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1.
J Microbiol Methods ; 120: 1-5, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26319185

ABSTRACT

The plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae were directly detected in extracts from infected rice seedlings by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method did not require culturing of the pathogens on artificial medium. In the MALDI-TOF MS analysis, peaks originating from bacteria were found in extracts from infected rice seedlings. The spectral peaks showed significantly high scores, in spite of minor differences in spectra. The spectral peaks originating from host plant tissues did not affect this direct MALDI-TOF MS analysis for the rapid identification of plant pathogens.


Subject(s)
Burkholderia gladioli/isolation & purification , Burkholderia/isolation & purification , Dickeya chrysanthemi/isolation & purification , Oryza/microbiology , Plant Diseases/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Bacterial Typing Techniques , Burkholderia/classification , Burkholderia/pathogenicity , Burkholderia gladioli/classification , Burkholderia gladioli/pathogenicity , Dickeya chrysanthemi/classification , Dickeya chrysanthemi/pathogenicity , Oryza/chemistry , Seedlings/microbiology
2.
mBio ; 6(3): e00353-15, 2015 Apr 28.
Article in English | MEDLINE | ID: mdl-25922390

ABSTRACT

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.


Subject(s)
DNA, Bacterial/genetics , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/pathogenicity , Gene Expression Regulation, Bacterial , Stress, Physiological , Virulence Factors/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromosomes, Bacterial , DNA, Bacterial/chemistry , DNA, Superhelical , Dickeya chrysanthemi/physiology , Genes, Bacterial , Lamiales/microbiology , Transcriptome , Virulence , Virulence Factors/metabolism
3.
Mol Plant Microbe Interact ; 27(7): 700-11, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24625032

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Dickeya chrysanthemi/physiology , Dickeya chrysanthemi/pathogenicity , Gene Expression Regulation, Bacterial/physiology , Plant Diseases/microbiology , Bacterial Proteins/genetics , Bacteriophages , Cichorium intybus/microbiology , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/virology , Solanum tuberosum/microbiology , Virulence
4.
PLoS One ; 7(1): e30702, 2012.
Article in English | MEDLINE | ID: mdl-22292023

ABSTRACT

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.


Subject(s)
Aphids/microbiology , Bacterial Toxins/metabolism , Dickeya chrysanthemi/physiology , Enterobacteriaceae Infections/microbiology , Sepsis/microbiology , Animals , Animals, Genetically Modified , Aphids/embryology , Aphids/genetics , Aphids/physiology , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/metabolism , Dickeya chrysanthemi/pathogenicity , Disease Vectors , Embryo, Nonmammalian/microbiology , Endotoxins/genetics , Endotoxins/metabolism , Enterobacteriaceae Infections/genetics , Enterobacteriaceae Infections/veterinary , Gene Expression Regulation , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Intestines/embryology , Intestines/microbiology , Pisum sativum/parasitology , Plant Diseases/microbiology , Sepsis/genetics , Sepsis/veterinary
5.
Physiol Plant ; 145(3): 406-25, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22268629

ABSTRACT

Erwinia chrysanthemi is a devastating bacterial pathogen in Phalaenopsis amabilis and causes soft-rotting disease by secretion of cell wall-degrading enzymes. However, the molecular mechanisms underlying the interaction of P. amabilis with E. chrysanthemi remain elusive. In this study, early molecular events of the plant in response to the pathogen attack were investigated. The alteration in reactive oxygen species accumulation and peroxidase activity occurred at the site of infection. Subsequently, a systematic sequencing of expressed sequence tags (ESTs) using suppression subtractive hybridization (SSH) was performed to obtain the first global picture of the assembly of genes involved in the pathogenesis. The majority of the SSH clones showed a high identity with genes coding for proteins that have known roles in redox homeostasis, responses to pathogens and metabolism. A notable number of the SSH clones were those encoding WRKY, MYB and basic leucine zipper transcription factors, indicating the stimulation of intracellular signal transduction. An orchid gene encoding trans-2-enoyl-CoA reductase (ECR) was the most abundant transcripts in the EST library. ECR is an enzyme catalyzing the very long chain fatty acids (VLCFAs) biosynthesis, and the full-length cDNA of the ECR gene (PaECR1) was obtained. Functional analysis of PaECR1 was conducted by virus-induced gene silencing to knock down the gene expression in P. amabilis. The PaECR1-silenced plants were more susceptible to E. chrysanthemi infection, implying potential roles for VLCFAs in the pathogenesis. In summary, the pathogen-responsive gene expression profiles facilitated a more comprehensive view of the molecular events that underlie this economically important plant-pathogen interaction.


Subject(s)
Dickeya chrysanthemi/pathogenicity , Gene Expression Profiling/methods , Host-Pathogen Interactions , Orchidaceae/microbiology , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Wall/metabolism , Cell Wall/microbiology , Enzyme Activation , Expressed Sequence Tags , Gene Expression Regulation, Plant , Gene Knockdown Techniques/methods , Gene Silencing , Genes, Plant , Homeostasis , Molecular Sequence Data , Orchidaceae/genetics , Orchidaceae/metabolism , Oxidation-Reduction , Oxidoreductases Acting on CH-CH Group Donors/genetics , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Peroxidase/genetics , Peroxidase/metabolism , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Reactive Oxygen Species/metabolism , Sequence Alignment , Signal Transduction
6.
Environ Microbiol ; 13(11): 2901-14, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21906221

ABSTRACT

Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence.


Subject(s)
Bacterial Proteins/metabolism , Dickeya chrysanthemi/genetics , Genes, Regulator , Plants/microbiology , Repressor Proteins/metabolism , Bacterial Proteins/genetics , Dickeya chrysanthemi/pathogenicity , Gene Regulatory Networks , Genes, Bacterial , Mutation , Quorum Sensing , Repressor Proteins/genetics , Virulence , Virulence Factors/genetics , Virulence Factors/metabolism
7.
J Bacteriol ; 193(4): 963-70, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21169494

ABSTRACT

The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.


Subject(s)
Bacterial Proteins/metabolism , Carboxylic Ester Hydrolases/metabolism , Coumaric Acids/metabolism , Dickeya chrysanthemi/enzymology , Gene Expression Regulation, Enzymologic , Polysaccharide-Lyases/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/genetics , Cichorium intybus/microbiology , Cytoplasm/chemistry , Cytoplasm/enzymology , Cytoplasm/genetics , Dickeya chrysanthemi/chemistry , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/pathogenicity , Extracellular Space/chemistry , Extracellular Space/enzymology , Extracellular Space/genetics , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Plant Diseases/microbiology , Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/genetics , Protein Transport , Sequence Alignment , Virulence
8.
Hybridoma (Larchmt) ; 29(5): 383-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-21050038

ABSTRACT

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.


Subject(s)
Ananas/microbiology , Antibodies, Monoclonal/immunology , Antibody Specificity , Dickeya chrysanthemi/immunology , Plant Diseases/immunology , Ananas/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity/physiology , Cell Line, Tumor , Diagnostic Techniques and Procedures , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/pathogenicity , Hybridomas/immunology , Hybridomas/metabolism , Mice , Plant Diseases/genetics , Plant Diseases/microbiology , Polymerase Chain Reaction/methods , Species Specificity
9.
Mol Plant Microbe Interact ; 23(10): 1345-55, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20831411

ABSTRACT

The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.


Subject(s)
Bacterial Proteins/metabolism , Dickeya chrysanthemi/enzymology , Exoribonucleases/metabolism , Gene Expression Regulation, Bacterial/physiology , RNA, Bacterial/metabolism , Bacterial Proteins/genetics , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/metabolism , Dickeya chrysanthemi/pathogenicity , Exoribonucleases/genetics , RNA, Bacterial/genetics
10.
Mol Plant Microbe Interact ; 23(7): 871-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20521950

ABSTRACT

The type III secretion system (T3SS) is considered one of the major virulence factors in many bacterial pathogens. This report demonstrates that RssB, ClpXP, and RpoS play a role in T3SS regulation of Dickeya dadantii 3937. ClpP is a serine-type protease which associates with the ClpX chaperone to form a functional Clp proteolytic complex for degradation of proteins. With the assistance of recognition factor RssB, ClpXP degrades the RpoS sigma factor. RpoS positively regulates the expression of the rsmA gene encoding an RNA-binding regulatory protein. By interacting with the hrpL mRNA, RsmA reduces HrpL production and downregulates the T3SS genes in the HrpL regulon. In addition, ClpXP, RssB, and RpoS affect pectinolytic enzyme production in D. dadantii 3937, probably through RsmA. The ClpXP and RssB proteins are essential for bacterial virulence.


Subject(s)
Dickeya chrysanthemi/enzymology , Endopeptidase Clp/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Brassica/microbiology , Dickeya chrysanthemi/metabolism , Dickeya chrysanthemi/pathogenicity , Endopeptidase Clp/genetics , Gene Expression Regulation, Bacterial/physiology , Virulence
11.
Phytopathology ; 100(2): 134-42, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20055647

ABSTRACT

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.


Subject(s)
Dickeya chrysanthemi/physiology , Host-Pathogen Interactions , Soil Microbiology , Solanum tuberosum/microbiology , Dickeya chrysanthemi/pathogenicity , Gentian Violet , Green Fluorescent Proteins/genetics , Microscopy, Confocal , Plant Diseases/microbiology , Plant Tubers/microbiology , Virulence
12.
J Bacteriol ; 191(22): 6960-7, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19734309

ABSTRACT

Erwinia chrysanthemi (Dickeya dadantii) is a plant pathogenic bacterium that has a large capacity to degrade the plant cell wall polysaccharides. The present study reports the metabolic pathways used by E. chrysanthemi to assimilate the oligosaccharides sucrose and raffinose, which are particularly abundant plant sugars. E. chrysanthemi is able to use sucrose, raffinose, or melibiose as a sole carbon source for growth. The two gene clusters scrKYABR and rafRBA are necessary for their catabolism. The phenotypic analysis of scr and raf mutants revealed cross-links between the assimilation pathways of these oligosaccharides. Sucrose catabolism is mediated by the genes scrKYAB. While the raf cluster is sufficient to catabolize melibiose, it is incomplete for raffinose catabolism, which needs two additional steps that are provided by scrY and scrB. The scr and raf clusters are controlled by specific repressors, ScrR and RafR, respectively. Both clusters are controlled by the global activator of carbohydrate catabolism, the cyclic AMP receptor protein (CRP). E. chrysanthemi growth with lactose is possible only for mutants with a derepressed nonspecific lactose transport system, which was identified as RafB. RafR inactivation allows the bacteria to the assimilate the novel substrates lactose, lactulose, stachyose, and melibionic acid. The raf genes also are involved in the assimilation of alpha- and beta-methyl-D-galactosides. Mutations in the raf or scr genes did not significantly affect E. chrysanthemi virulence. This could be explained by the large variety of carbon sources available in the plant tissue macerated by E. chrysanthemi.


Subject(s)
Dickeya chrysanthemi/metabolism , Melibiose/metabolism , Raffinose/metabolism , Sucrose/metabolism , Bacterial Proteins , Cichorium intybus/microbiology , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/pathogenicity , Melibiose/genetics , Models, Biological , Models, Genetic , Multigene Family/genetics , Multigene Family/physiology , Raffinose/genetics
13.
J Bacteriol ; 191(17): 5409-18, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19542281

ABSTRACT

SlyA, a MarR family transcriptional regulator, controls an assortment of biological functions in several animal-pathogenic bacteria. In order to elucidate the functions of SlyA in the phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) 3937, a slyA gene deletion mutant (denoted DeltaslyA) was constructed. The mutant exhibited increased sensitivity to sodium hypochlorite, the cationic antimicrobial peptide polymyxin B, and oxidative stress. The mutant showed reduced production of pectate lyase and exopolysaccharide and an inability to form a pellicle. The mutant lacking a functional slyA gene showed a significantly reduced ability to cause maceration of potato tubers. Accordingly, the mutant exhibited significantly reduced bacterial growth and failed to hyperinduce pectate lyase production in planta. Introduction of a plasmid containing slyA into the DeltaslyA mutant caused all of these phenotypes to recover to wild-type levels. These results suggest that SlyA plays an important role in virulence to plants by positively regulating the expression of multiple pathogenicity-related traits of D. dadantii 3937.


Subject(s)
Bacterial Proteins/physiology , Dickeya chrysanthemi/pathogenicity , Plant Diseases/microbiology , Solanum tuberosum/microbiology , Transcription Factors/physiology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/growth & development , Gene Deletion , Oxidants/pharmacology , Plant Roots/microbiology , Polymyxin B/pharmacology , Polysaccharide-Lyases/metabolism , Polysaccharides, Bacterial/metabolism , Sodium Hypochlorite/pharmacology , Transcription Factors/genetics , Virulence
14.
Microbiology (Reading) ; 155(Pt 2): 434-442, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19202091

ABSTRACT

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

Subject(s)
Chemotaxis , Dickeya chrysanthemi/physiology , Dickeya chrysanthemi/pathogenicity , Plant Diseases/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Flagella/genetics , Flagella/metabolism , Plant Leaves/microbiology , Signal Transduction , Virulence
15.
Mol Plant Microbe Interact ; 21(11): 1431-42, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18842093

ABSTRACT

The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control.


Subject(s)
Bacterial Proteins/genetics , Dickeya chrysanthemi/genetics , Gene Expression Regulation, Bacterial , Sigma Factor/genetics , Amino Acid Sequence , Bacterial Proteins/physiology , Dickeya chrysanthemi/pathogenicity , Models, Genetic , Molecular Sequence Data , Sequence Homology, Amino Acid , Sigma Factor/physiology , Temperature , Nicotiana/microbiology , Virulence/genetics
16.
Mol Plant Microbe Interact ; 21(11): 1471-81, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18842096

ABSTRACT

Analysis of the regulators of the LacI family was performed in order to identify those potentially involved in pathogenicity of Erwinia chrysanthemi (Dickeya dadantii). Among the 18 members of the LacI family, the function of 11 members is either known or predicted and only 7 members have, as yet, no proposed function. Inactivation of these seven genes, called lfaR, lfbR, lfcR, lfdR, lfeR, lffR, and lfgR, demonstrated that four of them are important for plant infection. The lfaR and lfcR mutants showed a reduced virulence on chicory, Saintpaulia sp., and Arabidopsis. The lfeR mutant showed a reduced virulence on Arabidopsis. The lfdR mutant was more efficient than the wild-type strain in initiating maceration on Saintpaulia sp. The genetic environment of each regulator was examined to detect adjacent genes potentially involved in a common function. Construction of transcriptional fusions in these neighboring genes demonstrated that five regulators, LfaR, LfcR, LfeR, LffR, and LfgR, act as repressors of adjacent genes. Analysis of these fusions also indicated that the genes controlled by LfaR, LfcR, LfgR, and LffR are expressed during plant infection. Moreover, addition of crude plant extracts to culture medium demonstrated that the expression of the LfaR- and LfgR-controlled genes is specifically induced by plant components.


Subject(s)
Bacterial Proteins/genetics , Dickeya chrysanthemi/genetics , Repressor Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Cichorium intybus/microbiology , Dickeya chrysanthemi/metabolism , Dickeya chrysanthemi/pathogenicity , Lac Repressors , Multigene Family/genetics , Mutation , Repressor Proteins/metabolism , Repressor Proteins/physiology , Virulence/genetics
17.
Mol Plant Pathol ; 9(1): 11-24, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18705880

ABSTRACT

In addition to the important role of abscisic acid (ABA) in abiotic stress signalling, basal and high ABA levels appear to have a negative effect on disease resistance. Using the ABA-deficient sitiens tomato (Solanum lycopersicum) mutant and different application methods of exogenous ABA, we demonstrated the influence of this plant hormone on disease progression of Erwinia chrysanthemi. This necrotrophic plant pathogenic bacterium is responsible for soft rot disease on many plant species, causing maceration symptoms mainly due to the production and secretion of pectinolytic enzymes. On wild-type (WT) tomato cv. Moneymaker E. chrysanthemi leaf inoculation resulted in maceration both within and beyond the infiltrated zone of the leaf, but sitiens showed a very low occurrence of tissue maceration, which never extended the infiltrated zone. A single ABA treatment prior to infection eliminated the effect of pathogen restriction in sitiens, while repeated ABA spraying during plant development rendered both WT and sitiens very susceptible. Quantification of E. chrysanthemi populations inside the leaf did not reveal differences in bacterial growth between sitiens and WT. Sitiens was not more resistant to pectinolytic cell-wall degradation, but upon infection it showed a faster and stronger activation of defence responses than WT, such as hydrogen peroxide accumulation, peroxidase activation and cell-wall fortifications. Moreover, the rapid activation of sitiens peroxidases was also observed after application of bacteria-free culture filtrate containing E. chrysanthemi cell-wall-degrading enzymes and was absent during infection with an out E. chrysanthemi mutant impaired in secretion of these extracellular enzymes.


Subject(s)
Abscisic Acid/pharmacology , Dickeya chrysanthemi/pathogenicity , Plant Diseases/microbiology , Solanum lycopersicum/metabolism , Solanum lycopersicum/microbiology , Cell Wall/metabolism , Cell Wall/microbiology , Colony Count, Microbial , Dickeya chrysanthemi/growth & development , Disease Susceptibility/microbiology , Genotype , Hydrogen Peroxide/metabolism , Solanum lycopersicum/drug effects , Solanum lycopersicum/genetics , Mutation , Peroxidases/metabolism , Plant Leaves/metabolism , Plant Leaves/microbiology
18.
Mol Microbiol ; 67(6): 1257-73, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18284573

ABSTRACT

The Erwinia chrysanthemi genome is predicted to encode three systems, Nif, Isc and Suf, known to assist Fe/S cluster biogenesis and the CsdAE cysteine desulphurase. Single iscU, hscA and fdx mutants were found sensitive to paraquat and exhibited reduced virulence on both chicory leaves and Arabidopsis thaliana. Depletion of the whole Isc system led to a pleiotropic phenotype, including sensitivity to both paraquat and 2,2'-dipyridyl, auxotrophies for branched-chain amino acids, thiamine, nicotinic acid, and drastic alteration in virulence. IscR was able to suppress all of the phenotypes listed above in a sufC-dependent manner while depletion of the Isc system led to IscR-dependent activation of the suf operon. No virulence defects were found associated with csdA or nifS mutations. Surprisingly, we found that the sufC mutant was virulent against A. thaliana, whereas its virulence had been found altered in Saintpaulia. Collectively, these results lead us to propose that E. chrysanthemi possess the Fe/S biogenesis strategy suited to the physico-chemical conditions encountered in its host upon infection. In this view, the IscR regulator, which controls both Isc and Suf, is predicted to play a major role in the ability of E. chrysanthemi to colonize a wide array of different plants.


Subject(s)
Adaptation, Physiological/physiology , Bacterial Proteins/metabolism , Dickeya chrysanthemi/metabolism , Iron-Sulfur Proteins/metabolism , Adaptation, Physiological/genetics , Arabidopsis/microbiology , Bacterial Proteins/genetics , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/metabolism , Cichorium intybus/microbiology , Chromosomes, Bacterial/genetics , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/pathogenicity , Gene Expression Regulation, Bacterial/drug effects , Genome, Bacterial , Iron-Sulfur Proteins/genetics , Models, Genetic , Mutation , Operon/genetics , Paraquat/pharmacology , Phenotype , Plant Leaves/microbiology , Virulence/genetics
19.
J Bacteriol ; 190(5): 1518-30, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18165304

ABSTRACT

During infection, the phytopathogenic enterobacterium Erwinia chrysanthemi has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. The E. chrysanthemi genome possesses two loci that could be devoted to iron storage: the bfr gene, encoding a heme-containing bacterioferritin, and the ftnA gene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike the bfr mutant, the ftnA mutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, the bfr ftnA mutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in the ftnA and bfr ftnA mutants. We found that ftnA gene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB. bfr gene expression is induced at the stationary phase of growth. The sigmaS transcriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence of E. chrysanthemi depending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.


Subject(s)
Bacterial Proteins/genetics , Cytochrome b Group/genetics , Dickeya chrysanthemi/genetics , Ferritins/genetics , Iron/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Base Sequence , Biological Transport , Blotting, Northern , Cichorium intybus/microbiology , Chlorides , Cytochrome b Group/metabolism , Cytochrome b Group/physiology , Dickeya chrysanthemi/metabolism , Dickeya chrysanthemi/pathogenicity , Ferric Compounds/metabolism , Ferritins/metabolism , Ferritins/physiology , Ferrous Compounds/metabolism , Gene Expression Regulation, Bacterial , Iron Radioisotopes/metabolism , Molecular Sequence Data , Mutation , Oxidative Stress , Plant Leaves/microbiology , Spectroscopy, Mossbauer , Virulence/genetics
20.
Environ Microbiol ; 10(3): 545-59, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18177376

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Dickeya chrysanthemi/pathogenicity , Genes, Regulator , Plant Diseases/microbiology , Repressor Proteins/metabolism , Transcription Factors/metabolism , Virulence Factors/genetics , Dickeya chrysanthemi/physiology , Gene Expression Regulation, Bacterial , Signal Transduction/genetics , Signal Transduction/physiology , Virulence Factors/physiology
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