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International audience; AbstractBacteria 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.

The soft rot Pectobacteriaceae (SRP) infect a wide range of plants worldwide and cause economic damage to crops and ornamentals but can also colonize other plants as part of their natural life cycle. They are found in a variety of environmental niches, including water, soil and insects, where they may spread to susceptible plants and cause disease. In this chapter, we look in detail at the plants colonized and infected by these pathogens and at the diseases and symptoms they cause. We also focus on where in the environment these organisms are found and their ability to survive and thrive there. Finally, we present evidence that SRP may assist the colonization of human enteric pathogens on plants, potentially implicating them in aspects of human/animal as well as plant health.

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 bacterial virulence. Its study in D. dadantii showed that it 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 are first addressed to the periplasm before their secretion by Out. No secretion signal present on the protein allows the identification of substrates of a T2SS. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein 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 showed 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.IMPORTANCEThe plant pathogen Dickeya rely on a type 2 secretion system named Out for their pathogenicity. Importance of plant cell wall degrading enzymes secreted by this system has been well studied. However, existence and role of other Out-secreted proteins has barely been investigated. By mining D. dadantii transcriptome data, we identified four new Out-secreted proteins. We showed that two of them, SvfA and SvfB, are necessary for the full virulence of the bacteria. These findings show that identification of all the proteins secreted by the Dickeya Out system is necessary for a better knowledge of the virulence of these bacteria.

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.

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 H2O2-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., The authors identify a family of iron-binding proteins that is present in phytopathogenic bacteria, fungi and oomycetes. Some of these proteins are secreted, bind metals, protect the pathogen from H2O2-induced death, and are involved in plant infection.

Two surveys conducted in 2017 and 2018 demonstrated Biomphalaria pfeifferi snails in Lake Malawi in Africa. Epidemiologic examination of 175 local children at 3 primary schools confirmed emergence of intestinal schistosomiasis. These findings highlight autochthonous transmission of Schistosoma mansoni flukes in Lake Malawi and the need to revise international travel advice.

Soft rot enterobacteria (DickeyaandPectobacterium) 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 summaryIdentification 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 ofDickeya dadantiiin 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 thatD. dadantiiflagellin 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.

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 -6dAlt p -(1→

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.

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.

The plant pathogenic bacteria Dickeya dadantii is also a pathogen of the pea aphid Acyrthosiphon pisum. The genome of the bacteria contains four cyt genes, encoding homologues of Bacillus thuringiensis Cyt toxins, which are involved in its pathogenicity to insects. We show here that these genes are transcribed as an operon, and we determined the conditions necessary for their expression. Their expression is induced at high temperature and at an osmolarity equivalent to that found in the plant phloem sap. The regulators of cyt genes have also been identified: their expression is repressed by H-NS and VfmE and activated by PecS. These genes are already known to regulate plant virulence factors, but in an opposite way. When tested in a virulence assay by ingestion, the pecS mutant was almost non-pathogenic while hns and vfmE mutants behaved in the same way as the wild-type strain. Mutants of other regulators of plant virulence, GacA, OmpR and PhoP, that do not control Cyt toxin production, also showed reduced pathogenicity. In an assay by injection of bacteria, the gacA strain was less pathogenic but, surprisingly, the pecS mutant was slightly more virulent. These results show that Cyt toxins are not the only virulence factors required to kill aphids, and that these factors act at different stages of the infection. Moreover, their production is controlled by general virulence regulators known for their role in plant virulence. This integration could indicate that virulence towards insects is a normal mode of life for D. dadantii.

Sialic acids are acidic sugars present mostly on vertebrate cell surfaces, which can be metabolized by bacteria and act as an inflammation signal. N-Acetylneuraminic acid, the most abundant sialic acid, can enter into Escherichia coli K12 through NanC, an N-acetylneuraminic acid-inducible outer-membrane channel. With its 215 residues, NanC belongs to the family of small monomeric KdgM-related porins. KdgM homologues are found in gammaproteobacteria, including major plant and human pathogens, and together they define a large family of putative acidic sugar/oligosaccharide transporters, which are as yet poorly characterized. Here, we present the first high-resolution structure of a KdgM family member. NanC folds into a 28-A-high, 12-stranded beta-barrel, resembling the beta-domain of autotransporter NalP and defining an open pore with an average radius of 3.3 A. The channel is lined by two strings of basic residues facing each other across the pore, a feature that appears largely conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.

The entry of oligogalacturonates into Dickeya dadantii occurs through the specific channel KdgM. The genome of the bacterium encodes a second member of this family of outer membrane proteins, KdgN. We showed that this protein is also involved in the uptake of oligogalacturonates. When KdgN was reconstituted in proteoliposomes, it formed channels with a conductance of about 450 pS at a positive potential. These channels had weak anionic selectivity. The regulation of kdgN is complex, and five genes controlling the expression of kdgN have been identified: kdgR , pecS , ompR , hns , and crp . Moreover, kdgN was regulated by growth phase but only when bacteria were grown in rich medium. Most of these regulators of kdgN also control kdgM expression, but some of them regulate kdgM in the opposite manner: while PecS and OmpR are repressors of kdgM , they are activators of kdgN . This pattern resembles the regulation of the Escherichia coli general porins OmpF and OmpC, but such opposite regulation of two specific outer membrane channels has never been described before. KdgN may allow the bacteria to collect oligogalacturonates under saprophytic conditions, when virulence genes, including kdgM , are not expressed.

International audience; 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 d-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. W eeds, pathogens and animal pests are potentially responsible for huge economic losses in agricultural production, and about 20% of these losses are due to animal pests 1. Among insects, aphids (Hemiptera: Aphidoidea) are one of the most injuring taxa for agricultural plants. They are difficult to control due to their specialized feeding mechanism and unusual reproductive biology 2,3. As a result, the management of aphid populations is quite challenging. Until now, most aphid pest control strategies rely on the use of specific sets of systemic chemical pesticides. But the extensive use of these pesticides had led to resistance to insecticides in several aphid species 4,5 , and cause significant environmental damage by targeting different guilds of beneficial insects (predators, parasitoids, and pollinators) 6,7. Therefore, it is highly desirable to develop biopesticides with low non-target effects. A substitute to current chemical pesticides is plant bioengineering; in order to be more selective to target pests, plants can be genetically modified to express insecticidal biomolecules within specific tissues 8. Nevertheless, as happened with conventional pesticides, plant bioengineering has also led to some field insect resistance 9. Hence, crops with more than one defensive protein, each with a different mechanism of action, have been proposed to delay insect resistance (gene pyramiding). However, very few genetically modified plants have yet been developed with resistance to sap-sucking insects, and none is used commercially 10–12. Therefore it seems essential to further develop suitable biopesticides, which could turn into candidate genes for the development of aphid-tolerant plants. In the track of such biopesticides, bacterial toxins did provide paradigmatic solutions, such as the crystal toxins encoded in plasmids of the soil bacterium Bacillus thuringiensis (Bt), which provided a vast diversity of Cry-like toxins for plant protection. Recently however, Bt-related toxins were found in genomes outside its original Gram1 bacterium: four genes namely cytA, cytB, cytC and cytD, encoding proteins homologous to Bacillus thuringiensis (Bt) Cyt toxins have been identified in the Dickeya dadantii (formerly Erwinia chrysanthemi) genome 13

Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA 83 ). In the absence of human donors, we immunized a macaque ( Macaca fascicularis ) with PA 83 to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (10 7 clones). We isolated a high-affinity (equilibrium dissociation constant [ K D ], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 ± 0.13 nM) Fab (designated 35PA 83 ) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA 83 and is part of the region which directly interacts with the cell receptor. 35PA 83 may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA 83 were similar to those of a human immunoglobulin germ line and were assigned to subgroups of human V, (D), or J genes by IMGT/V-QUEST analysis. The 35PA 83 framework regions were 92% identical to a representative allele of each subgroup. When compared to framework regions coded by related human germ line genes, only 2 of 74 (VH) or 75 (VK) analyzed amino acids of 35PA 83 have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA 83 well suited for expression as a whole primatized immunoglobulin G and demonstrates the practicality of using macaque Fabs when immunized human plasma cell donors are not available.

The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB , a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli . We show that nanC expression is induced by N -acetylneuraminic acid and modulated by N -acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control fimB expression. nanC expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an ompR background, where the general porins OmpF and OmpC are absent, NanC is required for growth of E. coli on N -acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an N -acetylneuraminic acid outer membrane channel protein.

The phytopathogenic Gram-negative bacteriumDickeya dadantii(Erwinia chrysanthemi) feeds on plant cell walls by secreting pectinases and utilizing the oligogalacturanate products. An outer membrane porin, KdgM, is indispensable for the uptake of these acidic oligosaccharides. Here, the crystal structure of KdgM determined to 1.9 Å resolution is presented. KdgM is folded into a regular 12-stranded antiparallel β-barrel with a circular cross-section defining a transmembrane pore with a minimal radius of 3.1 Å. Most of the loops that would face the cell exteriorin vivoare disordered, but nevertheless mediate contact between densely packed membrane-like layers in the crystal. The channel is lined by two tracks of arginine residues facing each other across the pore, a feature that is conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.

International audience; 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.

Two title compounds, methyl 3-deoxy- l -threo-2-hexulosonate 3 and methyl 3,5-dideoxy- d -glycero-2-hexulosonate 4, were prepared from d -glucono-1,5-lactone and proved to be gratuitous inducers of the expression of pectinase genes in the phytopathogenic bacteria Erwinia chrysanthemi.

The twin arginine translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii , the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of β-barrel proteins and lipoproteins. Thus, PnlH represents a new kind of outer membrane protein. In Escherichia coli , periplasmic chaperones SurA, Skp, and DegP work together with the β-barrel assembly machinery (Bam) to target and insert β-barrel proteins into the outer membrane. In this work, we showed that SurA is required for an efficient targeting of PnlH to the outer membrane. Moreover, we were able to detect an in vitro interaction between SurA and the PnlH signal sequence. Since the PnlH signal sequence contains a highly hydrophobic region, we propose that SurA protects it from the hydrophobic periplasm during targeting of PnlH to the outer membrane. We also studied the nature of the information carried by the PnlH signal sequence responsible for its targeting to the outer membrane after exportation by the Tat system.

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that “hydrobacteria” and “terrabacteria” might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land., Author Summary Genome sequencing and analysis of plant-associated beneficial soil bacteria Azospirillum spp. reveals that these organisms transitioned from aquatic to terrestrial environments significantly later than the suggested major Precambrian divergence of aquatic and terrestrial bacteria. Separation of Azospirillum from their close aquatic relatives coincided with the emergence of vascular plants on land. Nearly half of the Azospirillum genome has been acquired horizontally, from distantly related terrestrial bacteria. The majority of horizontally acquired genes encode functions that are critical for adaptation to the rhizosphere and interaction with host plants.

International audience; Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.

The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque ( Macaca fascicularis ) and was obtained by phage display. Panning of the library of 1.8 × 10 8 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ± 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

Dickeya dadantii ( Erwinia chrysanthemi ) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis , which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum . The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum , and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum , highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described ( Erwinia aphidicola ), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae . Previously characterized gut symbionts from thrips ( Erwinia / Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species ( Drosophila melanogaster , Sitophilus oryzae , and Spodoptera littoralis ). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.

Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls. We initiated a proteomic analysis to create a reference map of the E. chrysanthemi secretome. Extracellular proteins were isolated from E. chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis. By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified. In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis. We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris. After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX. In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD. Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system. The type I system Prt was necessary for the secretion of three proteases. No proteins secreted by the type III Hrp system could be detected in E. chrysanthemi supernatants. In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB, codes for a 433-amino-acid protein. The PemB N-terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB-like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non-ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.

The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system. Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other. We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins. We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins. The secretin is the major determinant of specificity of the Out system: OutD of E. carotovora changes the secretion specificity of E. chrysanthemi and enables it to secrete heterologous exoproteins. Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin. Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins. Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD. This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD.

OutB is a component of the Erwinia chrysanthemi Out secretion machinery. Homologues of OutB have been described in two other bacteria, Klebsiella oxytoca and Aeromonas hydrophila, but their requirement in the secretion process seems to be different. Study of OutB topology with the BlaM topology probe suggests that it is an inner-membrane protein with a large periplasmic domain. However, fractionation experiments indicate that it could be associated with the outer membrane through its C-terminal part. The secretion deficiency of an Erw. chrysanthemi outB mutant can be reversed by the addition of an inducer of the kdgR regulon. It was shown that this effect results from the increased expression of the secretin OutD and that secretion can be restored in an outB mutant by introducing the outD gene on a plasmid. Several experiments suggest an interaction between OutB and OutD. In Erw. chrysanthemi, the presence of OutD stabilizes OutB. OutD expressed in Escherichia coli can be protected from proteolytic degradation by the coexpression of OutB. This effect does not require the N-terminal, transmembrane segment of outB. OutB can be cross-linked with OutD by formaldehyde. These results indicate that OutB could act with OutD in the functioning of the Out secretion machinery.

The chromosome size of the principal intracellular symbiotic bacteria (endocytobiotes) of the weevil Sitophilus oryzae has been determined by pulsed field gel electrophoresis (PFGE). The restriction enzymes SpeI, SwaI and XbaI generated 22, 23 and 24 fragments, respectively. The fragment sizes ranged from 15 to 460 kb and the genome size of the symbiotic bacteria was estimated to be approximately 3.0 Mb. An additional fragment was seen when running total undigested DNA and should correspond to a plasmid of 138 kb. The principal symbiotic bacteria belong to the enterobacteriaceae family and are closely related to Escherichia coli . The difference between the genome size of E. coli (4.7 Mb) and symbiotic bacteria (3.0 Mb) was discussed and might be related to the intracellular evolution of the endocytobiotes within their host. No more than two rDNA (23S) loci were found with I-CeuI digestion, which argues against a serial fragment gene deletion in the course of evolution.

Erwinia chrysanthemi is an enterobacterium that causes various plant diseases. Its pathogenicity results from the secretion of pectinolytic enzymes responsible for the disorganization of the plant cell wall. The E. chrysanthemi strain 3937 produces two pectin methylesterases, at least seven pectate lyases, a polygalacturonase, and a pectin lyase. The extracellular degradation of the pectin leads to the formation of oligogalacturonides that are catabolized through an intracellular pathway. The pectinase genes are expressed from independent cistrons, and their transcription is favored by environmental conditions such as presence of pectin and plant extracts, stationary growth phase, low temperature, oxygen or iron limitation, and so on. Moreover, transcription of the pectin lyase gene responds to DNA-damaging agents. The differential expressions of individual pectinase genes presumably reflect their role during plant infection. The regulation of pel genes requires several regulatory systems, including the KdgR repressor, which mediates the induction of all the pectinolysis genes in the presence of pectin catabolites. KdgR also controls the genes necessary for pectinase secretion and other pectin-inducible genes not yet characterized. PecS, a cytoplasmic protein homologous to other transcriptional regulators, can bind in vitro to the regulatory regions of pectinase and cellulase genes. The PecT protein, a member of the LysR family of transcriptional regulators, represses the expression of some pectinase genes and also affects other metabolic pathways of the bacteria. Other proteins involved in global regulations, such as CRP or HNS, can bind to the regulatory regions of the pectinase genes and affect their transcription.

The bacteria Erwinia chrysanthemi secretes in the extracellular medium several pectinases of different types. A gene coding for a novel pectin methylesterase pemB has been cloned from an E. chrysanthemi strain 3937 gene library. The PemB sequence presents homology with other pectin methylesterases from bacteria and plants. PemB has been characterised as a bacterial lipoprotein. PemB is localised in the outer membrane and is not released into the extracellular medium. PemB was overproduced in E. coli and purified to homogeneity. It is activated by non-ionic detergents. PemB has a highest activity on methylated oligogalacturonides and this enzyme is necessary for the growth of the bacteria on this substrate as carbon source.

Four title compounds, 5-deoxy KDG Me ester, 5-epi KDG Me ester, 4-O-Me KDG Me ester and 4-deoxy KDG Me ester were prepared either from d-glucono-1,5-lactone or from 1,2:5,6 di-O-isopropylidene-d-mannitol. Biological tests performed on these molecules have shown that the compounds modified on the C-5 position (5-deoxy KDG Me ester and 5-epi KDG Me ester) are gratuitous inducers of the expression of pectinase genes in the phytopathogenic bacteria Erwinia Chrysanthemi when the C-4 modified molecules (4-O-Me KDG Me ester and 4-deoxy KDG Me ester) are not inducers.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.

Tn5 mutagenesis was performed on the Erwinia chrysanthemi strain 3937 with the transposon vector pMO194. The vector is spontaneously lost from the cell and Tn5 transposes randomly on the chromosome. A mutant with a derepressed expression of a kduD-lac fusion was obtained. In this mutant, Tn5 was inserted in a regulatory gene, named kdgR, resulting in constitutive expression of genes of the polygalacturonate degradative pathway (pelA, pelD, pelE, ogl, kduI, kduD, kdgT, kdgK and kdgA). Constitutive expression of these genes after inactivation of the kdgR gene by Tn5 insertion suggests that kdgR is a negative regulatory gene. The kdgR mutation was mapped near the trp locus on the E. chrysanthemi chromosome.

SUMMARY: 2-Keto-3-deoxygluconate : NAD 5-oxidoreductase (EC 1.1.1.127) is an enzyme used in pectinolysis by some phytopathogenic bacteria such as Pseudomonas and Erwinia. It converts 2,5-diketo-3-deoxygluconate (DKII) into 2-keto-3-deoxygluconate (KDG) with a concomitant oxidation of NADH. The reaction is reversible in the presence of NAD. The oxidoreductase isolated from Erwinia chrysanthemi was purified 40-fold by protamine treatment, (NH4)2SO4 fractionation, chromatography on DEAE-Sephadex and filtration on Sephadex G-200. The enzyme appeared relatively stable. There was no effect of EDTA or of added ions except Ag+. This ion and the thiol reagent p-chloromercuribenzoate inhibited the enzyme. Other uronic acids, sugars and organic acids tested were neither substrates nor inhibitors of its activity. The optimum pH was 10 for the oxidation of KDG and 7.5 for the reduction of DKII. Both oxidation and reduction reactions showed saturation kinetics with apparent K m values of 7.7 × 10-3 M for KDG and 4 × 10-4 M for NAD (at 37 °C, pH 8.6), and of 3.4 × 10-4 M for DKII and 0.3 × 10-4 M for NADH (at 37 °C and pH 7.0). The enzyme reaction was strongly inhibited by NADH at concentrations higher than 2 × 10-4 M.

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.

Pectate-lyases (PL) are extracellular enzymes secreted by Erwinia chysanthemi. By their attack of pectin, a constituent of plant cell walls, they are the major factor involved in the phytopathogenicity of this bacterial species. We described the isolation and characterization of mutants in the regulatory systems controlling the synthesis of PL. The regulation of these proteins appeared complex and submitted to at least two important controls. Three classes of mutants constitutively synthesizing PL were found after a chemical mutagenesis of the wild-type strain B374. The cri mutations (class IV) conferred a resistance to the catabolite repression exerted by glucose to various proteins of the cell such as PL, cellulases, proteases, B-galactosidase and the hexuronate transport system. In the cri mutants the synthesis of PL remained inducible by pectin degradative products but were no longer repressible in presence of glucose. In contrast, the gpiR mutants (class I) were always sensitive to glucose repression but no longer inducible in presence of pectin derivatives. In these mutants, the regulatory gene responsible for the induction by pectin derivatives was inactivated. This gene is probably gpiR itself. Moreover the gpiR mutation led to a synthesis of PL independent of the growth phase. In liquid cultures of E. chrysanthemi, PL synthesis increases only at the end of the exponential growth, while in the gpiR mutants the PL synthesis is proportional to the cell density.

The phytopathogenicity of Erwinia chrysanthemi seems to be related to its pectinolytic and cellulolytic activities. Polygalacturonate (PGA) can be degraded into galacturonate or 5-keto-4-deoxyuronate (DKI); 2-keto-3-deoxygluconate (KDG) is a common intermediate in the catabolism of these two compounds. The galacturonate catabolic pathway, first described in E. carotovora, is similar to that existing in Escherichia coli. As described for the PGA metabolism of a phytopathogenic Pseudomonas DKI is isomerized to 2,5-diketo-3-deoxygluconate (DKII) and then reduced to KDG. The enzyme 2-keto-3-deoxygluconate 2 NAD-oxidoreductase (KDGDH) that converts DKII to KDG has been purified and its biochemical properties analyzed. Mutants impaired in pectin degradation were isolated by chemical mutagenesis and Mu d (Ap lac) insertion. A mutation in the kduD gene coding for KDGDH prevented the growth of the bacteria on polygalacturonate. In the kduD-lac fusion strains, resulting from Mu d (Ap lac) insertion, β-galactosidase was induced by polygalacturonate, galacturonate and other intermediates of polygalacturonate catabolism. Galacturonate was inducer unless a mutation prevented its metabolization to KDG. Our results suggested that KDG and DKII are the true inducers of the synthesis of KDGDH and pectate lyases.

Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi . The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY of Yersinia pseudotuberculosis and pelB of Erwinia carotovora , which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW . PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca 2+ is unable to restore the activity of PelW or Ogl, while several other cations, including Co 2+ , Mn 2+ , and Ni 2+ , can activate both cytoplasmic lyases.