RESUMO
Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Erwinia/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Brassicaceae/genética , Brassicaceae/metabolismo , Erwinia/genética , Erwinia/patogenicidade , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Nicotiana/metabolismo , Nicotiana/microbiologia , Virulência/genética , Zea mays/metabolismo , Zea mays/microbiologiaRESUMO
The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.
Assuntos
Proteínas de Bactérias/genética , Erwinia/genética , Genes Bacterianos/genética , Óperon/genética , Fatores de Transcrição , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/biossíntese , Mapeamento Cromossômico , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Erwinia/patogenicidade , Teste de Complementação Genética , Janus Quinases , Dados de Sequência Molecular , Família Multigênica/genética , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Pseudomonas/genética , RNA Helicases/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Nicotiana/microbiologia , Zea mays/microbiologiaRESUMO
Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, DeltaesaR, and DeltaesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.
Assuntos
Enterobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica , Polissacarídeos Bacterianos/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Acilação , Proteínas de Bactérias/metabolismo , Erwinia/genética , Mutagênese , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Erwinia stewartii contains a large cluster of wts genes that are required by this bacterium for pathogenicity on corn plants. Three complementation groups within the right half of this cluster, wtsA, wtsC, and wtsB, were previously identified. In this study, WtsA was found to be a positive activator of wtsB::lacZ expression. The wtsA locus was sequenced and a single open reading frame is present within the wtsA locus, which has the capacity to encode a 323 amino acid polypeptide. A corresponding 38 kDa protein was observed in Escherichia coli minicells containing the cloned wtsA gene. The predicted WtsA polypeptide has significant similarity to HrpS from Pseudomonas syringae pv. phaseolicola, as well as other members of the NtrC class of prokaryotic regulatory proteins. Similar to other genes activated by NtrC regulators, wtsB::lacZ expression in E. coli was dependent upon rpoN.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA , Erwinia/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Família Multigênica/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cruzamentos Genéticos , Erwinia/patogenicidade , Escherichia coli/genética , Proteínas de Escherichia coli , Genes Reporter , Teste de Complementação Genética , Óperon Lac , Dados de Sequência Molecular , Mutagênese , Pseudomonas/genética , RNA Polimerase Sigma 54 , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fator sigma/genética , Especificidade da Espécie , Virulência/genética , Zea mays/microbiologiaRESUMO
We have previously cloned the genes for synthesis of capsular polysaccharide (cps) and slime from Erwinia stewartii in cosmid pES2144. In this study, pES2144 was shown to complement 14 spontaneous cps mutants. These mutants were characterized by probing Southern blots of mutant genomic DNA with pES2144; insertions were detected in four mutants and deletions in six mutants. Genetic and physical maps of the pES2144 cps region were constructed by subcloning, restriction analysis, and transposon mutagenesis with Tn5, Tn5lac, and Tn3HoHo1. Mutations affecting the ability of pES2144 to restore mucoidy to cps deletion mutants were located in five regions, designated cpsA to cpsE. None of the cps mutants were able to cause systemic wilting of corn plants, and mutations in cps regions B to E further abolished the ability of the bacterium to cause watersoaked lesions on seedlings. The gene for uridine-5'-diphosphogalactose 4-epimerase (galE) was linked to the cps genes on pES2144. In E. stewartii, galE was constitutively expressed, whereas the genes for galactokinase (galK) and galactose-1-phosphate uridyltransferase (galT) were inducible and not linked to galE. Thus, galE does not appear to be part of the gal operon in this species.
Assuntos
Erwinia/genética , Genes Bacterianos , Polissacarídeos Bacterianos/genética , Mapeamento Cromossômico , Cromossomos Bacterianos , Clonagem Molecular , Enzimas de Restrição do DNA , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Erwinia/metabolismo , Galactoquinase/genética , Galactose/genética , Galactose/metabolismo , Mutação , Hibridização de Ácido Nucleico , Óperon , Polissacarídeos Bacterianos/biossíntese , Racemases e Epimerases/genética , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Zea mays/microbiologiaRESUMO
A library of Erwinia stewartii DNA was constructed in cosmid pVK100 and used to complement spontaneous and Mu pf7701-induced (designated by the prefix MU) avirulent mutants. Plasmid pES4507 restored water-soaking ability and extracellular polysaccharide (EPS) synthesis to mutants MU14110 and MU2B70 (group I); pES1044 restored water-soaking ability to MU43, MU51, MU136, MU141, and RDF6011 (group II); and pES2144 complemented four spontaneous EPS- mutants (group III). Hybridization of labeled plasmid DNA to Southern blots of genomic DNA from the mutants revealed that a Mu pf7701 insertion was associated with the respective cloned region in all mutants except MU2B70 and MU223. In these strains, the plasmid may be suppressing the avirulent phenotype rather than complementing the mutation.
