Nature Microbiology

Compact viral genomes encode sophisticated strategies to reprogram host gene expression, often by co-opting rather than replacing the host RNA polymerase. How bacteriophages achieve such control in Mycobacteria remains largely unknown. Here, time-resolved interaction proteomics of mycobacteriophage D29 infection identifies a conserved two-protein module, gp53-gp52, that binds the host RNA polymerase. Affinity purification confirmed association of both proteins with the polymerase, while native mass spectrometry demonstrated that gp53 displaces the host sigma factor. Structural modelling places gp53 at the sigma-binding surface of RNA polymerase, consistent with molecular mimicry of sigma-factor engagement. The module is conserved throughout Cluster A mycobacteriophages and occurs in fused or split genomic architectures. These findings suggest sigma-factor displacement as a mechanism of transcriptional takeover in viruses of mycobacteria and show how a compact viral module can repurpose an essential host molecular machine

Bacteriophages are highly specific predators of bacterial hosts, but the molecular mechanisms governing their entry across the structurally complex mycobacterial cell envelope remain poorly defined. Utilizing a genome-wide CRISPR interference library in the model organism Mycobacterium smegmatis against a phylogenetically representative collection of 70 mycobacteriophages, we mapped the functional architecture of host susceptibility determinants. This parallelized genetic screen revealed a spatial continuum of infection requirements spanning from outer leaflet surface structures down to deep cytoplasmic machinery. The four major outer membrane Mycobacterium smegmatis porins (Msp) emerged as the dominant candidate receptor family for 46 diverse phages, representing the first protein receptors identified within this group. Interrogating the asymmetric outer bilayer also implicated cell envelope glycolipids as candidate receptors for 14 phages, resolving into glycopeptidolipid sub-lineages. Beneath the mycomembrane, arabinogalactan functions as a candidate secondary receptor for 5 phages, while the inner membrane lipid transporter MmpL11 and the global nucleoid-associated protein Lsr2 represent determinant factors for 14 and 31 phages respectively. While physical deletions of major porins trigger spontaneous regulatory pathways that upregulate silent backup paralogues, acute transcriptional knockdowns circumvent suppressor accumulation, revealing unmasked single-gene dependencies. Structural analysis demonstrated that host determinant usage decouples entirely from whole-genome viral phylogeny, indicating that standard phylogenetic groupings fail to predict surface recognition profiles. Ultimately, mapping this host interactome reveals an evolutionary strategy where phages target highly redundant or abundant host machinery to exploit fixed physiological liabilities, presenting a robust molecular blueprint for the rational engineering of escape-resistant therapeutic phage cocktails.

Prophages are increasingly recognized as drivers of bacterial adaptation, yet how domesticated prophage genes shape microbial symbiosis remains poorly understood. Here, we identify GDPDXhp1, a glycerophosphodiester phosphodiesterase encoded by prophage Xhp1 of Schaalia odontolytica XH001, as a metabolic regulator linking bacterial physiology, episymbiosis and phage fitness. Association with the epibiotic bacterium Nanosynbacter lyticus TM7x strongly induced gdpdXhp1 expression and triggered GDPDXhp1-dependent lipid droplet accumulation in XH001. GDPDXhp1 orchestrated glycerophospholipid remodeling, membrane biophysical properties, respiration and cell-surface glycan composition in XH001, thereby promoting TM7x episymbiosis. Remarkably, GDPDXhp1 was also required for productive Xhp1 infection by remodeling susceptible bacterial host envelope structures to facilitate phage-host interactions. Together, our findings reveal a domesticated prophage enzyme that contributes to bacterial metabolism, interspecies symbiosis and viral reproduction, highlighting prophage as an overlooked source of metabolic innovation in microbial symbiosis.

Cell surface lipoproteins (SLPs) play crucial roles in the pathogenesis and cell biology of gram-negative bacteria, yet the route by which they reach the cell surface has remained unknown except in special cases. Here we show that in Bacteroidota bacteria, a phylum that includes prominent human commensals and anaerobic pathogens, SLP export is carried out by the outer membrane lipopolysaccharide (LPS) translocon. Isolation and structural characterisation of LPS translocon complexes from two Bacteroidota species reveals that they are massively enlarged and elaborated relative to the Escherichia coli paradigm with up to eight additional subunits located within, and on either side of, the outer membrane. We establish a role for the universally conserved core LptD subunit and four of the additional Bacteroidota-specific subunits in SLP export. Expansion of the LPS translocon in the Bacteroidota may allow export of the unusually complex SLPs found in this phylum. Our discoveries raise the possibility that SLP export is a conserved second function of the LPS transport pathway across gram-negative bacteria.

Nucleus-forming jumbo phages import a phage-encoded non-virion RNA polymerase (nvRNAP) into a proteinaceous nucleus, but how it is selected for import is unknown. Here, we identify an import pathway that couples nvRNAP subunit interactions with phage-encoded import factors. Maximal nvRNAP import requires a novel factor Imp7 (gp166) and the essential import factor Imp1. Imp7 is essential at environmental temperatures (20 C), where impaired nvRNAP import causes a profound loss of middle and late transcription and blocks phage DNA replication. Import of individual nvRNAP subunits also depends on other subunits, consistent with import licensing at the level of an assembled complex. In the absence of middle transcription and DNA replication, the phage nucleus surprisingly still assembles and segregates from the cytoplasm, demonstrating that nvRNAP nuclear localization and activity are not a critical checkpoint for nucleus assembly. Together, these findings define a specialized pathway for nuclear import of the multi-subunit RNAP and reveal that nuclear compartment assembly precedes the transcriptional and replicative programs required for its maturation.

Cryptococcus, a human pathogenic yeast, can undergo unisexual reproduction in the absence of compatible mating partners, yet this developmental program remains poorly understood. Here, we report that tunicamycin, a natural product produced by Streptomyces species, induces a complete unisexual reproductive cycle in different Cryptococcus species. Bacterial confrontation or purified tunicamycin trigger hyphal differentiation, meiotic recombination, and basidiospore formation, as confirmed by whole-genome sequencing and SNP analyses. We further demonstrate that tunicamycin-induced unisexual development requires canonical sexual regulators and meiotic machinery. Transcriptomic profiling reveals staged developmental reprogramming characterized by early induction of stress- and cell cycle-associated genes, followed by enrichment of genes in the filamentation-related pathways. This capacity varies among natural isolates of C. neoformans and was conserved in Cryptococcus gattii. Together, our findings demonstrate that a bacterial metabolite can activate meiotically competent unisexual reproduction, highlighting interkingdom chemical signaling as a potential driver of fungal evolution in natural environments.

Giant endogenous viral elements (GEVEs) are ubiquitous in eukaryotic genomes, but how the virus adapts to the host and is capable of reactivation despite host defense mechanisms remains unclear. Here we examined this process in the green alga Chlamydomonas reinhardtii and its latent giant virus, punuivirus, which is integrated into the host genome. We reveal a chimeric genomic architecture in which punuivirus latency genes are enriched in spliceosomal introns and 6-methyladenine marks, mirroring host transcriptional regulation, while virulence genes are predominantly controlled by a conserved viral promoter. Using single-cell RNA-seq, we show that nearly 70% of cells express viral transcripts, but only a small subpopulation commits to viral production, expressing virulence genes with host transcriptional shutoff. Moreover, we find that the intact punuivirus GEVE remains refractory to host 5-methylcytosine (5mC) silencing, whereas a naturally occurring deletion variant is associated with widespread 5mC accumulation across the GEVE and elsewhere in the host genome. The deleted region encodes a highly expressed ten-eleven translocation dioxygenase (TET), and epigenetic profiling of intact and deletion strains implicates this GEVE gene in removing 5mC marks, thereby maintaining the virus in a transcriptionally permissive latent state. Our study provides novel insights into the molecular underpinnings of host-virus co-existence in a giant virus-alga interaction.

Bacterivorous protists expose bacteria to hostile stress conditions that resemble those encountered within macrophage phagosomes and may therefore act as training grounds for bacterial pathogens. However, the mechanisms enabling bacterial survival within protozoan phagosomes remain poorly understood. Here we characterized the transcriptome of the opportunistic pathogen Burkholderia cenocepacia during transit through phagosomes and expelled food vacuoles of the ubiquitous protozoan ciliate Tetrahymena elliotti. Our data revealed a coordinated response involving bacterial metabolic reprogramming, with the upregulation of NADPH-generating pathways and host nutrient uptake systems, alongside the activation of several stress responses and envelope remodeling to maintain outer membrane barrier function and repair phagosomal damage. Disruption of antioxidant defenses, the RpoE-dependent envelope stress response, the Mla pathway and other proteins required for outer membrane integrity severely impaired intracellular survival in both protozoa and macrophages, revealing conserved mechanisms required for persistence within evolutionarily distant phagocytes. This adaptive response not only contributed to survival but it also primed B. cenocepacia for increased chlorhexidine resistance and adhesion to collagen and human lung epithelial cells. Collectively, our findings reveal that protozoan phagosomes drive physiological adaptations with consequences extending beyond grazing resistance. Protozoa may therefore disseminate stress-adapted bacteria with enhanced resistance and colonization potential, providing a mechanistic link between adaptation to environmental predators and survival in the human host.

Bacteria deploy polymorphic toxins to inhibit competitors and rely on cognate immunity proteins to prevent self-intoxication. Immunity determinants act by binding and neutralizing incoming toxins, and in the rare cases where toxin-induced damage is also reversed, that activity resides within the immunity protein itself. Whether a conflict system can instead dedicate a separate gene to repairing the damage is unknown. Here we report that an operon in Bacillus subtilis encodes the toxin YwqJ, its cognate immunity protein YwqK, and EndoV an endonuclease that incises DNA at the lesion the toxin creates. YwqJ is a single-stranded DNA cytidine deaminase translocated by the type VIIb secretion system (T7SSb) to mediate contact-dependent interbacterial antagonism. Unexpectedly, EndoV exhibits a preference for uracil over the canonical hypoxanthine, processing genomic uracil in cells and protecting against YwqJ when immunity is absent. Because EndoV acts on the chemical lesion rather than the effector, its protection is indifferent to the source of deamination. EndoVBs accordingly restricts replication of PBS1, a bacteriophage whose genome naturally substitutes uracil for thymine. These findings establish DNA repair as a novel layer of defense within toxin-immunity loci and reveal a DNA repair mechanism that unifies interbacterial antagonism and antiviral immunity.

Shiga toxin-producing Escherichia coli (STEC) are virulent due to their production of lethal toxins, which are genetically encoded by viruses that exclusively infect these bacteria, called phages. After infection, children and immunocompromised patients are particularly at risk of developing life-threatening complications. Antibiotics are contraindicated here, because they increase phage activity, and this leads to increased toxin production. This limits treatments to fluid resuscitation and supportive care. Here, we describe how acid exposure and trimethoprim both potently suppress phage production ~100-fold, even in the presence of antibiotics that increase phage activity. We characterized these stimuli further, and found that they suppress phages independently of canonical bacterial SOS responses that are well-established to be involved in phage activity. Trimethoprim partially relies on the glutamate-dependent bacterial acid stress response to have this suppressive effect. It allows the earliest steps of phage induction to happen, but completely blocks their genomic replication. We translated these findings to a clinical STEC isolate and found that trimethoprim potently suppressed toxin production, again, even in the presence of antibiotics that increase phage activity. These findings form a key lead in finding a new option to treat these infections, without risking potentially lethal complications.

Bloodstream infection (BSI) is a life-threatening condition where the risk of dying is affected by both patient- and pathogen-related factors. Certain microbial traits (e.g., virulence genes) are thought to affect survival rates from BSIs, but the magnitude of their impact on human mortality remains unclear. To address this, we analyzed the microbial genomes, quantitative proteomes, and electronic medical records (EMRs) from 35,746 BSIs from Calgary, Canada (2006-2022). We show that EMR-based machine learning models accurately predict 30-day mortality (AUC=0.83), but surprisingly, integrating >70,000 microbial genes/proteins does not improve this performance. We demonstrate that microbial factors co-segregate with specific patient cohorts and are therefore not independent predictors of risk. We conclude that microbial virulence factors have minimal impact on mortality rates from BSIs.

The cell envelope of bacteria and fungi is a major source of microbial-associated molecular patterns (MAMPs), which are recognised by multiple host pattern recognition receptors (PRRs) such as TLRs and NODs. The key role of this recognition in protecting against infection and regulating diverse physiological processes has been studied for decades. In contrast, although the archaea are growingly recognised as an important component of the animal and human microbiome, their interaction with the innate immune system has been largely understudied. Here, we asked whether the archaeal cell envelope acts as MAMP. We selected 15 archaeal species from different environments, including the human microbiome, and harbouring diverse envelope characteristics, and we tested their ability to activate innate immune responses in human and murine immune cells. We first show that archaeal cells activate the innate immune transcription factor NF-kB and induce IL-6 secretion. Using whole archaeal cells, supernatants, and purified envelope components, we then find that none of the main PRR receptors which recognise the bacterial envelope (TLR4, TLR5, NOD1 and NOD2) are involved in this activation. We only detected activation of TLR2, regardless of the environmental origins of the archaea tested, although with overall lower activity compared to bacteria. We further demonstrate that different TLR2 heterodimers are involved, depending on the species, although the archaeal agonist remains unknown. Altogether, our results highlight a surprising paucity of interaction of archaeal cell envelope components with host PPRs, in contrast to bacteria, and open new avenues of research toward further investigation of archaea/host interplay.

Bacterial small-molecule messengers that function in diverse immune signaling pathways have been thought to derive strictly from biosynthesis using nucleotide and cofactor substrates. Here we report a nuclease-driven immune signaling pathway in which tRNA cleavage generates a specific small molecule that triggers cell death in response to RNA-guided transcript recognition in prokaryotes. We identify a class of type VI-B CRISPR systems in which the RNA-guided nuclease Cas13 and its accessory transmembrane protein, 2TM{beta}, are co-encoded in an operon and are jointly required for bacteriophage defense. Upon target RNA recognition, Cas13 activates 2TM{beta} by producing mono- and dinucleotide messengers bearing 2',3'-cyclic uridine phosphate ends by tRNA cleavage. Activated 2TM{beta} destroys infected cells by forming a multimeric complex that disrupts cell membranes to mediate cell death. These findings demonstrate RNA-programmed tRNA cleavage for second-messenger biogenesis, revealing a new logic for prokaryotic immune signaling.

Bacterial infochemicals can profoundly alter phytoplankton physiology, yet how these compounds influence viral infection outcomes remains poorly understood. The bacterial quorum sensing molecule 2-heptyl-4-quinolone (HHQ) enables the globally abundant coccolithophore, Gephyrocapsa huxleyi, to evade virus-induced mortality following infection by Emiliania huxleyi virus (EhV). HHQ reduced infectious virus production at 24 hours despite largely intact viral gene transcription, as revealed by RNA sequencing of host and viral responses during EhV-201 infection. Host transcriptomics instead revealed rapid and progressive HHQ-dependent physiological remodeling evidenced by changes in protein encoding genes associated with vesicular trafficking, and cytoskeletal organization. Transcriptional responses further implicated endolysosomal sorting, membrane remodeling, and protein modification as potential barriers to productive infection. These findings indicate that HHQ-mediated evasion of virus-induced mortality occurs downstream of viral entry and transcription, through remodeling of host processes required for virion assembly, maturation, trafficking, and release.

Understanding the complex interactions between phage, antibiotics and bacteria is vital for the development of rationale phage-antibiotic therapies. These interactions have traditionally been investigated via bulk assays, thus masking eventual cellular heterogeneities and the impact of spatial refuges on phage and antibiotic efficacy. Using microfluidics-based time-lapse microscopy, we reveal large phenotypic heterogeneity in the response of Burkholderia thailandensis to both the bacteriophage {Phi}Bp-AMP1 and sub-inhibitory concentrations of two selected antibiotics ciprofloxacin and trimethoprim. We discover that simultaneous exposure to ciprofloxacin and phage decreases the size of individual B. thailandensis cells, whereas phage and trimethoprim together slow down cell doubling. Exposure to ciprofloxacin also causes extended phage lysis of B. thailandensis which is preceded by a distinguishable pause in cell elongation, whereas exposure to trimethoprim constrains lysis. As a consequence, exposure to ciprofloxacin further hinders the growth of B. thailandensis in the presence of phage, whereas exposure to trimethoprim enhances growth in the presence of phage. These findings help understand the effects of the interactions between antibiotics and phage on bacterial subpopulations in spatial refuges that are notoriously difficult to treat with antibiotics.

Standard metagenomics resolves microbial communities only in relative terms and at coarse taxonomic resolution, obscuring how individual strains change in absolute abundance and evolve within a host. We present long-read quantiomics (LRQ), a quantitative metagenomics platform coupling long-read sequencing with plasmid-based internal standards to recover high-quality genomes, including from degraded or low-input stool, and measure absolute, genome-resolved strain abundances. We validate LRQ using large plasmid standards and bacterial spike-ins, then apply it to eight years of dense sampling from an individual with colonic Crohn's disease. LRQ resolves co-resident strains of the same species following opposing trajectories with inflammation. Escherichia coli, long treated as a single pathobiont, separates into two sub-populations with opposing inflammation responses; the inflammation-associated lineage carries an immune-evasion module of virulence-associated metal-acquisition, biofilm, capsule and toxin genes. We observe genome-wide adaptive sweeps on timescales matched to disease incidence. LRQ therefore converts long-read metagenomics from a genome-recovery tool into a quantitative framework providing absolute, strain-level measurement of microbial ecology and evolution.

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems. Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa. Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

Bacterial phytopathogens deploy diverse antimicrobial systems, including diffusible secondary metabolites and contact-dependent secretion apparatuses, to compete with coinhabiting microorganisms during host colonization. How the production of these distinct weapons is coordinated remains incompletely understood. Here, we identified a two-tier regulatory cascade by which the Hfq-dependent small RNA (sRNA) ArcZ controls both antibacterial and antifungal antagonism in the phytopathogen Dickeya solani. High-throughput RNA interactome analysis (RIL-seq) shows that ArcZ interacts broadly across the transcriptome and directly base-pairs with the 5' untranslated region of the mRNA encoding the MarR-family transcription factor SlyA, activating its translation. SlyA subsequently binds directly to selected promoter regions within the solanimycin biosynthetic cluster (sol) and the type VI secretion system (T6SS) loci and activates their transcription. The ArcZ-SlyA axis is required for solanimycin mediated antifungal activity against Kluyveromyces lactis and for T6SS-dependent killing of the sympatric pathogen Pectobacterium atrosepticum, demonstrating that the D. solani T6SS mediates interbacterial competition. A compensatory base-pairing mutation between ArcZ and slyA restores both competitive phenotypes, establishing this post-transcriptional interaction as a direct upstream determinant. Together, these results reveal an sRNA controlled regulatory axis that coordinates diffusible antifungal and contact-dependent antibacterial activities.

L-form bacteria are wall-deficient variants that proliferate without an intact cell wall and independently of the canonical cell division machinery. Despite increasing insights into how L-forms survive and proliferate without a cell wall, fundamental questions regarding how cell cycle processes, including DNA and RNA synthesis, are coordinated in the wall-less state remain poorly understood. Here, we demonstrate that fluorescence-activated cell sorting (FACS) provides a robust approach for quantitative analysis of Escherichia coli L-forms. Flow cytometry revealed two reversible subpopulations differing in intracellular nucleic acid content, which rapidly re-established their heterogeneous distribution following sorting. Remarkably, cells with initially low nucleic acid content underwent a synchronized, population-wide increase in nucleic acid synthesis within 24 hours, a phenomenon that was independently confirmed by time-lapse fluorescence microscopy. Analysis of liquid cultures stained with SYTO and DAPI dyes showed that this transient increase occurred during early exponential growth and was driven predominantly by RNA rather than DNA synthesis. Consistent with observations in walled E. coli, RNA levels peaked before declining as cultures transitioned towards nutrient limitation. Together, these findings establish FACS as a powerful tool for studying L-form biology and reveal that, despite the absence of an intact cell wall and canonical cell division, L-forms retain coordinated, population-wide regulation of nucleic acid synthesis.

The emerging neonatal bacterial pathogen Paenibacillus thiaminolyticus causes sepsis and postinfectious hydrocephalus, yet few tools are available to study or specifically detect it. We developed fluorescent probes based on cell wall-binding domains (CBDs) from bacteriophage-derived cell-wall hydrolases. CBDs can target structurally conserved cell-wall glycans, making them attractive affinity reagents for bacterial detection. None of the 11 probes we designed labeled intact cells under standard conditions, and we identified a surface layer (S-layer) that prevented access to their cell-wall targets. Brief acidic treatment (pH 2.0) released abundant high-molecular-mass proteins from the cell surface and enabled labeling by five distinct CBD probes, each containing a single C-terminal S-layer homology domain. Mass spectrometry identified the main released protein as an ortholog of the P. alvei SpaA S-layer protein, which we designate SlsA. Across 26 surveyed P. thiaminolyticus genomes, we identified four distinct SlsA variants with 58-79% pairwise amino acid identity, encoded within a conserved cluster homologous to the P. alvei S-layer-associated locus. After either the acidic treatment or a quick flame fixation, the lead probe mGL-BDPt9 labeled six P. thiaminolyticus strains, including three Ugandan clinical isolates, as well as a P. dendritiformis clinical isolate from a US infant, and three additional Paenibacillus species. mGL-BDPt9 also distinguished P. thiaminolyticus from five gram-positive sepsis pathogens in pairwise mixtures and detected it in a five-species suspension. Together, these findings establish the presence of an S-layer in P. thiaminolyticus and provide a rapid fluorescent labeling strategy for multiple Paenibacillus pathogens associated with neonatal paenibacilliosis.