Nature Microbiology

Temperate bacterial viruses (phages) can transition between lysis - replicating and killing the host, and lysogeny - existing as dormant prophages while keeping the host viable. It was recently shown that upon invading a naive cell, some phages communicate using a peptide signal, termed arbitrium, to control the decision of entering lysogeny. Whether communication can also serve to regulate exit from lysogeny (known as phage induction) remains unclear. Here we show that arbitrium-coding prophages continue to communicate from the lysogenic state by secreting and sensing the arbitrium signal. Signaling represses DNA-damage dependent phage induction, enabling prophages to reduce induction rate when surrounded by other lysogens. We show that the mechanism by which DNA damage and communication are integrated differs between distantly related arbitrium-coding phages. Additionally, signaling by prophages tilts the decision of nearby infecting phages towards lysogeny. Altogether, we find that phages use small molecule communication throughout their entire life-cycle to measure the abundance of lysogens in the population, thus avoiding wasteful attempts at secondary infections when they are unlikely to succeed.

Bacteriophages kill bacteria by osmotic lysis towards the end of the lytic cycle. In the case of Gram-positive bacteria, peptidoglycan-degrading endolysins released at the end of infection cycle cause explosive cell lysis not only of the infected host, but can also attack non-infected bystander cells. Here, we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

Despite extensive characterisation of envelope biogenesis systems in diderm bacteria, glycerophospholipid (GPL) trafficking remains poorly understood, and has only been studied in a handful of model species. Within the Proteobacteria, the maintenance of lipid asymmetry (Mla) system facilitates retrograde GPL trafficking via six proteins, MlaA-F. GPLs are extracted from the outer leaflet of the outer membrane by the lipoprotein MlaA which associates with porin trimers, then shipped through the periplasmic space by the chaperone MlaC, which finally delivers GPLs to the inner membrane complex formed by MlaBDEF. Here, we investigate GPL trafficking in Veillonella parvula, a diderm member of the Firmicutes which encodes an Mla system devoid of MlaA and MlaC. V. parvula {Delta}mla mutants display phenotypes characteristic of disrupted lipid asymmetry such as hypervesiculation and detergent hypersensitivity, and lipid content analysis from outer membrane vesicles reveals an enrichment for the major lipid component phosphatidylethanolamine. Interestingly, suppressor analysis identifies mutations in tamB that rescue detergent hypersensitivity and hypervesiculation of {Delta}mla strains, supporting the involvement of these two systems in antagonistic GPL trafficking functions across diverse bacterial lineages. A combination of structural modeling and subcellular localisation assays shows that MlaDVp is longer than in classical diderm models and forms a transenvelope bridge, encoding both an inner membrane-localised MCE domain and an outer membrane {beta}-barrel. These results strongly suggest that V. parvula possesses a minimal Mla system for GPL trafficking, replacing the need for chaperones and outer membrane lipoproteins by directly connecting the two membranes. Finally, phylogenomic analysis indicates that this MlaEFD self-contained architecture is widely distributed in diderm bacteria and most likely represents the ancestral functional core of the Mla system, which subsequently increased in complexity in Proteobacteria and closely related phyla following the emergence of MlaABC. Our work broadens the diversity of current models of GPL trafficking in diderm bacteria, challenging the paradigm set by classical models and shedding light on the evolution of a crucial system in the biogenesis and maintenance of the bacterial outer membrane.

Neisseria gonorrhoeae is a leading cause of sexually transmitted infection (STI) and a priority AMR pathogen. Two narrow host range plasmids, pbla and pConj, have contributed to ending penicillin and tetracycline therapy, respectively, and undermine current prevention strategies including Doxy-PEP. Here, we investigated the origin and evolution of the beta-lactamase plasmid, pbla. We show that the interplay between pbla and pConj influences their co-occurrence and the spread of pbla in the gonococcal population. We demonstrate that pbla was acquired by the gonococcus on at least two occasions from Haemophilus ducreyi, and describe the subsequent evolutionary pathways taken by the three major pbla variants. Changes that mitigate fitness costs of pbla and the emergence of TEM beta-lactamases which confer increased resistance have contributed to the success of pbla. In particular, TEM-135, which has arisen in certain pbla variants, increases resistance to beta-lactams and only requires one amino acid change to become an extended spectrum beta-lactamase (ESBL). The evolution of pbla underscores the threat of plasmid-mediated resistance to current therapeutic and preventive strategies against gonococcal infection. Given the close relationship between pbla and pConj, widespread use of Doxy-PEP is likely to promote spread of pConj and pbla, and emergence of plasmid-mediated ESBL in the gonococcus, with dire public health consequences.

The release of bacterial bioactive molecules across the gut barrier is a crucial yet poorly understood step in microbe-host communication. Here we unravel a phage-driven mechanism that enables this process in a nutritional symbiont. In Lactiplantibacillus plantarum NC8 (LpNC8), we identify a stress-inducible prophage, pp2, that undergoes genotoxic-stress-dependent activation and triggers holin-lysin-mediated lysis. This controlled lytic program produces phage particles together with extracellular, membrane-derived bacterial particles, and is strictly required for the ability of LpNC8 to promote juvenile growth in nutritionally challenged Drosophila melanogaster. Disruption of pp2-dependent lysis abolishes particle release in vitro and eliminates the growth-promoting effect in vivo, without altering bacterial abundance in the gut. The magnitude of phage release correlates with the extent of host growth promotion, indicating that prophage induction contributes to the export of symbiotic factors. These findings reveal prophage-induced lysis as a central and previously unrecognized mechanism by which a beneficial gut bacterium enhances host development under nutritional stress.

Antimicrobial resistance (AMR) is a global public health crisis with few options for control. As such early identification of emerging bacterial strains capable of rapidly evolving AMR is key. Although antimicrobial tolerance and persistence are precursor phenotypes for AMR, little evidence exists to support their importance in real-world settings. Here we used bacterial genome wide association on national genomic surveillance data of the diarrhoeal, and World Health Organisation AMR priority pathogen, Shigella sonnei (n=3745) to agnostically identify common genetic signatures among lineages convergently evolving toward AMR (n=15). This revealed an association of an AMR trajectory with a multi-and highly variable second copy of metG, borne by a phage-plasmid we called pWPMR2. Further analyses revealed that pWPMR2 was present across clinically relevant enteric pathogens globally, including past and contemporary outbreaks, and that the additional-metG mechanism was present across multiple bacterial phyla. Functional microbiology, experimental evolution, and single-cell physiology studies confirmed that the expression of auxiliary metG, particularly the mutated version on pWPMR2, created a sub population of cells predisposed to survival in, and evolving resistance against, third generation cephalosporins (3GC). Thus, we demonstrate a novel mechanism of auxiliary metG carriage that predisposes bacteria to AMR with real world impacts. Furthermore, our approach is a timely example of using genomic epidemiology to rapidly guide functional microbiology studies in the era of routine genomic surveillance and also highlights several deficiencies in current AMR surveillance practices.

Phage therapy can be used in combination with antibiotics to combat infections with bacterial pathogens1-3. However, bacteria can rapidly evolve phage resistance via receptor mutation, or using their CRISPR-Cas adaptive immune systems4, which insert short phage-derived sequences into CRISPR loci in the bacterial genome5 to guide sequence-specific cleavage of cognate sequences6. Unlike CRISPR-Cas immunity, mutation of the phage receptor leads to attenuated virulence when the opportunistic pathogen Pseudomonas aeruginosa is infected with its phage DMS3vir7, which underscores the need to predict how phage resistance evolves under clinically relevant conditions. Here, using eight antibiotics with various modes of action, we show that bacteriostatic antibiotics (which inhibit cell growth without killing) specifically promote evolution of CRISPR-Cas immunity in P. aeruginosa by slowing down phage development and providing more time for cells to acquire phage-derived sequences and mount an immune response. Our data show that some antimicrobial treatments can contribute to the evolution of phage-resistant pathogens with high virulence.

Phage therapy is an exciting strategy against antimicrobial-resistant bacterial infections, but critical knowledge gaps regarding its clinical application persist. Studying a patient with a life-threatening, chronic bacterial infection who failed phage therapy, we uncovered important biological concepts with direct translational impact. Using longitudinal clinical samples, we found that patients can harbour pre-existing antibodies against active prophages induced from the genome of the causative pathogen. Notably, these antibodies can contribute to clinical failure by cross-reacting with and effectively neutralising therapeutic phage. We also uncovered bacterial heteroresistance, characterised by bacterial subpopulations from the initial infection with reduced phage susceptibility, as a further contributor to treatment failure. These findings highlight the intricate interplay between host immunology, bacterial genetic diversity and phage biology, bearing broad significance for clinical phage therapy. Future phage therapy patients, especially those with chronic infections, should be screened for antiphage immunity and bacterial heteroresistance prior to phage treatment.

Bacteria undergo cycles of growth and starvation, to which they must adapt swiftly. One important strategy for adjusting growth rates relies on ribosomal levels. While high ribosomal levels are required for fast growth, their dynamics during starvation remain unclear. Here, we analyzed ribosomal RNA (rRNA) content of individual Salmonella cells using Fluorescence In-Situ Hybridization (rRNA-FISH). During the transition from exponential to stationary phase we measured a dramatic decrease in rRNA numbers only in a subpopulation, resulting in a bimodal distribution of cells with high and low rRNA content. We showed that the two subpopulations are phenotypically distinct when subjected to nutritional upshifts. Using a transposon screen coupled with rRNA-FISH, we identified two mutants acting on rRNA transcription shutdown and degradation, that abolished the formation of the subpopulation with low rRNA content. Our work suggests that Salmonella employs a bet-hedging strategy in regulating ribosomal levels that may be beneficial for survival.

The rise of antibiotic-resistant bacterial infections has driven renewed interest in bacteriophage therapy, where viruses that specifically kill bacteria are used as targeted antimicrobials. Pseudomonas aeruginosa, a WHO critical-priority pathogen that causes severe infections in hospitalized and immunocompromised patients, presents a major challenge for phage therapy because of its extraordinary genetic diversity. Phages effective against one bacterial strain often fail against others, and existing cross-resistance-profiling approaches require iterative empirical testing of each new patient isolate. To establish a genome-based framework for rapid phage-isolate matching, we assembled a collection of 95 genomically diverse P. aeruginosa phages representing 20 genera and tested each against 99 genetically diverse clinical isolates, generating 9,405 infection outcome measurements. Bacterial O-antigen serotype emerged as the dominant determinant of strain susceptibility, while defense systems, anti-defense systems, and prophage burden contributed smaller strain-specific effects. The full curated multivariate model explained 47% of strain-susceptibility variance. Machine-learning models integrating these features and pangenome-derived gene clusters reached a per-strain AUROC of 0.86. In an in vivo proof-of-concept test against a single held-out strain, the ML-designed cocktail produced a [~]12-fold greater median CFU reduction than the expert-designed cocktail (q = 0.045), with both cocktails substantially reducing burden relative to the untreated control ([~]113-fold for ML, [~]9-fold for CG; both q < 10{square}3). SHAP analysis of the model identified bacterial surface-architecture genes (LPS biosynthesis, outer membrane proteins, type IV pili) as the dominant predictors, with defense-system content modulating which specific phages succeed against a strain rather than uniformly damping susceptibility. Together, these results establish a genome-based framework for predicting phage susceptibility in genetically diverse clinical isolates.

Type IV secretion systems (T4SS) enable the spread of antibiotic resistance and other virulence factors. In Gram-positive bacteria, T4SSs have long been thought to lack VirB2-like proteins that form conjugative pili and instead rely on adhesins for cell-cell contacts. Yet, it has remained unclear how subsequent DNA transfer (conjugation) is physically mediated. Here we identify a VirB2-like protein, PrgFB2, from the clinically isolated conjugative plasmid pCF10 in Enteroccocus faecalis and show that it is essential for conjugation. Structural modeling confidently predicts a pilus-like assembly for PrgFB2. We validate this prediction through mutagenesis, conjugation assays, and targeted chemical labeling. By combining various machine learning bioinformatic techniques, we analysed >1000 Gram-positive conjugative plasmids from diverse species, including major pathogens, and identified pili forming VirB2-like proteins in almost all of them. Our findings overturn the prevailing view that Gram-positive T4SSs lack pili. This discovery provides a new framework for understanding horizontal gene transfer and highlights critical targets for combating antimicrobial resistance and virulence in Gram-positive bacteria.

Microbial community assembly is governed by trophic interactions that mediate the transfer of carbon sources and biomass building blocks between species. However, central metabolism corresponds to only a small fraction of the biosynthetic potential of microbes: metabolites such as antimicrobial compounds, signaling molecules, and co-factors are underexplored forces shaping microbial communities. Here, we focus on B vitamin exchange in coastal marine bacterial communities that degrade particulate organic matter and find that natural seawater communities are vitamin limited. While almost a third of bacterial isolates from these communities are B vitamin auxotrophs, the pioneering degraders that first arrive on particles are vitamin producers that likely support auxotrophs through cross-feeding. However, combining experiments and a resource-explicit model, we show that auxotroph growth is often not restored by coculture with vitamin producers, but rather requires lysis and subsequent vitamin recycling. Our results highlight the importance of vitamin auxotrophies and lysis-mediated cross-feeding as important factors controlling microbial community assembly and succession on marine particles.

Horizontal gene transfer (HGT) in bacteria is shaped by restriction-modification systems (RMSs) that define genetic barriers between species and lineages. Neisseria gonorrhoeae harbours numerous RMS, most of which are part of its core genome. Here, we show that phase variation in these RMSs can limit interspecies and intraspecies plasmid exchange. While NlaIV likely influenced the acquisition of pConj from the meningococcus, phase variation of the NgoAV RMS produces epigenetically heterogeneous populations that present variable bottlenecks to within-species plasmid transmission, influencing the spread of anti-microbial-resistance plasmids, pConj and pbla. A LEAT sequence extends or retracts a molecular ruler within the HsdS specificity subunit, altering spacing of recognition motifs, while truncation of the protein leads to a switch from non-palindromic to palindromic recognition motifs. Thus, while the repertoire of gonococcal RMSs is largely conserved, phase-variation dynamically modulates gene flow, shaping plasmid transmission, and the evolution and spread of antimicrobial resistance.

Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture, comprised of two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extra-cellular chitinases excreted and left behind by detached cells. The "inheritance" of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a tradeoff between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

Lactobacillus species dominance of the vaginal microbiome is a hallmark of vaginal health. Pathogen displacement of vaginal lactobacilli drives innate immune activation and mucosal barrier disruption which increases the risks of STI acquisition and, in pregnancy, of preterm birth. Using cell reporter systems, we describe differential TLR mediated activation of the proinflammatory transcription factor NF-{kappa}B by vaginal pathogens and commensals. Vaginal Lactobacillus strains associated with optimal health were shown to selectively interact with anti-inflammatory innate immune receptors whereas species associated with suboptimal health including L. iners and Gardnerella vaginalis interacted with both pro- and anti-inflammatory receptors. Anti-inflammatory action of L. crispatus was regulated by surface layer protein (SLPs)-mediated shielding of TLR ligands and selective interaction with anti-inflammatory receptor, DC-SIGN. In pregnant women, cervicovaginal SLPs were predominately associated with Lactobacillus-enriched microbiota. These data offer new mechanistic insights into how vaginal microbiota modulate host immune response and influences risk of preterm birth.

Predicting which receptor a phage binds to from genome sequence alone has remained an intractable challenge, principally because the experimental phenotypic data required to train and validate predictive models have not been available at sufficient scale. Here we address this by conducting 1,050 genome-wide genetic screens across 255 taxonomically diverse Escherichia coli dsDNA phages, assigning host receptors to 193 phages across 19 receptor classes. Comparative genomics and AlphaFold3 structural modelling resolved the sequence determinants of specificity to defined receptor-binding protein domains and individual residues. Machine learning models trained on this dataset predicted host receptor identity from phage genome sequence alone without prior annotation of receptor-binding genes, achieving perfect precision and greater than 80% recall on 49 independently validated phages, and yielding predictions for 1,050 of 1,875 E. coli phage genomes in NCBI. Domain swaps redirected receptor specificity as predicted, and a single amino acid substitution proved both necessary and sufficient to switch recognition between two distinct porins. These results demonstrate that systematic phenotyping at scale makes sequence-based prediction of molecular interaction specificity tractable, with direct implications for phage-based medicine, microbiome engineering and the broader challenge of inferring host-pathogen interaction outcomes from sequence.

Antibiotic resistant pathogens are an increasing public health threat, as development of novel therapeutics is outpaced by resistance emergence and dissemination. Approaches to slow down or even revert antibiotic resistance are necessary to maintain efficacy of both existing and new antibiotics. Such approaches exploit the fitness cost of resistance elements, but have largely relied on assessing this cost in laboratory conditions that poorly reflect the native context in which pathogens reside. Here we present a method that allows to investigate the influence of personalized human gut microbiota compositions on the competitive fitness of antibiotic resistant pathogens. Using fecal matter-derived microbiomes we identify a specific community that selects for a carbapenem-resistant Klebsiella pneumoniae strain. This selective advantage is due to mutations arising in a LacI-type transcriptional regulator, GlyR. We show that upregulation of the downstream glycoporin GlyP is causing the effect. By deconvoluting the microbiome composition, we identify a focal E. coli strain as a central driver of the selection, which is further modulated by other microbiota members. We demonstrate that the selective advantage is due to carbohydrate competition, and in particular for glycerol-containing compounds. Importantly, glyR mutations are under strong positive but conditional selection in clinical K. pneumoniae isolates. This implies a reduced competitiveness in other environments, which we experimentally validate in vitro. Overall, this study offers a path to identify microbiome-specific interactions that modulate the competitiveness of antibiotic resistant pathogens.

Ancient bacteria largely lived and flourished as free-living cells till the rise of eukaryotes triggered their adaptation to a new habitat: the intracellular milieu. Rickettsiales bacteria are the most prevalent intracellular microorganisms discovered and the culprits behind some of mankinds worst pestilential diseases. Here, we show that intracellularity is not a defining feature of the group and describe the eco-evolutionary processes that transformed harmless free-living bacteria into obligate intracellular symbionts and parasites. We found that the evolution of free-living lineages towards enhanced cross-feeding interactions with microbial eukaryotes trapped them in a nutritional bind to their trophic partners. We discovered that the oldest Rickettsiales lineages are the closest relatives to modern eukaryotes and are enriched in proteins predicted to have been present in the mitochondrial ancestor. This study not only opens avenues for the detection and surveillance of emerging diseases but also expands our understanding of the origins of complex life. One-Sentence SummaryCross-feeding interactions shaped Rickettsiales genomic architectures along the parasite/free-living spectrum

Bacterial growth patterns are generally considered to be constrained and species-specific. Here, we show that Sphingobium yanoikuyae exhibits exceptional plasticity in envelope architecture and morphogenesis driven by a glycosphingolipid (GSL)-based outer membrane and an atypical peptidoglycan structure. At 30{degrees}C, cells expand asymmetrically via a rare bipolar envelope synthesis mode, whereas growth at 37{degrees}C triggers a transition toward longitudinal elongation accompanied by increased outer membrane vesiculation, indicating a reversible reprogramming of morphogenesis under host-like conditions. This switch is supported by rapid outer membrane dynamics, including high membrane fluidity and fast redistribution of envelope components. Despite these pronounced morphological changes, peptidoglycan composition remains conserved and unusually short, suggesting that growth plasticity is governed primarily by spatial regulation rather than changes in cell wall chemistry. Genetic analyses further identify essential roles for proteins involved in outer membrane-peptidoglycan and outer membrane-inner membrane coupling, as well as GSL transport systems, and core cell wall synthesis machinery, while revealing extensive redundancy in envelope remodeling enzymes. Together, these results establish S. yanoikuyae as a model for extreme envelope adaptability, where a highly fluid outer membrane and structurally unconventional peptidoglycan enable reversible transitions between distinct growth programs, potentially shaping environmental fitness and host-associated interactions.

Archaeal symbioses remain among the least understood cellular interactions. Ultra-small Nanobdellati (initially called DPANN) archaea rely on larger hosts for survival, yet their effects on host eco-physiology and their interactions with infecting viruses have not yet been examined experimentally. Here, we established the first stable tripartite co-culture comprising a Nanobdellales symbiont (YN4), its archaeal host (YN4HA), and a virus (MTIV4). We analyzed this system using physiological, genomic, transcriptomic, glycoproteomic, and cryo-electron tomography (cryoET) approaches. CryoET analysis revealed that the Nanobdellales archaeon formed cone-like structures to contact the host, as has been observed in other Nanobdellati-host systems. In this system, the host is infected by the virus. The Nanobdellales archaeon mitigated virus-induced growth inhibition of the host without any detectable fitness cost, indicating a defensive mutualism rather than a strictly parasitic relationship. This protective effect may involve Nanobdellati-driven remodeling of host cell surface glycans, suggesting a previously unrecognized glycan-mediated defense strategy. Overall, our tripartite system provides new insight into complex archaeal interactions in extreme environments that cannot be captured through conventional binary co-culture systems.