Understanding how complex, multi-gene systems evolve and function across genetic backgrounds is a central question in molecular evolution. While such systems often impose costs through epistatic interactions, some may behave as modular, "plug-and-play" units that retain function with minimal disruption. Here, we tested this using the polysaccharide capsule locus of Klebsiella pneumoniae, a highly exchangeable and fast-evolving locus, as a model. We genetically engineered capsule exchanges (swaps) across diverse genetic backgrounds and combined transcriptomics, fitness assays, and evolution experiments to show that capsule exchange has negligible effects on global expression and only marginal fitness costs, regardless of capsule type (or K type). Adaptation to capsule-costly environments consistently reduced capsule production regardless of K type, revealing shared adaptive trajectories rather than K type-specific pathways. Moreover, K type-specific traits involved in bacterial virulence, such as biofilm formation and hypermucoviscosity, were conserved across genetic backgrounds. This reveals that capsule swapping can directly shape host-pathogen interactions and influence within-patient evolution. Our findings provide strong evidence that capsule loci display plug-and-play dynamics: they are transferable, functional across contexts, and minimally disruptive to the host genome. This allows capsules to be seamlessly swapped, and help explain the evolutionary success, ecological versatility, and pervasive exchangeability of capsules in K. pneumoniae.

Phage-plasmids (P-Ps) are temperate phages that replicate as plasmids during lysogeny. Despite their high diversity, they carry genes similar to phages and plasmids. This leads to gene exchanges and to the formation of hybrid or defective elements, which limits accurate detection of P-Ps. To address this challenge, we developed tyPPing, an easy-to-use method that efficiently detects and types P-Ps with high accuracy. It searches for distinct frequencies and sets of conserved proteins to separate P-Ps from plasmids and phages. tyPPing's strength comes from both its precise predictions and its ability to systematically type P-Ps, including the assignment of confidence levels. We tested tyPPing on several databases and a collection of incomplete (draft) genomes. While predictions rely on the quality of assemblies, we detected high-quality P-Ps and experimentally proved them to be functional. Compared to other classification methods, tyPPing is designed to detect distinct P-P types and surpasses other tools in terms of sensitivity and scalability. P-Ps are highly diverse, making the systematic identification of new types a difficult task. By combining tyPPing with other tools, however, we show a valuable foundation for addressing this challenge. How to use tyPPing and other approaches is documented in our GitHub repository: github.com/EpfeiferNutri/Phage-plasmids/.IMPORTANCEMobile genetic elements, such as phages and plasmids, are diverse and drive bacterial evolution through horizontal gene transfer. Phage-plasmids, of which many carry antibiotic resistance genes or virulence factors, are both phages and plasmids and have life cycles of temperate phages and plasmids. This makes accurate classification difficult as current computational tools typically classify them as one or the other. We addressed this problem by developing tyPPing, a new and highly precise method, to systematically identify, separate, and catalog phage-plasmids. We demonstrated that tyPPing is highly accurate and broadly compatible. It provides a reliable foundation for all future studies involving phages and plasmids, ranging from agriculture environments to pathogenic strains of clinical settings.

Abstract Interactions between bacteria, bacteriophages, and their satellites are shaped by a myriad of defence and counter-defence mechanisms. Here, we identified and characterized the defence hotspots of thousands of P2-like phages and P4-like satellites to elucidate the origins and evolutionary dynamics of defence systems. Both P4 and P2 encode a broad diversity of recognizable defence systems. Defences are a substantial, yet likely underestimated, share of the elements’ pangenomes, as shown by novel antiviral functions discovered in P4 loci lacking known defence genes. Defence loci are very rapidly swapped, without pseudogenization, suggesting defences are replaced before becoming non-adaptive. This intense local recombination melds components of distinct systems into novel functional chimeras. Systems swap so rapidly that many elements with identical core genes have completely different defences. Surprisingly, despite P4 and P2’s concomitant replication and packaging, they almost never exchange defence genes. In contrast, near identical defence systems can be found in distinct types of MGEs and in cryptic chromosomal locations. Our findings highlight P4 and P2 as mobile platforms driving the modular diversification of bacterial antiviral repertoires. Hence, bacterial defences change quickly by phage and satellite turnover, and by the quick swap of defences within these elements.

Abstract Bacteriophages-bacteria interactions drive rapid evolution of both partners in laboratory studies. To understand how these dynamics unfold in natural environments, we re-sampled a population of Vibrio crassostreae and their phages in an open, animal-associated marine system four years apart. Analysis of over 1000 predominantly virulent phages revealed rapid change of some lineages, but persistence of others, with genomes highly conserved between years. This pattern is consistent with low substitution rates in persistent lineages and may reflect phages overwintering in wild oysters, slow virion decay, and for temperate phages, lysogeny within hosts. Over 600 V. crassostreae strains recovered at both time points assorted into the same major clades. Oyster-associated vibrios have larger genomes and more abundant and diverse mobile genetic elements suggesting that oysters are hotspots for genetic exchange and horizontal gene transfer. Their genomes encode virulence plasmids, prophages carrying anti-phage systems, phage-plasmids, and phage satellites that persist intracellularly as plasmids. Time series analyses revealed weak correlations between phage and bacterial abundances, a pattern compatible with cryptic population dynamics arising from genetic diversity. Together, these results indicate that natural coevolving phage-bacteria populations can exhibit complex dynamics, with rapid replacement of some lineages alongside multi-year persistence of others.

Exchange of genetic information by natural transformation shapes bacterial evolution. In Helicobacter pylori it is thought to drive its unusually high recombination rate, which has a crucial role in the evolution of virulence and the propagation of antibiotics resistance genes. While in most cases uptake of the incoming DNA into the periplasm is mediated by type IV pili, in H. pylori this initial step of natural transformation requires ComB, a unique competence-specific type IV secretion system (T4SS). The mechanisms by which ComB mediates DNA uptake are still poorly understood, since T4SS are usually involved in an opposite process of DNA export. Here, we identify a gene (hp1421) that is absolutely required for uptake of the transforming DNA into the periplasm, although distant from the comB operons. We show that hp1421 codes for a hexameric ATPase from the VirB11 family. HP1421 is present in the cytoplasm and interacts with ComB4, another ATPase of the T4SS inner membrane subcomplex. The structural modelling and functional analysis of HP1421 and its interaction with ComB4 indicate that HP1421 is a missing component of the ComB inner-membrane subcomplex that we propose to name ComB11. Phylogenetic analyses show that comB11 is a H. pylori core gene and suggest that the competence-dedicated ComB T4SS was a recent acquisition within Helicobacteraceae. Hence, co-option of the T4SS for DNA transformation requires nearly all the proteins that were previously essential for DNA conjugation.

Virulent bacteriophages infecting Klebsiella pneumoniae often show capsule-driven host tropism due to the presence of capsule-specific depolymerases. Yet for temperate phages the genetic and functional basis of such capsular specificity remains less well understood. Depolymerases appear unexpectedly rare in prophage genomes, raising unresolved questions about which prophage genes mediate capsular specificity, whether this apparent scarcity reflects biological or ecological differences versus annotation limitation, and whether prophage-encoded receptor-binding proteins (RBPs) are functionally active. To address these questions, we analysed 3,900 Klebsiella genomes from diverse ecological niches to identify prophage-encoded proteins mediating capsular specificity. We conducted a genome-wide association study (GWAS) correlating prophage protein clusters (from 8,105 prophages) with confidently assigned bacterial K-loci. GWAS revealed statistically supported predictors of capsular specificity for 16 of the 35 most diverse K-loci analysed. These predictors were dominated by diverse RBPs, including classical [Formula: see text]-helix depolymerases (6 predictors), SGNH-domain hydrolases predicted to deacetylate polysaccharides (6 predictors), and structurally novel RBPs lacking known depolymerase folds (2 predictors). Nearly one-third of K-loci yielded no statistically significant predictors. A targeted experimental screen of 50 candidate prophage depolymerases showed that 34 failed to yield detectable recombinant expression, and neither sequence similarity, structural prediction, nor prophage genomic context reliably predicted activity. Of the 14 active enzymes, 5 targeted a K-type different from that predicted of their bacterial host, and enzyme specificity was not consistently explained by sequence or structural homology. Comparison with GWAS predictions revealed that 10 of the 12 strongest GWAS predictors were experimentally validated, while 2 remained inconclusive. Together, these results highlight the intrinsic difficulty of predicting activity and capsular specificity of prophage-encoded RBPs from genomic information alone. Finally, analysis of 4,598 high-completeness prophages revealed that SGNH-domain hydrolases are among the most prevalent enzymatic domains in prophage RBPs. Two SGNH-domain RBPs identified by GWAS were experimentally confirmed as active esterases, supporting capsule deacetylation as a widespread alternative to polysaccharide depolymerisation in temperate phages. Our findings reveal that Klebsiella prophages encode structurally diverse RBPs, suggesting temperate phages may rely not only on depolymerisation but also on capsule modification-such as deacetylation-for infection. This also suggests that capsule modification may contribute to phage-host interactions in ways not fully captured by current K-locus assignments, with potential implications for phage specificity, competition and vaccine design.

Phenotypic heterogeneity allows bacteria to adapt fast to changing environments. Extracellular capsules are well-known virulence factors, but also increase the cell adaptability and prevalence under hostile conditions. To limit their cost, some species regulate capsule production by genetic phase variation. Here, we demonstrated that phenotypic heterogeneity is a major mechanism controlling capsule production in Klebsiella and Acinetobacter species. We designed a method to agnostically measure heterogeneity and show that 71% of Klebsiella pneumoniae strains can be heterogeneous. This is mostly associated with K. pneumoniae strains that do not encode rmp, a genetic determinant of hypervirulence. Capsule serotype exchanges across several genetic backgrounds revealed that heterogeneity depends on specific genome-capsule locus interactions. Importantly, we showed that heterogeneity provides a fitness advantage especially in conditions where the capsule is costly, as estimated by comparing non-heterogeneous and heterogeneous strains during competition with their non-capsulated variants. Finally, heterogeneity impacts phage adsorption patterns, and could thus alter the rate of horizontal gene transfer events. This unsuspected heterogeneity may help understand the transition from commensalism to pathogenesis and can have important implications in virulence, environmental survival and evolution of some ESKAPE pathogens.IMPORTANCEThe polysaccharidic capsule is present in ~50% of species across the bacterial phylogeny, including all ESKAPE microorganisms, the six most significant multidrug-resistant (MDR) nosocomial pathogens. It is also an important virulence factor and a major target for both phage therapy and the development of vaccines. Here, we reveal that in two major genera of ESKAPE pathogens, Klebsiella spp. and Acinetobacter spp., capsule production within clonal populations is heterogeneous, leading to mixed populations of hyper-, hypo-, and intermediate-capsulated cells. Such heterogeneity responds to different environmental cues, including changes in nutrient availability and spatial structure. We show that this plasticity, known to enable faster, more efficient adaptation to environmental changes, limits capsule costs and could explain Klebsiella and Acinetobacter resilience. Finally, capsule heterogeneity can play a major role in bacterial evolution, as a driver of horizontal gene transfer, and in treatment failure. Thus, it should be taken into account in the design of prophylactic strategies and antimicrobial therapy.

Cyanobacteria played a pivotal role in shaping Earth's early history and today are key players in many ecosystems. As versatile and ubiquitous phototrophs, they are used as models for oxygenic photosynthesis, nitrogen fixation, circadian rhythms, symbiosis, and adaptations to harsh environments. Cyanobacterial genomes and metagenomes exhibit high levels of genomic diversity partly driven by gene flow within and across species. Processes such as recombination and horizontal transfer of novel genes are facilitated by the mobilome that includes plasmids, transposable elements, and bacteriophages. We review these processes in the context of molecular mechanisms of gene transfer, barriers to gene flow, selection for novel traits, and auxiliary metabolic genes. Additionally, Cyanobacteriota are unique because ancient evolutionary innovations, such as oxygenic photosynthesis, can be corroborated with fossil and biogeochemical records. At the same time, sequencing of extant natural populations allows the tracking of recombination events and gene flow over much shorter timescales. Here, we review the challenges of assessing the impact of gene flow across the whole range of evolutionary timescales. Understanding the tempo and constraints to gene flow in Cyanobacteriota can help decipher the timing of key functional innovations, analyze adaptation to local environments, and design Cyanobacteriota for robust use in biotechnology.

Plasmids play key roles in the spreading of many traits, ranging from antibiotic resistance to varied secondary metabolism, from virulence to mutualistic interactions, and from defense to antidefense. Our understanding of plasmid mobility has progressed extensively in the last few decades. Conjugative plasmids are still often the textbook image of plasmids, yet they are now known to represent a minority. Many plasmids are mobilized by other mobile genetic elements, some are mobilized as phages, and others use atypical mechanisms of transfer. This review focuses on recent advances in our understanding of plasmid mobility, from the molecular mechanisms allowing transfer and evolutionary changes of plasmids to the ecological determinants of their spread. In this emerging, extended view of plasmid mobility, interactions between mobile genetic elements, whether involving exploitation, competition, or elimination, affect plasmid transfer and stability. Likewise, interactions between multiple cells and their plasmids shape the latter patterns of transfer through transfer-mediated bacterial predation, interference, or eavesdropping in cell communication, and by deploying defense and antidefense activity. All these processes are relevant for microbiome intervention strategies, from plasmid containment in clinical settings to harnessing plasmids in ecological or industrial interventions.

The use of antibiotics disrupts the gut microbiota, potentially leading to long-term health issues and the spread of resistance. To investigate the impact of antibiotics on phage populations, we followed 22 healthy individuals two weeks before and up to six months after a three-day course of 3rd-generation cephalosporins. The populations of phages encoded very rarely antibiotic resistance genes and were mostly temperate including many phage-plasmids. Moreover, gut phages remained individual-specific even after microbiome perturbation by antibiotics. Yet, we found a 20% decline in phage diversity and abundance, which took 30 days to recover, and a few (mostly virulent) phages burst the day after treatment. We suggest they contribute to the recovery of gut bacterial diversity, since several targeted Parabacteroides distasonis, a bacterium thriving after cephalosporin treatment, which only proliferated in the absence of these phages. Our findings point out that phages play a crucial role in the gut microbiota's response to antibiotics by restoring microbial balance and diversity. ### Competing Interest Statement The authors have declared no competing interest.

Antibiotic use disrupts the gut microbiota, posing risks of long-term health issues and resistance. To study its impact on gut phages, we followed 22 healthy individuals 2 weeks before and up to 6 months after a 3-day course of 3rd-generation cephalosporins. Our results show that gut phages rarely encode antibiotic resistance genes and are mostly temperate, including many phage plasmids. Furthermore, phage populations remain individual-specific even after microbiome perturbation. Yet, we report a 20% decline in phage diversity the day after treatment, alongside blooms of a few, mostly virulent, phages. We suggest that some of these phages contribute to the recovery of bacterial diversity via "kill-the-winner" dynamics. This is supported by (temporarily) dominant phages targeting Parabacteroides distasonis, a bacterium that thrives post-treatment only in the absence of these phages. Our findings suggest gut phages are crucial to the microbiome response to antibiotics, aiding the restoration of balance and diversity.

Integrons are genetic systems that drive bacterial adaptation by acquiring, expressing, and shuffling gene cassettes. While mobile integrons are well known for spreading antibiotic resistance genes, the functions of the hundreds of cassettes carried by sedentary integrons remain largely unexplored. We show that many of these cassettes encode small variants of known antiphage systems that favor their inclusion in the integron. We also demonstrate that nearly 10% of the integron cassettes in the pandemic Vibrio cholerae strain encode novel antiphage functions. Most of these novel systems have little or no similarity to previously known ones, with several providing defense through cell lysis or growth arrest. Our work highlights the stabilization and prevalence of small antiphage systems within integrons, making them an untapped biobank of defense mechanisms.

The host range of a bacteriophage-the diversity of hosts it can infect-is central to understanding phage ecology and applications. Whereas most well-characterized phages have narrow host ranges, broad-host-range phages represent an intriguing component of marine ecosystems. The genetic and evolutionary mechanisms driving their generalism remain poorly understood. In this study, we analyzed Schizotequatroviruses and their Vibrio crassostreae hosts, collected from an oyster farm. Schizotequatroviruses exhibit broad host ranges, large genomes (~252 kbp) encoding 26 transfer ribonucleic acids, and conserved genomic organization interspersed with recombination hotspots. These recombination events, particularly in regions encoding receptor-binding proteins and antidefense systems, highlight their adaptability to host resistance. Some lineages demonstrated the ability of receptor-switching between OmpK and LamB. Despite their broad host range, Schizotequatroviruses were rare in the environment. Their scarcity could not be attributed to burst size, which was comparable to other phages in vitro, but may result from ecological constraints or fitness trade-offs, such as their preference for targeting generalist vibrios in seawater rather than the patho-phylotypes selected in oyster farms. Our findings clarify the genetic and ecological variables shaping Schizotequatrovirus generalism and provide a foundation for future phage applications in aquaculture and beyond.

Microorganisms endure novel challenges for which other microorganisms in other biomes may have already evolved solutions. This is the case of nosocomial bacteria under antibiotic therapy because antibiotics are of ancient natural origin and resistances to them have previously emerged in environmental bacteria. In such cases, the rate of adaptation crucially depends on the acquisition of genes by horizontal transfer of plasmids from distantly related bacteria in different biomes. We hypothesized that such processes should be driven by plasmids among the most mobile and evolvable. We confirmed these predictions by showing that plasmid species encoding antibiotic resistance are very mobile, have broad host ranges, while showing higher rates of homologous recombination and faster turnover of gene repertoires than the other plasmids. These characteristics remain outstanding when we remove resistance plasmids from our dataset, suggesting that antibiotic resistance genes are preferentially acquired and carried by plasmid species that are intrinsically very mobile and plastic. Evolvability and mobility facilitate the transfer of antibiotic resistance, and presumably of other phenotypes, across distant taxonomic groups and biomes. Hence, plasmid species, and possibly those of other mobile genetic elements, have differentiated and predictable roles in the spread of novel traits.

Natural transformation is the only process of gene exchange under the exclusive control of the recipient bacteria. It has often been considered as a source of novel genes, but quantitative assessments of this claim are lacking. To investigate the potential role of natural transformation in gene acquisition, we analyzed a large collection of genomes of Acinetobacter baumannii (Ab) and Legionella pneumophila (Lp) for which transformation rates were experimentally determined. Natural transformation rates are weakly correlated with genome size. But they are negatively associated with gene turnover in both species. This might result from a negative balance between the transformation's ability to cure the chromosome from mobile genetic elements (MGEs), resulting in gene loss, and its facilitation of gene acquisition. By comparing gene gains by transformation and MGEs, we found that transformation was associated with the acquisition of small sets of genes per event, which were also spread more evenly in the chromosome. We estimated the contribution of natural transformation to gene gains by comparing recombination-driven gene acquisition rates between transformable and non-transformable strains, finding that it facilitated the acquisition of ca. 6.4% (Ab) and 1.1% (Lp) of the novel genes. This moderate contribution of natural transformation to gene acquisition implies that most novel genes are acquired by other means. Yet, 15% of the recently acquired antibiotic resistance genes in A. baumannii may have been acquired by transformation. Hence, natural transformation may drive the acquisition of relatively few novel genes, but these may have a high fitness impact.

Understanding host-range determinants in temperate bacteriophages is critical for elucidating phage-host co-evolution and advancing phage therapies.We analysed 3,900 Klebsiella genomes from diverse ecological niches to identify prophage-encoded proteins mediating capsule tropism. We applied a genome-wide association study (GWAS) correlating prophage protein clusters (from 8,105 prophages) with confidently assigned bacterial K-loci. GWAS identified high-confidence predictors for 16 out of 35 most diverse K-loci, of which 14 were receptor-binding proteins (RBPs) belonging to classical depolymerases ( n = 6), SGNH hydrolases which deacetylate polysaccharides ( n = 6), and structurally novel RBPs ( n = 2). When we relaxed the filtering thresholds, we identified 26 putative depolymerases, of which 12 were deemed as strong predictions against 10 K-types. In parallel, 50 depolymerases manually found in prophages from the representative subset of 99 bacterial isolates, together with an additional 10 depolymerases based on GWAS predictions, were prepared as recombinant proteins and tested on a Klebsiella reference panel of 119 K-types. Most predicted prophage depolymerases (34/60) failed to yield soluble products, 6 were not active on the K-types panel, and 5/14 targeted a different K-type than their bacterial host, highlighting the unpredictability of prophages as a source of functional enzymes. A comparison of GWAS-predicted enzymes, from the manual search and from virulent phages showed that depolymerase specificity was often difficult to infer from sequence or structure alone. Our findings reveal that Klebsiella prophages encode structurally diverse RBPs, suggesting temperate phages may rely not only on depolymerisation but also on capsule modification—such as deacetylation—for infection. This suggests capsule diversity in K. pneumoniae may be substantially underestimated, with implications for phage specificity, competition and vaccine design. ### Competing Interest Statement The authors have declared no competing interest.

Bacterial genomes contain a plethora of secondary replicons of divergent size. Circular replicons must carry a system for resolving dimeric forms, resulting from recombination between sister copies. These systems use site-specific recombinases. Among these, the XerCD recombinase resolves dimers of chromosomes and certain plasmids, using different modes of regulation. We have analyzed the dimer resolution functions in enterobacterial secondary replicons and show that, in addition to the main chromosomes, XerCD is preferentially used by small plasmids and by the largest secondary replicons, megaplasmids and secondary chromosomes. Indeed, all replicons longer than 250 kb host an active XerCD recombination site. These sites, in contrast to those of small plasmids, use the same control as chromosomes, coupled to cell division by the FtsK protein. We conclude that a chromosome-like mode of dimer resolution is mandatory for the faithful inheritance of large plasmids and chromids, its acquisition being a prerequisite for the genesis of secondary chromosomes from plasmids.

Phage satellites are defined as viruses that have a life cycle dependent on a helper virus. Thus, they are often considered as parasites of parasites, although recent work suggests it may be more accurate to consider them as symbionts that evolved along a parasitism–mutualism continuum. Over the past years, multiple studies have examined the fascinating life cycle of these elements, focusing on the characterization of the molecular mechanisms they use to hijack the helper phage machinery for their own packaging and transfer. As some phage satellites encode toxins and other virulence and resistance genes, the impact of these elements on bacterial virulence has also been extensively analysed. Recent studies suggest that satellites have unprecedented roles in the ecology and evolution of bacteria and their mobile genetic elements. In this Review, we explore the genetics and the life cycle of these elements, with special emphasis on the new mechanisms involved in their spread in nature. We discuss the unexpected impact of these elements on the evolution of other mobile genetic elements and their host bacteria, and examine their potential origins. In this Review, Penadés et al. explore the genetics, potential origins and life cycle of phage satellites, and they discuss the impact of these elements on the evolution of other mobile genetic elements and their host bacteria.

Natural transformation is a widespread molecular pathway of horizontal gene transfer involving the uptake and recombination of exogenous DNA. Exogenous DNA follows a pathway involving genes sequentially required for its capture, internalization, protection, and recombination with the chromosome. Most of these genes were identified through the isolation of transformation-defective mutants and/or based on their expression preceding natural transformation. Yet, genes required for key steps of the pathway remain elusive. We sought to identify any missing component by comparing Tn-seq data obtained in two distantly-related transformable diderm species, the human pathogen Legionella pneumophila and the cyanobacterium Synechococcus elongatus . We identified yraN , a widespread and highly conserved gene of unknown function required for natural transformation. We provide evidence that YraN is a nuclease associated with the ComM helicase, which cooperate to process the D-loop formed by the invasion of the transforming DNA in the chromosomal DNA strands. We propose a model in which cleavage of the displaced strand by YraN can promote the recombination of transforming DNA, leading to extended recombination events. The identification of this YraN/ComM nuclease/helicase system supports the hypothesis that bacteria possess a conserved pathway for the transport and recombination of exogenous DNA.Significance Many bacteria use a dedicated pathway to internalize and integrate extracellular DNA into their chromosome. This allows them to naturally acquire genes, or gene variants, that can confer them new traits, hence the term of natural transformation. Although reported nearly a century ago by Frederick Griffith, several aspects of the mechanism of natural transformation remain elusive. Specifically, it is not clear whether or not all the conserved molecular players of the pathway have been identified. We queried two distinct bacterial species for any gene that would be required for natural transformation. We confirmed all core players of the pathway, but also uncovered a highly conserved one, YraN. We provide evidence that YraN is an endonuclease, which in association with the ComM helicase, promotes the efficient integration of the extracellular DNA in the bacterial chromosome.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACT Conjugative plasmids are important drivers of bacterial evolution, but most plasmids lack genes for conjugation. It is currently not known if the latter can transfer because origins of transfer by conjugation ( oriT ), which would allow their mobilization by conjugative plasmids, are poorly known. Here, we identify and characterize occurrences of known oriT families across thousands of plasmids confirming that most conjugative and mobilizable plasmids still lack identifiable families of oriTs . They reveal clear patterns in terms of intergenic position, distance to the relaxases, and MOB-type association. This allowed to develop a computational method to discover novel oriT s. As a proof of concept, we identify 21 novel oriTs from the nosocomial pathogens Escherichia coli , Klebsiella pneumoniae , and Acinetobacter baumannii , some of them responsible for the mobility of critical antimicrobial resistance genes. These 21 oriT families share key characteristics of the others and fill most of the missing diversity of oriTs in relaxase-encoding plasmids both in terms of frequency and phylogeny. We confirmed experimentally the function of six of them. The ability to identify novel oriT s paves the way to explore conjugation across bacterial plasmids, notably among the majority lacking conjugation-related genes.