RESUMEN

BACKGROUND: Erwinia chrysanthemi (Ec) is a destructive pathogen which causes soft-rot diseases in diverse plant species including orchids. We investigated whether colonization of Oncidium roots by the endophytic fungus Piriformospora indica (Pi) restricts Ec-induced disease development in leaves, and whether this might be related to the regulation of nucleotide binding site-leucine rich repeat (NBS-LRR) Resistance (R) genes. RESULTS: Root colonization of Oncidium stackings by Pi restricts progression of Ec-induced disease development in the leaves. Since Pi does not inhibit Ec growth on agar plates, we tested whether NBS-LRR R gene transcripts and the levels of their potential target miRNAs in Oncidium leaves might be regulated by Pi. Using bioinformatic tools, we first identified NBS-LRR R gene sequences from Oncidium, which are predicted to be targets of miRNAs. Among them, the expression of two R genes was repressed and the accumulation of several regulatory miRNA stimulated by Ec in the leaves of Oncidium plants. This correlated with the progression of disease development, jasmonic and salicylic acid accumulation, ethylene synthesis and H2O2 production after Ec infection of Oncidium leaves. Interestingly, root colonization by Pi restricted disease development in the leaves, and this was accompanied by higher expression levels of several defense-related R genes and lower expression level of their target miRNA. CONCLUSION: Based on these data we propose that Pi controls the levels of NBS-LRR R mRNAs and their target miRNAs in leaves. This regulatory circuit correlates with the protection of Oncidium plants against Ec infection, and molecular and biochemical investigations will demonstrate in the future whether, and if so, to what extent these two observations are related to each other.

Asunto(s)

Basidiomycota/fisiología , Dickeya chrysanthemi/fisiología , Genes de Plantas , Orchidaceae/genética , Orchidaceae/microbiología , Enfermedades de las Plantas/microbiología , Endófitos/fisiología , MicroARNs/genética , MicroARNs/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Raíces de Plantas/genética , Raíces de Plantas/microbiología , ARN de Planta/genética , ARN de Planta/metabolismo

RESUMEN

N-Acyl-homoserine lactones (AHLs)-dependent quorum sensing (QS) system(s) is recruited by the soft rot bacterium Dickeya chrysanthemi for coordinating its social activities such as secretion of plant cell wall-degrading enzymes, while the main signal molecule and quantity dependence of virulence to QS in this bacterium have not been clarified. To do this end, the involvement of AHLs in African violet leaves and potato tuber maceration; swarming motility; pectate lyase and polygalacturonase enzymes production and in planta expression of virulence genes including pelE, pehX and pemA by electroporating two quorum-quenching vectors. The expression of two types of AHL-lactonase expressing vector caused dramatic decrease in swarming motility, production of pectinolytic enzymes and macerating of plant tissues. The maximum ability of quenching of QS in repression of D. chrysanthemi virulence was assessed quantitatively by q-RT-PCR, as expression of pelE, pehX and pemA genes were decreased 90.5-92.18 % in quenched cells. We also showed that virulence and pathogenicity of this bacterium was under the control of DHL-dependent QS system and that the existence of second DHL operating system is probable for this bacterium. Thus, this signal molecule would be the key point for future research to design DHL-specific lactonase enzymes using bioinformatics methods.

Asunto(s)

Proteínas Bacterianas/genética , Dickeya chrysanthemi/genética , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/microbiología , Percepción de Quorum , Solanum tuberosum/microbiología , Factores de Virulencia/genética , Acil-Butirolactonas/metabolismo , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Dickeya chrysanthemi/fisiología , Poligalacturonasa/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Factores de Virulencia/metabolismo

RESUMEN

UNLABELLED: Recent studies strongly suggest that the gene expression sustaining both normal and pathogenic bacterial growth is governed by the structural dynamics of the chromosome. However, the mechanistic device coordinating the chromosomal configuration with selective expression of the adaptive traits remains largely unknown. We used a holistic approach exploring the inherent relationships between the physicochemical properties of the DNA and the expression of adaptive traits, including virulence factors, in the pathogen Dickeya dadantii (formerly Erwinia chrysanthemi). In the transcriptomes obtained under adverse conditions encountered during bacterial infection, we explored the patterns of chromosomal DNA sequence organization, supercoil dynamics, and gene expression densities, together with the long-range regulatory impacts of the abundant DNA architectural proteins implicated in pathogenicity control. By integrating these data, we identified transient chromosomal domains of coherent gene expression featuring distinct couplings between DNA thermodynamic stability, supercoil dynamics, and virulence traits. IMPORTANCE: We infer that the organization of transient chromosomal domains serving specific functions acts as a fundamental device for versatile adjustment of the pathogen to environmental stress. We believe that the identification of chromosomal "stress-response" domains harboring distinct virulence traits and mediating the cellular adaptive behavior provides a breakthrough in understanding the control mechanisms of bacterial pathogenicity.

Asunto(s)

ADN Bacteriano/genética , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/patogenicidad , Regulación Bacteriana de la Expresión Génica , Estrés Fisiológico , Factores de Virulencia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , ADN Bacteriano/química , ADN Superhelicoidal , Dickeya chrysanthemi/fisiología , Genes Bacterianos , Lamiales/microbiología , Transcriptoma , Virulencia , Factores de Virulencia/metabolismo

RESUMEN

Bacteria from the genus Dickeya (formerly Erwinia chrysanthemi) are plant pathogens causing severe diseases in many economically important crops. A majority of the strains responsible for potato disease in Europe belong to a newly identified Dickeya solani species. Although some ecological and epidemiological studies have been carried out, little is known about the regulation of D. solani virulence. The characterization of four D. solani strains indicates significant differences in their virulence on potato, although they are genetically similar based on genomic fingerprinting profiles. A phenotypic examination included an analysis of virulence on potato; growth rate in culture; motility; Fe3+ chelation; and pectate lyase, cellulase, protease, biosurfactant, and blue pigment production. Mutants of four D. solani strains were constructed by inactivating the genes coding either for one of the main negative regulators of D. dadantii virulence (kdgR, pecS, and pecT) or for the synthesis and perception of signaling molecules (expI and expR). Analysis of these mutants indicated that PecS, PecT, and KdgR play a similar role in both species, repressing, to different degrees, the synthesis of virulence factors. The thermoregulator PecT seems to be a major regulator of D. solani virulence. This work also reveals the role of quorum sensing mediated by ExpI and ExpR in D. solani virulence on potato.

Asunto(s)

Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/fisiología , Dickeya chrysanthemi/patogenicidad , Regulación Bacteriana de la Expresión Génica/fisiología , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/genética , Bacteriófagos , Cichorium intybus/microbiología , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/virología , Solanum tuberosum/microbiología , Virulencia

RESUMEN

The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinic-receptor superfamily.

Asunto(s)

Proteínas Bacterianas/genética , Dickeya chrysanthemi/genética , Canales Iónicos Activados por Ligandos/genética , Mutación , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Cristalografía por Rayos X , Cisteamina/farmacología , Dickeya chrysanthemi/fisiología , Femenino , Células HEK293 , Humanos , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/fisiología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Modelos Moleculares , Oocitos/metabolismo , Oocitos/fisiología , Conformación Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Factores de Tiempo , Xenopus laevis

RESUMEN

Dickeya dadantii (syn. Erwinia chrysanthemi) is a plant pathogenic bacteria that harbours a cluster of four horizontally-transferred, insect-specific toxin genes. It was recently shown to be capable of causing an acute infection in the pea aphid Acyrthosiphon pisum (Insecta: Hemiptera). The infection route of the pathogen, and the role and in vivo expression pattern of these toxins, remain unknown. Using bacterial numeration and immunolocalization, we investigated the kinetics and the pattern of infection of this phytopathogenic bacterium within its insect host. We compared infection by the wild-type strain and by the Cyt toxin-deficient mutant. D. dadantii was found to form dense clusters in many luminal parts of the aphid intestinal tract, including the stomach, from which it invaded internal tissues as early as day 1 post-infection. Septicemia occurred soon after, with the fat body being the main infected tissue, together with numerous early infections of the embryonic chains showing embryonic gut and fat body as the target organs. Generalized septicemia led to insect death when the bacterial load reached about 10(8) cfu. Some individual aphids regularly escaped infection, indicating an effective partial immune response to this bacteria. Cyt-defective mutants killed insects more slowly but were capable of localisation in any type of tissue. Cyt toxin expression appeared to be restricted to the digestive tract where it probably assisted in crossing over the first cell barrier and, thus, accelerating bacterial diffusion into the aphid haemocel. Finally, the presence of bacteria on the surface of leaves hosting infected aphids indicated that the insects could be vectors of the bacteria.

Asunto(s)

Áfidos/microbiología , Toxinas Bacterianas/metabolismo , Dickeya chrysanthemi/fisiología , Infecciones por Enterobacteriaceae/microbiología , Sepsis/microbiología , Animales , Animales Modificados Genéticamente , Áfidos/embriología , Áfidos/genética , Áfidos/fisiología , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Dickeya chrysanthemi/patogenicidad , Vectores de Enfermedades , Embrión no Mamífero/microbiología , Endotoxinas/genética , Endotoxinas/metabolismo , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/veterinaria , Regulación de la Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Intestinos/embriología , Intestinos/microbiología , Pisum sativum/parasitología , Enfermedades de las Plantas/microbiología , Sepsis/genética , Sepsis/veterinaria

RESUMEN

During infection, the phytopathogenic enterobacterium Dickeya dadantii has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. A tight control of the bacterial intracellular iron content is necessary for full virulence of D. dadantii: previous studies have shown that the ferritin FtnA and the bacterioferrtin Bfr, devoted to iron storage, contribute differentially to the virulence of this species. In this work, we investigated the role of the Dps miniferritin in iron homeostasis in D. dadantii. We constructed a Dps-deficient mutant by reverse genetics. This mutant grew like the wild-type stain under iron starvation and showed no decreased iron content. However, the dps mutant displayed an increased sensitivity to hydrogen peroxide in comparison to the wild-type strain. This hydrogen peroxide susceptibility only occurs when bacteria are in the stationary phase. Unlike the bfr and the ftnA mutants, the dps mutant is not affected in its pathogenicity on host plants. The dps gene expression is induced at the stationary phase of growth. The Sigma S transcriptional factor is necessary for this control. Furthermore, dps expression is positively regulated by the oxidative stress response regulator OxyR during the exponential growth phase, after hydrogen peroxide treatment. These results indicate that the Dps miniferritin from D. dadantii has a minor role in iron homeostasis, but is important in conferring tolerance to hydrogen peroxide and for survival of cells that enter the stationary phase of growth.

Asunto(s)

Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Dickeya chrysanthemi/fisiología , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Unión al ADN/genética , Ferritinas/genética , Hierro/metabolismo , Datos de Secuencia Molecular , Mutación , Estrés Oxidativo

RESUMEN

ABSTRACT Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

Asunto(s)

Dickeya chrysanthemi/fisiología , Interacciones Huésped-Patógeno , Microbiología del Suelo , Solanum tuberosum/microbiología , Dickeya chrysanthemi/patogenicidad , Violeta de Genciana , Proteínas Fluorescentes Verdes/genética , Microscopía Confocal , Enfermedades de las Plantas/microbiología , Tubérculos de la Planta/microbiología , Virulencia

RESUMEN

Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94%), cheY (80%), cheW (74%), cheB (54%) and cheZ (48%). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA

Asunto(s)

Quimiotaxis , Dickeya chrysanthemi/fisiología , Dickeya chrysanthemi/patogenicidad , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Flagelos/genética , Flagelos/metabolismo , Hojas de la Planta/microbiología , Transducción de Señal , Virulencia

RESUMEN

Pathogenicity of the enterobacterium Erwinia chrysanthemi (Dickeya dadantii), the causative agent of soft-rot disease in many plants, is a complex process involving several factors whose production is subject to temporal regulation during infection. PecS is a transcriptional regulator that controls production of various virulence factors. Here, we used microarray analysis to define the PecS regulon and demonstrated that PecS notably regulates a wide range of genes that could be linked to pathogenicity and to a group of genes concerned with evading host defenses. Among the targets are the genes encoding plant cell wall-degrading enzymes and secretion systems and the genes involved in flagellar biosynthesis, biosurfactant production, and the oxidative stress response, as well as genes encoding toxin-like factors such as NipE and hemolysin-coregulated proteins. In vitro experiments demonstrated that PecS interacts with the regulatory regions of five new targets: an oxidative stress response gene (ahpC), a biosurfactant synthesis gene (rhlA), and genes encoding exported proteins related to other plant-associated bacterial proteins (nipE, virK, and avrL). The pecS mutant provokes symptoms more rapidly and with more efficiency than the wild-type strain, indicating that PecS plays a critical role in the switch from the asymptomatic phase to the symptomatic phase. Based on this, we propose that the temporal regulation of the different groups of genes required for the asymptomatic phase and the symptomatic phase is, in part, the result of a gradual modulation of PecS activity triggered during infection in response to changes in environmental conditions emerging from the interaction between both partners.

Asunto(s)

Proteínas Bacterianas/fisiología , Dickeya chrysanthemi/fisiología , Magnoliopsida/microbiología , Proteínas Represoras/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Huella de ADN , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

The goal of this study is to investigate the impact of biofilm physical and biological properties on bacterial transport and deposition in porous media. Experiments were performed in packed columns to examine the removal of Erwinia chrysanthemi (Ech3937), a phytopathogen, from the bulk fluid due to its attachmentto glass beads coated with Pseudomonas aeruginosa biofilms. Two isogenic P. aeruginosa strains, PAO1 and PD0300, with different EPS secretion capabilities and EPS compositions, were used to culture biofilms. The Ech3937 transport and distribution in packed columns were studied in both upflow and downflow cell injection modes over a range of solution ionic strengths. The results show that the presence of biofilm strongly interferes with the deposition behavior of Ech3937 in porous media. The spatial variation of deposited Ech3937 cells contradicts the log-linear pattern predicted by the classic filtration theory, indicating that the biofilm physical structure and polymeric interactions between the biofilm EPS and Ech3937 cell surface are the main mechanisms that control bacterial deposition. When the biofilm accumulation is relatively small, bacterial adhesion onto biofilm-coated porous media is mainly inhibited by steric forces. By contrast, cell deposition is enhanced by the reduced porous media porosity when biofilm is more abundant.

Asunto(s)

Biopelículas , Dickeya chrysanthemi/fisiología , Pseudomonas aeruginosa/fisiología , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Concentración Osmolar , Plantas/parasitología , Porosidad , Ácidos Urónicos/metabolismo

RESUMEN

Pathogenicity of the phytopathogenic enterobacterium Erwinia chrysanthemi, the causal agent of soft rot disease on many plants, is a complex process involving several factors whose production is regulated by a complex, intertwined regulatory network. In this work we characterized the GacA regulator, member of the GacS-GacA two-component system, as a global regulator which is required for disease expression but not for bacterial multiplication in planta during the first stages of the plant infection. GacA was shown to control the expression of plant cell wall-degrading enzymes and hrp genes in vitro. Analysis of virulence gene expression during infection of Arabidopsis thaliana revealed a coordinated expression of these virulence genes at 12 h post infection and showed that GacA is required for the appropriate production of virulence factors in planta. GacA might partly act by negatively controlling the expression of the pecT gene encoding the global repressor PecT, indicating a hierarchy in the pathways involved in the E. chrysanthemi regulatory network.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Dickeya chrysanthemi/patogenicidad , Genes Reguladores , Enfermedades de las Plantas/microbiología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Factores de Virulencia/genética , Dickeya chrysanthemi/fisiología , Regulación Bacteriana de la Expresión Génica , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Virulencia/fisiología

RESUMEN

Erwinia chrysanthemi pv. zeae is one of the Erwinia chrysanthemi pathovars that infects on both dicotyledons and monocotyledons. However, little is known about the molecular basis and regulatory mechanisms of its virulence. By using a transposon mutagenesis approach, we cloned the genes coding for an E. chrysanthemi pv. zeae synthase of acyl-homoserine lactone (AHL) quorum-sensing signals (expI(Ecz)) and a cognate response regulator (expR(Ecz)). Chromatography analysis showed that expI(Ecz) encoded production of the AHL signal N-(3-oxo-hexanoyl)-homoserine lactone (OHHL). Null mutation of expI(Ecz) in the E. chrysanthemi pv. zeae strain EC1 abolished AHL production, increased bacterial swimming and swarming motility, disabled formation of multicell aggregates, and attenuated virulence of the pathogen on potato tubers. The mutation also marginally reduced the inhibitory activity of E. chrysanthemi pv. zeae on rice seed germination. The mutant phenotypes were rescued by either exogenous addition of AHL signal or in trans expression of expI(Ecz). These data demonstrate that the AHL-type QS signal plays an essential role in modulation of E. chrysanthemi pv. zeae cell motility and the ability to form multicell aggregates and is involved in regulation of bacterial virulence.

Asunto(s)

Acil-Butirolactonas/metabolismo , Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/fisiología , Dickeya chrysanthemi/patogenicidad , Regulación Bacteriana de la Expresión Génica , Percepción de Quorum , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/genética , Secuencia de Bases , Movimiento Celular , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Datos de Secuencia Molecular , Oryza/microbiología , Semillas/microbiología , Análisis de Secuencia de ADN , Solanum tuberosum/microbiología , Transactivadores/genética , Transactivadores/metabolismo , Virulencia

RESUMEN

Dickeya dadantii (Erwinia chrysanthemi 3937) secretes exoenzymes, including pectin-degrading enzymes, leading to the loss of structural integrity of plant cell walls. A type III secretion system (T3SS) is essential for full virulence of this bacterium within plant hosts. The GacS/GacA two-component signal transduction system participates in important biological roles in several gram-negative bacteria. In this study, a gacA deletion mutant (Ech137) of D. dadantii was constructed to investigate the effect of this mutation on pathogenesis and other phenotypes. Compared with wild-type D. dadantii, Ech137 had a delayed biofilm-pellicle formation. The production of pectate lyase (Pel), protease, and cellulase was diminished in Ech137 compared with the wild-type cells. Reduced transcription of two endo-Pel genes, pelD and pelL, was found in Ech137 using a green fluorescence protein-based fluorescence-activated cell sorter promoter activity assay. In addition, the transcription of T3SS genes dspE (an effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) was reduced in Ech137. A lower amount of rsmB regulatory RNA was found in gacA mutant Ech137 compared with the wild-type bacterium by quantitative reverse-transcription polymerase chain reaction. Compared with wild-type D. dadantii, a lower amount of hrpL mRNA was observed in Ech137 at 12 h grown in medium. Although the role of RsmA, rsmB, and RsmC in D. dadantii is not clear, from the regulatory pathway revealed in E. carotovora, the lower expression of dspE, hrpA, and hrpN in Ech137 may be due to a post-transcriptional regulation of hrpL through the Gac-Rsm regulatory pathway. Consequently, the reduced exoenzyme production and Pel gene expression in the mutant may be sue partially to the regulatory role of rsmB-RsmA on exoenzyme expression. Similar to in vitro results, a lower expression of T3SS and pectinase genes of Ech137 also was observed in bacterial cells inoculated into Saintpaulia ionantha leaves, perhaps accounting for the observed reduction in local maceration. Interestingly, compared with the wild-type D. dadantii, although a lower concentration of Ech137 was observed at day 3 and 4 postinoculation, there is no significant difference in bacterial concentration between the wild-type bacterium and Ech137 in the early stage of infection. Finally, the nearly abolished systemic invasion ability of Ech137 suggests that GacA of D. dadantii is essential for the pathogenicity and systemic movement of the bacterium in S. ionantha.

Asunto(s)

Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/patogenicidad , Regulación Bacteriana de la Expresión Génica , Poligalacturonasa/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Biopelículas , Dickeya chrysanthemi/enzimología , Dickeya chrysanthemi/fisiología , Redes Reguladoras de Genes , Genes Bacterianos , Magnoliopsida/microbiología , Mutación/genética , Enfermedades de las Plantas/microbiología , Polisacárido Liasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espectrofotometría

RESUMEN

The PhoPQ two-component system regulates virulence factors in Erwinia chrysanthemi, a pectinolytic enterobacterium that causes soft rot in several plant species. We characterized the effect of a mutation in phoQ, the gene encoding the sensor kinase PhoQ of the PhoPQ two-component regulatory system, on the global transcriptional profile of E. chrysanthemi using cDNA microarrays and further confirmed our results by quantitative reverse transcription-PCR analysis. Our results indicate that a mutation in phoQ affects transcription of at least 40 genes, even in the absence of inducing conditions. Enhanced expression of several genes involved in iron metabolism was observed in the mutant, including that of the acs operon that is involved in achromobactin biosynthesis and transport. This siderophore is required for full virulence of E. chrysanthemi, and its expression is governed by the global repressor protein Fur. Changes in gene expression were also observed for membrane transporters, stress-related genes, toxins, and transcriptional regulators. Our results indicate that the PhoPQ system governs the expression of several additional virulence factors and may also be involved in interactions with other regulatory systems.

Asunto(s)

Proteínas Bacterianas/fisiología , Dickeya chrysanthemi/fisiología , Regulación Bacteriana de la Expresión Génica , Factores de Virulencia/biosíntesis , Toxinas Bacterianas/genética , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/patogenicidad , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Bacterianos , Genes Reguladores , Proteínas de Transporte de Membrana/genética , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Operón , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sideróforos/biosíntesis , Sideróforos/genética , Sideróforos/metabolismo , Factores de Virulencia/genética

RESUMEN

Erwinia chrysanthemi 3937 (Ech3937) is a phytopathogenic bacterium with a wide host range. The pectinolytic enzymes secreted by the bacterium and the type III secretion system (T3SS) are essential for full virulence. We used the green fluorescent protein gene as a reporter to investigate the expression of dspE (a putative T3SS effector) and pelD (a major pectin-degrading enzyme) in populations of Ech3937 under different conditions. Gene expression was analyzed by measuring the fluorescence intensity of individual cells with a fluorescence-activated cell sorter. Ech3937 dspE was induced in minimal medium (MM) with only a portion of Ech3937 cells (43.03%) expressing dspE after 12 h of culture. The nutrient-rich King's medium B did not fully eliminate the expression of dspE; a small percentage of Ech3937 cells (5.55%) was able to express dspE after 12 h of culture in this medium. In all, 68.95% of Ech3937 cells expressed pelD after 12 h of culture in MM supplemented with polygalacturonic acid (PGA). However, 96.34% of Echl31 cells (an hrpL deletion mutant of Ech3937) expressed pelD after 12 h of culture in MM supplemented with PGA. In potato tubers, 6.32% of the bacterial cells expressed dspE 2 h after inoculation, whereas only 0.25% of the cells expressed pelD. However, after 24 h, the percentage of cells expressing pelD (68.48%) was approximately 3.5 times that of cells expressing dspE (19.39%). In contrast to potato tubers, similar proportion of Ech3937 cells expressing dspE (39.34%) and pelD (40.30%) were observed in Chinese cabbage 24 h after inoculation. From promoter activity and real-time quantitative results, the expression of pelD in Ech3937 was demonstrated to be downregulated by HrpL in MM supplemented with PGA.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/fisiología , Regulación Bacteriana de la Expresión Génica , Polisacárido Liasas/metabolismo , Brassica/microbiología , Tubérculos de la Planta/microbiología , Polisacárido Liasas/genética , Regiones Promotoras Genéticas , Solanum tuberosum/microbiología

RESUMEN

The hypersensitive response elicitor harpin (HrpN) of soft rot pathogen Erwinia chrysanthemi strains 3937 and EC16 is secreted via the type III secretion system and remains cell surface bound. Strain 3937 HrpN is essential for cell aggregation, but the C-terminal one-third of the protein is not required for aggregative activity.

Asunto(s)

Adhesión Bacteriana/fisiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Dickeya chrysanthemi/fisiología , Secuencia de Aminoácidos , Adhesión Bacteriana/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Homología de Secuencia de Aminoácido

RESUMEN

The tol-pal genes are necessary for maintaining the outer-membrane integrity of Gram-negative bacteria. These genes were first described in Escherichia coli, and more recently in several other species. They are involved in the pathogenesis of E. coli, Haemophilus ducreyi, Vibrio cholerae and Salmonella enterica. The role of the tol-pal genes in bacterial pathogenesis was investigated in the phytopathogenic enterobacterium Erwinia chrysanthemi, assuming that this organism might be a good model for such a study. The whole Er. chrysanthemi tol-pal region was characterized. Tol-Pal proteins, except TolA, showed high identity scores with their E. coli homologues. Er. chrysanthemi mutants were constructed by introducing a uidA-kan cassette in the ybgC, tolQ, tolA, tolB, pal and ybgF genes. All the mutants were hypersensitive to bile salts. Mutations in tolQ, tolA, tolB and pal were deleterious for the bacteria, which required high concentrations of sugars or osmoprotectants for their viability. Consistent with this observation, they were greatly impaired in their cell morphology and division, which was evidenced by observations of cell filaments, spherical forms, membrane blebbing and mislocalized bacterial septa. Moreover, tol-pal mutants showed a reduced virulence in a potato tuber model and on chicory leaves. This could be explained by a combination of impaired phenotypes in the tol-pal mutants, such as reduced growth and motility and a decreased production of pectate lyases, the major virulence factor of Er. chrysanthemi.

Asunto(s)

Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Dickeya chrysanthemi/patogenicidad , Dickeya chrysanthemi/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Cichorium intybus/microbiología , Clonación Molecular , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/fisiología , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Movimiento , Mutación , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Raíces de Plantas/microbiología , Análisis de Secuencia de ADN , Solanum tuberosum/microbiología , Virulencia

RESUMEN

A collection of 75 strains of Pectobacterium chrysanthemi (including all biovars and pathovars) and the type strains of Brenneria paradisiaca (CFBP 4178(T)) and Pectobacterium cypripedii (CFBP 3613(T)) were studied by DNA-DNA hybridization, numerical taxonomy of 121 phenotypic characteristics, serology and 16S rRNA gene-based phylogenetic analyses. From analysis of 16S rRNA gene sequences, it was deduced that P. chrysanthemi strains and B. paradisiaca CFBP 4178(T) formed a clade distinct from the genera Pectobacterium and Brenneria; therefore, it is proposed to transfer all the strains to a novel genus, Dickeya gen. nov. By DNA-DNA hybridization, the strains of P. chrysanthemi were distributed among six genomic species: genomospecies 1 harbouring 16 strains of biovar 3 and four strains of biovar 8, genomospecies 2 harbouring 16 strains of biovar 3, genomospecies 3 harbouring two strains of biovar 6 and five strains of biovar 5, genomospecies 4 harbouring five strains of biovar 2, genomospecies 5 harbouring six strains of biovar 1, four strains of biovar 7 and five strains of biovar 9 and genomospecies 6 harbouring five strains of biovar 4 and B. paradisiaca CFBP 4178(T). Two strains of biovar 3 remained unclustered. Biochemical criteria, deduced from a numerical taxonomic study of phenotypic characteristics, and serological reactions allowed discrimination of the strains belonging to the six genomic species. Thus, it is proposed that the strains clustered in these six genomic species be assigned to the species Dickeya zeae sp. nov. (type strain CFBP 2052(T)=NCPPB 2538(T)), Dickeya dadantii sp. nov. (type strain CFBP 1269(T)=NCPPB 898(T)), Dickeya chrysanthemi comb. nov. (subdivided into two biovars, bv. chrysanthemi and bv. parthenii), Dickeya dieffenbachiae sp. nov. (type strain CFBP 2051(T)=NCPPB 2976(T)), Dickeya dianthicola sp. nov. (type strain CFBP 1200(T)=NCPPB 453(T)) and Dickeya paradisiaca comb. nov., respectively.

Asunto(s)

Dickeya chrysanthemi/clasificación , Enterobacteriaceae/clasificación , Animales , Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/fisiología , Enterobacteriaceae/genética , Enterobacteriaceae/fisiología , Genes de ARNr , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Conejos , Serotipificación

RESUMEN

Ferritins are multimeric iron storage proteins encoded by a four-member gene family in Arabidopsis (AtFer1-4). To investigate whether iron sequestration in ferritins is a part of an iron-withholding defense system induced in response to bacterial invasion, we used Arabidopsis thaliana as a susceptible host for the pathogenic bacterium Erwinia chrysanthemi. In this study, we used a T-DNA insertion mutant line to show that the lack of a functional AtFer1 gene resulted in an enhanced susceptibility of Arabidopsis plants to E. chrysanthemi. We found that the AtFer1 gene is upregulated during infection, with a biphasic accumulation of the transcript at critical time points 0.5 and 24 h post-infection (p.i.). The activation of AtFer1 expression observed at 24 h p.i. was independent of the iron-dependent regulatory sequence (IDRS) known to mediate the transcriptional response of the AtFer1 gene to iron excess and to nitric oxide. Upregulation of AtFer1 gene expression was compromised after inoculation with an E. chrysanthemi siderophore null mutant. Infiltration of the purified siderophores chrysobactin and desferrioxamine strongly increased AtFer1 transcript abundance and it did not occur with the iron-loaded forms of these siderophores. We found that neither oxidative stress nor nitric oxide was involved in the plant response to chrysobactin. Our data show that ferritin accumulation during infection of Arabidopsis by E. chrysanthemi is a basal defense mechanism which is mainly activated by bacterial siderophores. The potential role of siderophores in this process is discussed.

Asunto(s)

Proteínas de Arabidopsis/biosíntesis , Arabidopsis/metabolismo , Arabidopsis/microbiología , Dickeya chrysanthemi/fisiología , Ferritinas/biosíntesis , Regulación de la Expresión Génica de las Plantas/fisiología , Sideróforos/fisiología , Proteínas de Arabidopsis/genética , Mapeo Cromosómico , Cromosomas de las Plantas , Ferritinas/genética , Mutación , Enfermedades de las Plantas/microbiología , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Regiones Promotoras Genéticas , Regulación hacia Arriba