Tailocins, phage-tail-derived bacteriocins, are increasingly recognized as potent mediators of microbial antagonism, yet their ecological scope beyond kin-targeting remains poorly understood. Here, we investigated whether P2D1, a tailocin produced by the plant pathogen Dickeya dadantii 3937, can act against environmental bacteria phylogenetically distant from Dickeya spp. Screening 480 soil and rhizosphere isolates from three distinct plant-associated habitats in Poland, we identified nine Pseudomonas spp. strains susceptible to tailocin P2D1. Whole-genome sequencing and phenotype profiling revealed that these isolates spanned multiple clades, including taxa related to P. germanica, P. tensinigenes, and P. parakoreensis. The D. dadantii mutant lacking genes encoding tailocin sheath and tube proteins lost antagonistic activity against Pseudomonas isolates, confirming that tailocins alone mediate the observed killing. Plant tissue assays revealed that six of the P2D1-susceptible strains were nonpathogenic and could mitigate D. dadantii-induced soft rot on potato. In contrast, three isolates related to P. tensinigenes were able to cause rot on their own under permissive conditions. Together, these findings demonstrate that P2D1 tailocin extends its activity to ecologically co-occurring but taxonomically distant Pseudomonas, suggesting that conserved receptors underline cross-genus targeting. More broadly, our results add to the limited evidence for tailocin activity beyond kin killing and therefore challenge the prevailing paradigm of kin-restricted tailocin specificity. They further suggest that tailocins may influence microbial community assembly across taxonomic boundaries, while their in vivo roles remain understudied.

Transposon sequencing (Tn-seq) is a powerful technique for defining the essential genes required for bacterial survival. However, gene essentiality can vary significantly across taxonomic levels, and comparing large Tn-seq datasets from multiple strains presents considerable analytical challenges. To address this, we developed TNSEEK, a fully automated bioinformatics pipeline for the systematic and comparative analysis of transposon sequencing experiments. We applied TNSEEK to analyze six Soft Rot Pectobacteriaceae (SRP) strains, encompassing species from the Dickeya and Pectobacterium genera, grown in a rich medium. This approach identified a core essentialome of 225 genes, primarily involved in fundamental cellular maintenance, conserved across all six strains, a set comparable in size to that of the broader Enterobacteriaceae family. Only a few genus-specific essential genes were found highlighting interesting distinct metabolic capabilities between Dickeya and Pectobacterium genera. In striking contrast, we discovered a large variable essentialome comprising 181 strain-specific genes, many of which of unknown function. A portion of these strain-specific essential genes are components of defense systems and prophage genomic regions. The unexpected essentiality of these modules suggests they form a constitutively active frontline defense. Furthermore, a comparison with the E. coli essentialome demonstrates that discrepancies in gene essentiality can often be attributed to differences in growth conditions, particularly temperature, as well as variations in genetic redundancy. In conclusion, the TNSEEK pipeline is a robust tool for exploring functional genomics across multiple strains.

Industrial and urban activities release toxic chemical waste into the environment. Pseudomonas putida, a soil bacterium, is known to degrade hydrocarbons and xenobiotics, and possesses numerous genes associated with heavy metal tolerance. Most studies on metal tolerance in P. putida focus solely on over- or underexpressed genes, potentially overlooking important genes with unchanged expression. This study employed a Tn-seq approach to identify the essential genes required for P. putida growth under metal stress. This method enables the identification of mutants with altered fitness in the presence of excess metals. The screen successfully identified a number of known genes implicated in metal resistance, including czcA-1, cadA-3, cadR, and pcoA2, thereby validating the approach. Further analyses using targeted mutagenesis and complementation assays revealed PP_5337 as a putative transcriptional regulator involved in copper tolerance and the two-component system RoxSR (PP_0887/PP_0888) as a key determinant of cadmium tolerance. Additionally, PP_1663 and PP_5002 were identified as contributing to cadmium and cobalt tolerance, respectively. This study provides the first evidence linking these genes to metal tolerance, highlighting gaps in our understanding of metal tolerance mechanisms in P. putida and demonstrating the utility of Tn-seq for identifying novel tolerance determinants.

Little is known about the role of antibiotics in microbial ecosystems in the absence of clinical antibiotic pressure. The soft rot Pectobacteriaceae (SRP) species complex comprises 37 bacterial species that are collectively responsible for severe plant decay in many crops. Within this complex, Pectobacterium versatile strains harbour the BlaPEC-1 β-lactamase. The aim of our work was to analyse the role of BlaPEC-1 during plant infection. To this end, two blaPEC-1-deleted strains were compared with their wild-type counterparts in vitro and in mono- or mixed infections of potato tubers with different SRP strains. In vitro, BlaPEC-1 enables P. versatile to resist ampicillin or the carbapenem produced by Pectobacterium brasiliense. In mono-infections of potato tubers, blaPEC-1-deleted strains were not affected in virulence, fitness or association with bacterial commensals. In mixed infections, BlaPEC-1 was required for the coexistence of P. versatile with the carbapenem-producing strain and was necessary to rescue carbapenem-sensitive strains both in vitro and in planta. Protection was observed even when BlaPEC1-expressing bacteria were a minority within the symptom. These results indicate that BlaPEC-1 exerts a true β-lactamase function during the infection process and acts as a public good of the SRP species complex to maintain SRP strain diversity. ### Competing Interest Statement The authors have declared no competing interest.

Intra-strain variation in model bacterial pathogens can compromise experimental reproducibility and obscure biological interpretations. Dickeya solani, a necrotrophic potato pathogen, is widely studied using the type strain IPO 2222. However, phenotypic discrepancies among laboratories led us to investigate the genetic integrity of this reference stock. We identified at least three distinct IPO 2222 variants co-existing in the original stock, differing solely by mutations in the gene encoding the small regulatory RNA (sRNA) ArcZ. These findings resolve conflicting reports of antimicrobial activity in this strain described by Brual et al. (PLOS Genetics 19, e1010725, 2023) and Matilla et al. (mBio, e02472-22, 2022). We demonstrate that these are adaptive mutations rapidly selected in planta during host infection. Crucially, rather than systematically inactivating the gene, these mutations modulate the cellular levels of processed ArcZ. This modulation can uncouple virulence from antimicrobial activity. These variants behave as social cheaters, exhibiting a frequency-dependent fitness advantage over the cooperative wild-type strain during co-infection. These findings provide evidence that remodeling of a pleiotropic sRNA drives the emergence of bacterial cheaters within a plant host. The speed at which these mutants sweep through the population underscores the intense selective pressure acting on regulatory networks during infection, identifying sRNA modulation as a pivotal mechanism for rapid short-term adaptation. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-22-CE35-0017

Conjugative plasmids are the main drivers of antibiotic resistance dissemination contributing to the emergence and extensive spread of multidrug resistance clinical bacterial pathogens. pOXA-48 plasmids, belonging to the IncL group, emerge as the primary vehicle for carbapenem resistance in Enterobacteriaceae. Despite the problematic prevalence of pOXA-48, most research focus on epidemiology and genomics, leaving gaps in our understanding of the mechanisms behind its propagation. In this study, we use a transposon sequencing approach to identify genetic elements critical for plasmid stability, replication, and conjugative transfer. Our results identify a novel type I toxin-antitoxin system, uncharacterized essential maintenance factors, and components of the type IV secretion system and regulatory elements crucial for conjugation. This study advances our understanding of pOXA-48 biology, providing key insights into the genetic factors underlying its successful maintenance and spread in bacterial populations.

ArcZ is a small regulatory RNA conserved in Enterobacterales It is an Hfq-dependent RNA that is cleaved by RNase E in a processed form of 55-60 nucleotides. This processed form is highly conserved for controlling the expression of target mRNAs. ArcZ expression is induced by abundant oxygen levels and reaches its peak during the stationary growth phase. This control is mediated by the oxygen-responsive two-component system ArcAB, leading to the repression of arcZ transcription under low-oxygen conditions in most bacteria in which it has been studied. ArcZ displays multiple targets, and it can control up to 10% of a genome and interact directly with more than 300 mRNAs in Escherichia coli and Salmonella enterica ArcZ displays a multifaceted ability to regulate its targets through diverse mechanisms such as RNase recruitment, modulation of ribosome accessibility on the mRNA, and interaction with translational enhancing regions. By influencing stress response, motility, and virulence through the regulation of master regulators such as FlhDC or RpoS, ArcZ emerges as a major orchestrator of cell physiology within Enterobacterales.

Chemical waste with toxic effects is released into the environment by industrial and urban activities. Pseudomonas putida , a rhizosphere bacterium, harbors a wide variety of genes capable of degrading hydrocarbons and xenobiotic compounds in its natural environment. This bacterium harbors also a large set of metal resistance genes. Most studies that identify genes involved in metal resistance in P. putida focus on over/underexpressed genes and may miss other genes important for metal resistance whose expression does not change. In this study, we used a Tn-seq approach to determine the essential genome of P. putida required for growth in the presence of an excess of metals in a culture medium. Tn-seq enables the detection of mutants with reduced or increased fitness in the presence of metal excess. We validated our screen by identifying known metal resistance gene such as czcA-1 ( PP\_0043 ), cadA-3 ( PP\_5139 ), cadR ( PP\_5140 ) and pcoA2 ( PP\_5380 ). Their mutants were underrepresented in the presence of zinc, cadmium (for cadA-3 and cadR ) or copper respectively. In this study, we demonstrate by targeted mutagenesis and complementation assay that PP\_5337 and PP\_0887 are putative transcriptional regulators involved in copper and cadmium resistance, respectively, in P. putida . The study revealed the role of two genes, PP\_1663 and PP\_5002 , in cadmium and cobalt resistance respectively. This is the first evidence linking these genes to metal resistance and highlights the incomplete understanding of metal resistance mechanisms in P. putida . ### Competing Interest Statement The authors have declared no competing interest.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum. They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions. Common genes included acr genes involved in the detoxification of plant defense phytoalexins and kduD, kduI, eda (=kdgA), gudD, garK, garL, and garR genes involved in the assimilation of pectin and galactarate. In root colonization, Tn-seq highlighted 83 genes, all different from those in stem and tuber lesion conditions. They encode the exploitation of organic and mineral nutrients (dpp, ddp, dctA, and pst) including glucuronate (kdgK and yeiQ) and synthesis of metabolites: cellulose (celY and bcs), aryl polyene (ape), and oocydin (ooc). We constructed in-frame deletion mutants of bcsA, ddpA, apeH, and pstA genes. All mutants were virulent in stem infection assays, but they were impaired in the competitive colonization of roots. In addition, the ΔpstA mutant was impaired in its capacity to colonize progeny tubers. Overall, this work distinguished two metabolic networks supporting either an oligotrophic lifestyle on roots or a copiotrophic lifestyle in lesions. This work revealed novel traits and pathways important for understanding how the D. solani pathogen efficiently survives on roots, persists in the environment, and colonizes progeny tubers.

The successful infection of a host plant by a phytopathogenic bacterium depends on a finely tuned molecular cross talk between the two partners. Thanks to transposon insertion sequencing techniques (Tn-seq), whole genomes can now be assessed to determine which genes are important for the fitness of several plant-associated bacteria in planta. Despite its agricultural relevance, the dynamic molecular interaction established between the foliar hemibiotrophic phytopathogen Xanthomonas hortorum pv. vitians and its host, lettuce (Lactuca sativa), remains completely unknown. To decipher the genes and functions mobilized by the pathogen throughout the infection process, we conducted a Tn-seq experiment in lettuce leaves to mimic the selective pressure occurring during natural infection. This genome-wide screening identified 170 genes whose disruption caused serious fitness defects in lettuce. A thorough examination of these genes using comparative genomics and gene set enrichment analyses highlighted that several functions and pathways were highly critical for the pathogen's survival. Numerous genes involved in amino acid, nucleic acid, and exopolysaccharide biosynthesis were critical. The xps type II secretion system operon, a few TonB-dependent transporters involved in carbohydrate or siderophore scavenging, and multiple genes of the carbohydrate catabolism pathways were also critical, emphasizing the importance of nutrition systems in a nutrient-limited environment. Finally, several genes implied in camouflage from the plant immune system and resistance to immunity-induced oxidative stress were strongly involved in host colonization. As a whole, these results highlight some of the central metabolic pathways and cellular functions critical for Xanthomonas host adaptation and pathogenesis. IMPORTANCE Xanthomonas hortorum was recently the subject of renewed interest, as several studies highlighted that its members were responsible for diseases in a wide range of plant species, including crops of agricultural relevance (e.g., tomato and carrot). Among X. hortorum variants, X. hortorum pv. vitians is a reemerging foliar hemibiotrophic phytopathogen responsible for severe outbreaks of bacterial leaf spot of lettuce all around the world. Despite recent findings, sustainable and practical means of disease control remain to be developed. Understanding the host-pathogen interaction from a molecular perspective is crucial to support these efforts. The genes and functions mobilized by X. hortorum pv. vitians during its interaction with lettuce had never been investigated. Our study sheds light on these processes by screening the whole pathogen genome for genes critical for its fitness during the infection process, using transposon insertion sequencing and comparative genomics.

Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA(Pch) induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.

The Vfm quorum sensing (QS) system is preponderant for the virulence of different species of the bacterial genus Dickeya. The vfm gene cluster encodes 26 genes involved in the production, sensing or transduction of the QS signal. To date, the Vfm QS signal has escaped detection by analytical chemistry methods. However, we report here a strain-specific polymorphism in the biosynthesis genes vfmO and vfmP, which is predicted to be related to the production of different analogues of the QS signal. Consequently, the Vfm communication could be impossible between strains possessing different variants of the genes vfmO/P. We constructed three Vfm QS biosensor strains possessing different vfmO/P variants and compared these biosensors for their responses to samples prepared from 34 Dickeya strains possessing different vfmO/P variants. A pattern of specificity was demonstrated, providing evidence that the polymorphism in the genes vfmO/P determines the biosynthesis of different analogues of the QS signal. Unexpectedly, this vfmO/P-dependent pattern of specificity is linked to a polymorphism in the ABC transporter gene vfmG, suggesting an adaptation of the putative permease VfmG to specifically bind different analogues of the QS signal. Accordingly, we discuss the possible involvement of VfmG as co-sensor of the Vfm two-component regulatory system.

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on targeted-antibacterial-plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step toward the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.

The essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome of Xanthomonas hortorum pv. vitians , a gammaproteobacterial plant pathogen of lettuce ( Lactuca sativa L.) which belongs to the plant-pathogen reservoir genus Xanthomonas and is affiliated to the family Xanthomonadaceae . No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome of X. hortorum pv. vitians LM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features of Xanthomonas or Xanthomonadaceae species, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study of X. hortorum pv. vitians .

Bacteria of the genus Dickeya are phytopathogens characterised by a wide host range and large geographical distribution. The disease is named soft rot because the plant material is liquefied by bacterial enzymes. The massive production of pectate lyases is typical of Dickeya. Secretion by a specialised system named Out enables the export of pectinases that cause soft rot by cleaving pectin present in plant cell walls. The 10 species of the genus Dickeya were isolated from diseased plants or water. They attack a variety of crops and ornamental plants in tropical, subtropical and temperate climates. Dickeya solani and Dickeya dianthicola are the two main species responsible for potato diseases, leading to significant economic losses. Recent diseases on fruit trees are also due to Dickeya. These bacteria have developed different mechanisms to cope with adverse conditions and compete with other microorganisms. In addition, they use a complex regulatory network to adjust the production of pectate lyases during the successive phases of plant infection. Key Concepts Dickeya members are plant pathogens characterised by a broad-host range and a large geographical distribution. The genus Dickeya is subdivided into ten species, seven acting as phytopathogens and three found in water. Dickeya species are ‘brute force’ pathogens as they use a set of plant cell wall degrading enzymes as their primary weapon. Massive production of pectate lyases and accessory pectinases is the main virulence signature of the Dickeya species; they cause the symptom of rotting of the plant tissues. Since antibiotics are not allowed in agriculture, there is currently no effective chemical control for Dickeya. Dickeya has developed mechanisms to compete with other microorganisms and to cope with stressful conditions found in the environment or during plant colonisation. The infectious process begins with an asymptomatic phase, corresponding to the asymptomatic presence of Dickeya in plant tissues, followed by the symptomatic phase corresponding to soft-rot appearance. The disease appears when environmental parameters, mainly temperatures and humidity, become favourable. During the plant infection, the expression of pectate lyase genes is finely controlled by a complex regulatory network. Besides pectate lyase secretion, many other functions favour the bacterial virulence, such as protein secretion systems, motility, chemotaxis, iron capture and envelope components.

Pseudomonas chlororaphis is a promising biocontrol agent promoting plant-growth and providing protection against pest insects and phytopathogenic fungi. We have identified in the genome of P. chlororaphis PA23 an operon encoding the toxin Exolysin (ExlA) and its outer-membrane transporter, ExlB. We found that P. chlororaphis producing ExlA (ExlA Pch ) is cytotoxic towards murine macrophages and human epithelial cells at 30 °C. P. chlororaphis PA23 provoked shrinkage of epithelial cell, leakage of cytoplasmic components and subsequent cell death. During infection, ExlA Pch incorporated into epithelial cell membranes within detergent-resistant lipid rafts, suggesting the same mechanisms of cell destruction by pore-formation as reported for P. aeruginosa toxin. ExlA Pch was not involved in the capacity of the strain to kill fungi, amoeba or other bacteria. The contribution of ExlA in insecticidal activity of P. chlororaphis was evaluated in the wax moth larvae Galleria mallonella and in Drosophila melanogaster flies. The impact of the deletion of a gene encoding exlA homologue was tested in the natural fly pathogen P. entonomophila . In both models, the ExlA absence delayed killing, suggesting the contribution of the toxin in bacteria-insect pathogenic interactions.