinant of host range, while capsule K-serotype was the strongest host-side determinant of susceptibility; aggregate defense, prophage, plasmid, and antimicrobial-resistance features contributed comparatively little. A genome-guided machine learning model predicted interactions without curated host annotations (AUROC, 0.882; AUPR, 0.765), outperforming a model based only on phage genus and K-serotype and modestly exceeding a curated genomic baseline. The model recovered capsule- and lipopolysaccharide-biosynthesis genes, canonical receptors and defense-associated features as major predictors using SHAP analysis. Feasibility tests of expert- and model-selected cocktails exposed a translational constraint. Although all formulations suppressed growth in vitro, only the specific cocktail whose phages replicated robustly within the murine gut reduced colonization, suggesting in vivo amplification rather than predicted host range as the limiting factor for therapeutic efficacy. Together with the activity of a model-selected cocktail built for an isolate completely excluded from training, these results provide a species-wide resource for K. pneumoniae phage matching and support a hybrid workflow combining genome-based ranking with targeted phenotypic validation.

Many bacterial immune defenses transmit recognition of phage infection via the generation of diverse cyclic nucleotide second messengers. Phage have evolved to subvert this kind of immunity by sequestering, degrading, or inhibiting synthesis of these signaling molecules. Consequently, bacteria have evolved counter-mechanisms to detect disruption of cyclic nucleotide signaling and induce another layer of immune protection. Here we detail our discovery of the PanDA defense system (Panoptes-like DisA), an antiphage defense which detects sequestration of 3'3'-c-di-AMP by phage sponge proteins. PanDA consists of two proteins, PanS and PanE, which are both necessary for defense. PanS contains a minimal diadenylate cyclase (DAC) domain that constitutively generates the cyclic dinucleotide 3'3'-c-di-AMP which binds to and represses a toxic effector, the 2TM-{beta} family protein PanE. When a cell is infected by a phage encoding the sponge protein Acb4 (anti-CBASS protein 4), PanE is activated and induces membrane permeability. This work represents the first confirmed use of 3'3'-c-di-AMP as an immune second messenger in bacteria, facilitated by the exaptation of a DAC domain which has thus far only been best understood for its non-immune signaling roles.

ESKAPE pathogens drive clinical antibiotic resistance and intractable infections, severely compromising antimicrobial therapies. Bacteriophages are promising alternatives to antibiotics, yet their diversity, function and ecological impacts in ESKAPE pathogens remain poorly defined, hindering phage therapy translation. Here, we integrated 11,947 high-quality ESKAPE genomes with global metagenomic viral data to construct a comprehensive non-redundant virome of 14,496 ESKAPE-associated viruses, including four unreported viral clades. We found pervasive competition among mobile genetic elements (MGEs) in the ESKAPE mobilome, where nested MGE architectures empower low-mobility antibiotic resistance genes (ARGs) with horizontal transfer ability to fuel resistance dissemination. Unlike ARG-rich MGEs, ESKAPE phages carry minimal ARGs and antagonize plasmids to constrain ARG propagation, confirming their biosafety for therapy. We further revealed distinct phage-host arms races, typically virulent phages enrich anti-defense genes to evade bacterial immunity, and novel viruses hijack host methyltransferases targeted by CRISPR-Cas systems. This study establishes a systematic ESKAPE virome resource, demonstrates phages dual roles in targeting resistant pathogens and curbing resistance spread, and provides mechanistic support for phage therapy clinical application.

Plasmids drive horizontal gene transfer and the spread of antibiotic resistance, yet their distribution across microbial genomes is highly uneven and often viewed as environmentally driven, leaving unresolved whether host chromosomal architecture imposes predictable constraints. Here, using machine learning on 52,393 complete prokaryotic genomes, we show that chromosomal gene content encodes predictive information for multiple dimensions of plasmid biology: carriage status, quantitative load, and mobility potential. Remarkably, highly compressed chromosomal signatures-- as few as 15 genes or the coarse-grained composition of seven major enzyme classes--suffice for robust prediction. Moreover, different functional cargoes carried by plasmids, including antibiotic resistance classes, can also be predicted from host chromosomal signatures. These findings establish that plasmid-host compatibility is systematically encoded in host chromosomes, reframing plasmid ecology from environment-driven to host-constrained--a shift with direct implications for combating resistance and engineering stable microbial chassis.

Asgard archaea are the closest known relatives of eukaryotes and are central to models of eukaryogenesis involving archaeal-bacterial symbiosis, yet the contribution of viruses remains unexplored. Here, for the first time, we visualised unique viruses associated with Asgard archaeal cells. Additionally, we identified the first putative Asgard-archaeal virus satellite, which exhibited genomic interactions involving a bacterium, Stromatodesulfovibrio nilemahensis, providing evidence of a virus-mediated interaction between an Asgard archaeon and a bacterium. Additionally, novel proviruses of S. nilemahensis displayed distinct genomic features where predictions of alternate recombination sites suggested the acquisition of horizontally acquired genes associated with biofilm formation. Further, we comprehensively characterise (pro)viruses associated with this co-culture using high resolution cryo-electron tomography, proximity ligation (Hi-C), and metagenomics. Together, these findings expand the known diversity of Asgard archaeal viruses and establish a foundation for investigating the role of viruses in microbial symbiosis relevant to the emergence of eukaryotic life.

Herpesvirus virions form by remodeling of intracellular virus-host interaction networks into evolutionarily conserved particle architectures. Here, we define a virion-wide spatial and quantitative protein proximity map of herpes simplex virus 1 (HSV-1) by combining cross-linking mass spectrometry with quantitative proteomics. Integration with intracellular interaction maps reveals that maturation acts as a selective filter, transforming broad virus-host associations into an organized virion network. This process depletes biosynthetic and nuclear components while enriching interactions involved in tegument organization and envelope acquisition around the viral protein UL49. Comparison with analogous maps of human cytomegalovirus (HCMV) identifies HSV-1-UL49 and HCMV-UL32 as functionally equivalent network hubs despite lacking evolutionary relatedness. Both hubs converge on shared phosphoregulatory host factors, short linear interaction motifs, and liquid-liquid phase separation. At the virion surface, the host complement regulator CD59 protects particles from complement-mediated inactivation. Together, these findings show how conserved organizational principles shape virus-specific virion interaction networks during herpesvirus maturation.

Bacterial sRNAs are pervasive post-transcriptional regulators, yet how they arise, evolve, and decay remains poorly understood. Here, we provide a high-resolution transcriptome map and curated sRNA set for the pathogen Vibrio parahaemolyticus. We identify over 100 sRNAs, including broadly conserved, lineage-specific, and previously unidentified transcripts, as well as dual-function regulatory/coding sRNAs. Functional analysis of several examples highlights conserved and lineage-specific regulators of metabolism and flagella. Broadly conserved VcrX represses chitin utilization genes and may regulate Vibrio Spot 42, which we confirm is translated. We expand on FlaX regulation of polar flagella across the genus by demonstrating that the sRNA differentially activates/represses downstream flagellins, with a potential FlaX sponge mediating feedback in specific clades. We further show that V. parahaemolyticus, but not V. cholerae, RyhB is translated into a Cys-rich small protein that could regulate related pathways. Together, these findings establish a resource for Vibrio and a platform for comparative studies of post-transcriptional regulation, enabling investigation of how sRNAs and their regulatory networks evolve.

Temperate Bacillus phages use arbitrium peptides to coordinate lysis-lysogeny decisions, but whether the mature communication peptide can be sensed directly by Bacillus subtilis and affect its physiology and behavior is unknown. Here we show that the {varphi}3T arbitrium peptide SAIRGA elicits a sequence- and stereochemistry-dependent response in Bacillus subtilis that is strongly expressed in surface-grown colony biofilms but is not accompanied by comparable changes in planktonic growth or static-liquid pellicle morphology. The response persists in the absence of AimR, the canonical arbitrium receptor. Within colonies, SAIRGA alters spatial PtapA activity and increases heat-resistant spore formation without increasing total viable cell yield. Untargeted metabolomics reveals broad dose-dependent remodeling that tracks peptide activity, while program-level proteomics independently converges on late-sporulation and mature-spore-associated states. This study highlights how a phage-derived peptide may act as a signal, enabling the host to pivot toward a survival-focused developmental state.

Bacteriophages that infect Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever, are regarded as specialized, because all previously characterized phages depend on the Vi capsular polysaccharide for infection. Whether capsule-independent infection strategies exist has remained unclear. Here we identify environmental phages that infect S. Typhi both in the presence and absence of Vi. Screening 140 urban wastewater samples from Dhaka, Bangladesh, where typhoid is endemic, we recovered phages infecting a Vi-deficient S. Typhi strain from 41 samples (29%). All 41 phages also infected the isogenic Vi-expressing host, although 28 did so with 10- to 105-fold lower infection efficiency, and suppressing capsule expression increased susceptibility to 23, indicating an inhibitory effect of Vi on infection by many these phages. All 41 phages infected S. Paratyphi A and nine infected a monophasic S. Typhimurium, a broader host range than the Vi-dependent phages, which were restricted to Vi-expressing Typhi. Across 26 circulating genotypes, capsule suppression increased susceptible genotypes per phage by 1.51 on average (Wilcoxon p = 5.76 x 10-6), though four genotypes remained resistant to all phages tested, indicating additional determinants of susceptibility. Whole-genome sequencing of 27 phages identified three genera in two families, predominantly Teetrevirus (19/27); TerL phylogeny separated these from classical Vi-dependent phage lineages. Together, these findings reveal a broader-host-range component of Typhi phage ecology and show that Vi dependence is not a universal feature of phages capable of infecting S. Typhi.

Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) exhibit distinct seasonal epidemiology, with RSV circulating in early autumn and hMPV peaking in midwinter, yet the structural basis for this niche partitioning remains undefined. Here, we integrate in situ cryo-electron tomography and functional virology to decode the architectural logic governing their entry dynamics. RSV employs a matrix (M)-regulated prefusion F (pre-F) organization, partitioning trimers into stabilizing hexagonal superlattices and fusion-competent pools to maintain superior thermotolerance. By contrast, hMPV compensates for its intrinsically unstable, monomeric pre-F with extreme ribonucleoprotein polyploidy, packaging [~]4-fold more genome equivalents to ensure productive infection. Fusion events localize exclusively to M-depleted, non-arrayed membrane regions, establishing a spatial checkpoint for activation. These findings reveal a conserved trade-off between environmental resilience and genomic redundancy that dictates divergent pneumoviral entry strategies, explaining their distinct seasonal ecological niches.

Transcription underpins the expression of genetic information and is viewed as a series of independent initiation and termination events. In bacteria, promoters and terminators of transcription are therefore considered distinct regulatory elements. Here, we challenge this view. Genome-scale analyses reveal that promoters and terminators are extensively coupled. Thus, initiation and termination sites, for adjacent transcription units, frequently overlap. This conserved organisation arises because the DNA sequences, which direct termination, also contribute to promoter function. This couples neighbouring transcription units and generates regulatory interference between RNA polymerases. These findings define a universal mechanism for coordinating transcription across bacterial genomes.

Bacterial diadenylate cyclase (DAC) enzymes synthesize the nucleotide signal 3'3' cyclic di-AMP (3'3'-c-di-AMP) to control osmoregulation, cell-wall homeostasis, and DNA-damage responses. Here we discover specialized roles for DAC enzymes in bacterial immunity and define a Panoptes-like system we name Panoptoo as a DAC-containing anti-phage defense that guards against viral immune evasion. The Panoptoo protein PanS is a minimal DAC that constitutively synthesizes 3'3' cyclic UMP-AMP (3'3'-cUA) or 3'3'-c-di-AMP to negatively regulate a partnering PanE S2TM{beta} membrane-targeting effector. We show that Panoptoo decoy signaling acts as a counter-defense to detect phage immune evasion proteins that inhibit nucleotide immune signals. A 1.5 [A] crystal structure of PanS in complex with 3'3'-cUA explains how a symmetry break in the canonical DAC active site enables synthesis of asymmetric signaling molecules. Together, our results uncover a role for DAC domains in bacterial anti-phage defense and expand our understanding of nucleotide signaling in antiviral immunity.

Vibrio parahaemolyticus (V. para) is an enteric pathogen that establishes a protected intracellular niche using its second type III secretion system. However, how this bacterium adapts to the host cytoplasm while overcoming cellular defenses has remained unclear. To define these mechanisms, we performed dual-transcriptomic profiling of both pathogen and host during invasion, intracellular replication, and late infection. Our analyses revealed extensive metabolic reprogramming by V. para, including induction of diverse nutrient transporters and metabolic pathways. We discovered that because mammalian cells are auxotrophic for aromatic amino acids, V. para must activate its own unique biosynthetic machinery, a requirement that proved essential for intracellular growth. Infected host cells mounted a sustained NF-kappaB response. Both heightened NF-kappaB activation and disruption of canonical NF-kappaB signaling restricted bacterial expansion, indicating that V. para exploits a finely tuned Goldilocks level of immune signaling to promote survival and replication. Together, these findings uncover fundamental metabolic and immune adaptations that drive pathogenesis.

Collateral sensitivity (CS) - resistance to one antibiotic inducing hypersensitivity to another - offers an evolutionary trap for multidrug-resistant pathogens. A strongly connected component (SCC) in the directed CS graph is a closed cycle in which every drug is reachable from every other. Prior evidence for CS SCCs is exclusively in vitro. We mined 104,337 susceptibility records from BV-BRC spanning four WHO critical-priority pathogens (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa; 18,821 isolates), using Fisher's exact tests with Benjamini-Hochberg FDR correction, Tarjan's algorithm, and permutation testing (n = 1,000). Two species yielded qualifying SCCs. In K. pneumoniae (4,286 isolates), a 3-node SCC - imipenem, meropenem, tetracycline - was detected (empirical p = 0.001); both carbapenem-tetracycline edges are bidirectional (OR = 1.81-1.82, q < 0.002, n > 850 per edge). In E. coli (6,720 isolates), a bidirectional 2-node SCC links colistin and cefotaxime (OR = 10.13, 95% CI 2.82-46.12, q = 0.042, n = 87; permutation p = 0.008); with a fragility index of 1, we report it as a hypothesis, not an established effect size. The carbapenem signal is tetracycline-specific: tigecycline shows co-resistance (OR < 0.35), as its distinct RamA/AcrAB-TolC mechanism predicts. ORs of 2.2-2.7 persisted across independent year bands (2009-2014). S. aureus returned no qualifying SCC, but that null is power-limited: only 8% of testable pairs could detect the K. pneumoniae effect size. Prior clinical analyses characterised pairwise and three-way collateral effects; to our knowledge these are the first closed CS cycles identified in clinical surveillance data, motivating experimental follow-up.

Influenza A virus replicates its segmented RNA genome in the nucleus in the context of viral ribonucleoprotein (vRNP) complexes. Newly synthesised vRNPs are exported to the cytoplasm for virion assembly, whereas structurally similar complementary RNPs (cRNPs), which function as replication intermediates, are not incorporated into virions. The molecular basis for this selectivity remains unclear. Here, combining promoter mutagenesis, single-molecule fluorescence in situ hybridisation (smFISH), virus-like particle (VLP) assays and cryo-electron microscopy (cryo-EM), we find that cRNPs are retained in the nucleus and that export competence is encoded by promoter architecture, with the length of the single-stranded 3' promoter region as the principal contributing feature and promoter sequence and duplex architecture further modulating export efficiency. cRNPs carrying export-competent promoters are incorporated into virus-like particles, demonstrating a functional link between nuclear export and packaging competence. Cryo-EM analyses reveal that both 3' vRNA and cRNA promoters bind a common polymerase surface site but are associated with distinct polymerase conformational landscapes, with only the vRNA promoter being compatible with an encapsidase conformation of the polymerase. Together, our findings identify a promoter-dependent checkpoint that links RNA promoter architecture and polymerase conformation to RNP export and packaging. These results demonstrate that influenza virus initiates genome selection in the nucleus and reveal nuclear export as an early step in genome selection.

Streptomycetes are prevalent members of soil and plant microbiomes, yet how they cope with toxins produced by root-infecting fungal pathogens remains poorly understood. Plant pathogenic Fusarium species produce the mycotoxin fusaric acid (FA) that contributes to virulence and perturbs rhizosphere microbiome dynamics. Here, we show that root-colonising Streptomyces sp. ATMOS43 neutralizes FA through amino acid conjugation. Metabolomics revealed the formation of single amino acid and dipeptidyl conjugates of FA, with FA-Ser as a major conjugate that lacked detectable toxicity in in vitro and in planta assays. Proteomics and physiological analyses revealed that FA toxicity involves, in part, zinc chelation, which is abolished upon conjugation of FA to Ser. Co-cultivation experiments further showed that Streptomyces sp. ATMOS43 restores growth of FA-sensitive streptomycetes, indicating that conjugation can mitigate the impact of FA on plant microbiome assembly. Together, our findings show that plant-associated streptomycetes can protect plants by directly inhibiting Fusarium growth and by neutralizing its toxic virulence factor FA.

Bacterial toxin-antitoxin (TA) systems are classically viewed as stress-activated toxic switches. Specifically, ribonucleolytic toxins are thought to indiscriminately cleave RNA to halt cellular growth. We recently showed that the MazF toxin of Bacillus subtilis targets an unusually strict 6bp RNA cleavage sequence, but the implications of this stringent specificity were unknown. Here, we demonstrate that the MazEF system functions as a non-lethal post-transcriptional regulator in B. subtilis. Using a specialized single cell fluorescent reporter and transcriptome profiling, we show that MazF is uniformly activated across the population upon entry into the stationary phase, where it cleaves a narrow mRNA regulon to reshape gene expression. Rather than inhibiting growth, MazF activation tunes down the Spo0A stress response by repressing the mRNA level of its kinases. Reduced stress leads to an adaptive shortening of the lag phase upon nutrient replenishment. Furthermore, MazEFs structural architecture, cleavage specificity, and impact on growth recovery are highly conserved across Gram-positive bacteria. Altogether, our findings redefine a paradigmatic toxin as a precision global mRNA stress regulator that primes cells for rapid regrowth. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743204v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1790c77org.highwire.dtl.DTLVardef@215962org.highwire.dtl.DTLVardef@1734aa4org.highwire.dtl.DTLVardef@296b45_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pellicles, expansive bacterial communities that form on liquid surfaces, contain residents that incorporate asynchronously and exist in distinct physiological states. How interactions between cell populations convert segregated founder pellicle microcolonies into a contiguous, macroscale structure is unknown. Using high resolution timelapse microscopy of Vibrio cholerae pellicle formation, we show that the confluent community forms through the recruitment of individual planktonic cells to regions between founder microcolonies. In addition to Type IV MSHA pili, individual planktonic V. cholerae cells attach to the air-liquid interface using their cell-surface-bound vibrio polysaccharide (VPS), which binds to the Bap1 and RbmC adhesins secreted by existing pellicle microcolonies. Planktonic V. cholerae cells readily attach to the interface by VPS-adhesin binding when they exist in the low-cell-density quorum-sensing state because this is the mode that promotes VPS production. Single-molecule FISH of V. cholerae pellicles reveals that regions near pellicle microcolonies, where secreted Bap1 and RbmC adhesin levels are the highest, recruit a higher proportion of VPS-producing planktonic cells than do more distant regions. Thus, existing pellicle microcolony inhabitants in an advanced phase of sessile growth prime the surface for new colonizer cell attachment, driving a spatial pattern of gene expression within the pellicle community.

Outer-membrane permeability is a major determinant of antibiotic susceptibility in Gram-negative bacteria and is generally thought to be controlled through transcriptional regulation of porin expression. Here we identify the small periplasmic protein PtrA as a regulator of OprD-dependent carbapenem permeability in Pseudomonas aeruginosa. PtrA promotes imipenem resistance without altering OprD abundance, associates with OprD-containing membrane complexes and reduces intracellular imipenem entry. Using zinc and copper as complementary physiological signals, we show that PtrA-mediated permeability control is mechanistically distinct from CzcRS-dependent repression of oprD and precedes transcriptional porin depletion. These findings define a two-phase mechanism in which rapid periplasmic regulation provides an immediate adaptive response before transcriptional remodeling of the outer membrane. Our work identifies adaptive impermeability as a previously unrecognized mechanism linking environmental sensing to dynamic control of bacterial outer-membrane permeability.

Immediate-early viral proteins must rapidly counteract host defenses directed at incoming viral genomes, yet the protein interaction networks that mediate this remain unresolved. This gap persists largely because immediate-early proteins are expressed at low abundance during this stage of infection, making them challenging to investigate with standard approaches. Here, we present a virus-driven proximity proteomics framework to investigate immediate-early phase virus-host interactions in an authentic infection context. An isogenic P2A control virus untethers a miniTurbo biotin ligase from the viral protein under investigation, effectively modeling bait abundance and background changes from a matched infection context. Continuous biotin labeling from the earliest hours of infection further amplifies detection of transient, low-abundance interactions characteristic of this phase. Using the adenovirus E1A hub protein as a benchmark, we recovered the majority of known E1A interactors and identified over 150 high-confidence interactions. Applying this strategy to the immediate-early phase, we resolved the E1A interactome and identified the p400 chromatin remodeling complex as its dominant target. We show p400 associates with incoming viral genomes, represses gene expression, and regulates persistent infection. These data identify p400 as a poised host restriction factor and establish a temporally resolved proximity proteomics strategy transferable to other immediate-early viral proteins.