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.

Symbiosis is widespread in nature and plays a fundamental role in organism adaptation and evolution. In nutritional endosymbiosis, host cells accommodate intracellular bacteria and act as a "metabolic factory," requiring extensive metabolic exchanges between host and endosymbiont. To investigate the mechanisms supporting these exchanges, we used the association between the bacterium Sodalis pierantonius and the insect Sitophilus spp. that thrives on an exclusive cereal diet. Volume electron microscopy uncovered that endosymbionts generate complex membranous tubular networks (tubenets) that connect bacteria and drastically increase their exchange surface with the host cytosol. In situ high spatial resolution chemical analysis indicated that tubenets are enriched in carbohydrates, which are the main substrate used by bacteria to generate nutrients for the host. Multiple membranous structures favoring nutrient absorption are described in multicellular organisms. This work demonstrates that bacteria have convergently evolved a similar "biostrategy" that enhances nutrient acquisition by increasing membrane interface.

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.

AbstractBackgroundInsects living in nutritionally poor environments often establish long-term relationships with intracellular bacteria that supplement their diets and improve their adaptive and invasive powers. Even though these symbiotic associations have been extensively studied on physiological, ecological and evolutionary levels, few studies have focused on the molecular dialogue between host and endosymbionts to identify genes and pathways involved in endosymbiosis control and dynamics throughout host development.ResultsWe simultaneously analyzed host and endosymbiont gene expression during the life cycle of the cereal weevilSitophilus oryzae, from larval stages to adults, with a particular emphasis on emerging adults where the endosymbiontSodalis pierantoniusexperiences a contrasted growth-climax-elimination dynamics. We unraveled a constant arms race in which different biological functions are intertwined and coregulated across both partners. These include immunity, metabolism, metal control, apoptosis, and bacterial stress response.ConclusionsThe study of these tightly regulated functions, which are at the center of symbiotic regulations, provides evidence on how hosts and bacteria finely tune their gene expression and respond to different physiological challenges constrained by insect development in a nutritionally limited ecological niche.Graphical Abstract

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence. Previous studies showed that the D. dadantii T2SS secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins possess a signal peptide and are first addressed to the periplasm before their recruitment by Out. T2SS-specific secretion signals remain unknown which prevents in silico identification of T2SS substrates. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced and encoding proteins with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

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.

Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H 2 O 2 -induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.

Summary The identification of the virulence factors of plant‐pathogenic bacteria has relied on the testing of individual mutants on plants, a time‐consuming process. Transposon sequencing (Tn‐seq) is a very powerful method for the identification of the genes required for bacterial growth in their host. We used this method in a soft‐rot pathogenic bacterium to identify the genes required for the multiplication of Dickeya dadantii in chicory. About 100 genes were identified showing decreased or increased fitness in the plant. Most had no previously attributed role in plant–bacterium interactions. Following our screening, in planta competition assays confirmed that the uridine monophosphate biosynthesis pathway and the purine biosynthesis pathway were essential to the survival of D. dadantii in the plant, as the mutants ∆carA, ∆purF, ∆purL, ∆guaB and ∆pyrE were unable to survive in the plant in contrast with the wild‐type (WT) bacterium. This study also demonstrated that the biosynthetic pathways of leucine, cysteine and lysine were essential for bacterial survival in the plant and that RsmC and GcpA were important in the regulation of the infection process, as the mutants ∆rsmC and ∆gcpA were hypervirulent. Finally, our study showed that D. dadantii flagellin was glycosylated and that this modification conferred fitness to the bacterium during plant infection. Assay by this method of the large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.

O-polysaccharides were isolated from lipopolysaccharides obtained from four different strains of plant pathogenic bacteria belonging to the species Dickeya solani: two of them were isolated in Poland (IFB0099 and IFB0158), the third in Germany (IFB0223) and the last one, D. solani Type Strain IPO2222, originated from the Netherlands. In addition, the O-polysaccharide of a closely related species D. dadantii strain 3937 was isolated. The purified polysaccharides of the five strains were analyzed using NMR spectroscopy and chemical methods. Sugar and methylation analyses, including absolute configuration assignment, together with NMR data revealed that all O-polysaccharides tested are homopolymers of 6-deoxy-d-altrose (d-6dAlt) the following structure: →2)-β-d-6dAltp-(1→.

ABSTRACT Soft rot enterobacteria ( Dickeya and Pectobacterium ) are major pathogens that cause diseases on plants of agricultural importance such as potato and ornamentals. Long term studies to identify virulence factors of these bacteria focused mostly on plant cell wall degrading enzymes secreted by the type II secretion system and the regulation of their expression. To identify new virulence factors we performed a Tn-seq genome-wide screen of a transposon mutant library during chicory infection followed by high-throughput sequencing. This allowed the detection of mutants with reduced but also increased fitness in the plant. Virulence factors identified differed from those previously known since diffusible ones (secreted enzymes, siderophores or metabolites) were not detected by this screen. In addition to genes encoding proteins of unknown function that could be new virulence factors, others could be assigned to known biological functions. The central role of the FlhDC regulatory cascade in the control of virulence was highlighted with the identification of new members of this pathway. Scarcity of the plant in certain amino acids and nucleic acids required presence of the corresponding biosynthetic genes in the bacteria. Their products could be targets for the development of antibacterial compounds. Among the genes required for full development in chicory we also identified six genes involved in the glycosylation of the flagellin FliC, glycosylation, which in other plant pathogenic bacteria contributes to virulence. Author summary Identification of virulence factors of plant pathogenic bacteria has relied on the test of individual mutants on plants, a time-consuming method. New methods like transcriptomic or proteomic can now be used but they only allow the identification of genes induced during the infection process and non-induced genes may be missed. Tn-seq is a very powerful method to identify genes required for bacterial growth in their host. We used for the first time this method in a plant pathogenic bacteria to identify genes required for the multiplication of Dickeya dadantii in chicory. We identified about 100 genes with decreased or increased fitness in the plant. Most of them had no previously described role in bacterial virulence. We unveiled important metabolic genes and regulators of motility and virulence. We showed that D. dadantii flagellin is glycosylated and that this modification confers fitness to the bacteria during plant infection. Our work opens the way to the use of Tn-seq with bacterial phytopathogens. Assay by this method of large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.

ABSTRACT Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves the products of the dltXABCD genes. These genes are widespread in Gram-positive bacteria, and they are also found in a few Gram-negative bacteria. Notably, these genes are present in all soft-rot enterobacteria (Pectobacterium and Dickeya) whose dltDXBAC operons have been sequenced. We studied the function and regulation of these genes in Dickeya dadantii. dltB expression was induced in the presence of the AMP polymyxin. It was not regulated by PhoP, which controls the expression of some genes involved in AMP resistance, but was regulated by ArcA, which has been identified as an activator of genes involved in AMP resistance. However, arcA was not the regulator responsible for polymyxin induction of these genes in this bacterium, which underlines the complexity of the mechanisms controlling AMP resistance in D. dadantii. Two other genes involved in resistance to AMPs have also been characterized, phoS and phoH. dltB, phoS, phoH, and arcA but not dltD mutants were more sensitive to polymyxin than the wild-type strain. Decreased fitness of the dltB, phoS, and phoH mutants in chicory leaves indicates that their products are important for resistance to plant AMPs. IMPORTANCE Gram-negative bacteria can modify their lipopolysaccharides (LPSs) to resist antimicrobial peptides (AMPs). Soft-rot enterobacteria (Dickeya and Pectobacterium spp.) possess homologues of the dlt genes in their genomes which, in Gram-positive bacteria, are involved in resistance to AMPs. In this study, we show that these genes confer resistance to AMPs, probably by modifying LPSs, and that they are required for the fitness of the bacteria during plant infection. Two other new genes involved in resistance were also analyzed. These results show that bacterial resistance to AMPs can occur in bacteria through many different mechanisms that need to be characterized.

In the track of new biopesticides, four genes namely cytA , cytB , cytC and cytD encoding proteins homologous to Bacillus thuringiensis ( Bt ) Cyt toxins have been identified in the plant pathogenic bacteria Dickeya dadantii genome. Here we show that three Cyt-like δ-endotoxins from D. dadantii (CytA, CytB and CytC) are toxic to the pathogen of the pea aphid Acyrthosiphon pisum in terms of both mortality and growth rate. The phylogenetic analysis of the comprehensive set of Cyt toxins available in genomic databases shows that the whole family is of limited taxonomic occurrence, though in quite diverse microbial taxa. From a structure-function perspective the 3D structure of CytC and its backbone dynamics in solution have been determined by NMR. CytC adopts a cytolysin fold, structurally classified as a Cyt2-like protein. Moreover, the identification of a putative lipid binding pocket in CytC structure, which has been probably maintained in most members of the Cyt-toxin family, could support the importance of this lipid binding cavity for the mechanism of action of the whole family. This integrative approach provided significant insights into the evolutionary and functional history of D. dadantii Cyt toxins, which appears to be interesting leads for biopesticides.

Pectate lyases are enzymes involved in plant cell wall degradation. They cleave pectin using a β-elimination mechanism, specific for acidic polysaccharides. They are mainly produced by plant pathogens and plant-associated organisms, and only rarely by animals. Pectate lyases are also commonly produced in the bacterial world, either by bacteria living in close proximity with plants or by gut bacteria that find plant material in the digestive tract of their hosts. The role of pectate lyases is essential for plant pathogens, such as Dickeya dadantii, that use a set of pectate lyases as their main virulence factor. Symbiotic bacteria produce their own pectate lyases, but they also induce plant pectate lyases to initiate the symbiosis. Pectin degradation products may act as signals affecting the plant–bacteria interactions. Bacterial pectate lyases are also essential for using the pectin of dead or living plants as a carbon source for growth. In the animal gut, Bacteroides pectate lyases degrade the pectin of ingested food, and this is particularly important for herbivores that depend on their microflora for the digestion of pectin. Some human pathogens, such as Yersinia enterocolitica, produce a few intracellular pectate lyases that can facilitate their growth in the presence of highly pectinolytic bacteria, at the plant surface, in the soil or in the animal gut.

Type II secretion systems (T2SSs) generally release their substrates into the culture medium. A few T2SS substrates remain anchored to or bound at the surface of the bacteria after secretion. Since they handle already folded proteins, T2SSs are the best way for bacteria to target, at their surface, proteins containing a cofactor, proteins that have to be folded in the cytoplasm or in the periplasm, or multimeric proteins. However, how a T2SS deals with membrane-anchored proteins is not yet understood. While this type of protein has until now been overlooked, new proteomic approaches will facilitate its identification.

The plant pathogenic bacterium Dickeya dadantii has recently been shown to be able to kill the aphid Acyrthosiphon pisum. While the factors required to cause plant disease are now well characterized, those required for insect pathogeny remain mostly unknown. To identify these factors, we analyzed the transcriptome of the bacteria isolated from infected aphids. More than 150 genes were upregulated and 300 downregulated more than 5-fold at 3 days post infection. No homologue to known toxin genes could be identified in the upregulated genes. The upregulated genes reflect the response of the bacteria to the conditions encountered inside aphids. While only a few genes involved in the response to oxidative stress were induced, a strong defense against antimicrobial peptides (AMP) was induced. Expression of a great number of efflux proteins and transporters was increased. Besides the genes involved in LPS modification by addition of 4-aminoarabinose (the arnBCADTEF operon) and phosphoethanolamine (pmrC, eptB) usually induced in Gram negative bacteria in response to AMPs, dltBAC and pbpG genes, which confer Gram positive bacteria resistance to AMPs by adding alanine to teichoic acids, were also induced. Both types of modification confer D. dadantii resistance to the AMP polymyxin. A. pisum harbors symbiotic bacteria and it is thought that it has a very limited immune system to maintain these populations and do not synthesize AMPs. The arnB mutant was less pathogenic to A. pisum, which suggests that, in contrast to what has been supposed, aphids do synthesize AMP.