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1.
Phytopathology ; 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38749069

RESUMO

The previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either T6SS or T4SS. This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with stains defective in T6SS or/and T4SS confirm that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose-deposition in the course of infection. Notably, this redundant secretion mechanism between T6SS and T4SS is believed to be the first of its kind in bacteria.

2.
Microbiology (Reading) ; 168(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35917166

RESUMO

Pectobacterium atrosepticum is part of a larger family of soft rot bacteria (Pectobacteriaceae) that cause disease on a wide range of crops worldwide. They are closely related to members of the Enterobacteriaceae and, as the plant pathogens and plant associated members of the group, form part of a continuum towards opportunistic and more devastating animal and human pathogens. Many of the horizontally acquired islands present in the genome of P. atrosepticum are directly responsible for life on plants. These include genes for a plethora of plant cell wall degrading enzymes, plant toxins, siderophores etc., which are exported by multiple secretion systems under a highly coordinated regulation system.


Assuntos
Pectobacterium , Solanum tuberosum , Enterobacteriaceae , Humanos , Pectobacterium/genética , Doenças das Plantas/microbiologia , Plantas , Solanum tuberosum/microbiologia
3.
Microbiology (Reading) ; 167(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34319868

RESUMO

Arabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l-arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l-arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l-arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l-arabinose metabolism (araBA) and transport (araF) genes were activated at 18 °C in vitro by l-arabinose and expressed over prolonged periods in planta. Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro, and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l-arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum, reflective of distinct plant-associated lifestyles.


Assuntos
Arabinose/metabolismo , Escherichia coli O157/metabolismo , Plantas Comestíveis/microbiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Contagem de Colônia Microbiana , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microbiologia de Alimentos , Lactuca/microbiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Spinacia oleracea/microbiologia
4.
J Biol Chem ; 289(49): 34349-65, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25320086

RESUMO

Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.


Assuntos
Adesinas Bacterianas/genética , Arabinose/química , Parede Celular/química , Escherichia coli O157/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Polissacarídeos/química , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Arabinose/metabolismo , Aderência Bacteriana , Parede Celular/metabolismo , Parede Celular/microbiologia , Escherichia coli O157/química , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Células Vegetais/química , Células Vegetais/metabolismo , Células Vegetais/microbiologia , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spinacia oleracea/química , Spinacia oleracea/metabolismo , Spinacia oleracea/microbiologia
5.
Mol Plant Microbe Interact ; 28(4): 420-31, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25411959

RESUMO

Type VI secretion systems (T6SSs) are a class of macromolecular machines that are recognized as an important virulence mechanism in several gram-negative bacteria. The genome of Pantoea ananatis LMG 2665(T), a pathogen of pineapple fruit and onion plants, carries two gene clusters whose predicted products have homology with T6SS-associated gene products from other bacteria. Nothing is known regarding the role of these T6SS-1 and T6SS-3 gene clusters in the biology of P. ananatis. Here, we present evidence that T6SS-1 plays an important role in the pathogenicity of P. ananatis LMG 2665(T) in onion plants, while a strain lacking T6SS-3 remains as pathogenic as the wild-type strain. We also investigated the role of the T6SS-1 system in bacterial competition, the results of which indicated that several bacteria compete less efficiently against wild-type LMG 2665(T) than a strain lacking T6SS-1. Additionally, we demonstrated that these phenotypes of strain LMG 2665(T) were reliant on the core T6SS products TssA and TssD (Hcp), thus indicating that the T6SS-1 gene cluster encodes a functioning T6SS. Collectively, our data provide the first evidence demonstrating that the T6SS-1 system is a virulence determinant of P. ananatis LMG 2665(T) and plays a role in bacterial competition.


Assuntos
Sistemas de Secreção Bacterianos/genética , Interações Hospedeiro-Patógeno/genética , Pantoea/genética , Pantoea/patogenicidade , Doenças das Plantas/microbiologia , Virulência/genética , Sistemas de Secreção Bacterianos/fisiologia , Técnicas de Inativação de Genes , Genes Bacterianos , Interações Hospedeiro-Patógeno/fisiologia , Família Multigênica , Mutação , Cebolas/microbiologia , Pantoea/fisiologia , Virulência/fisiologia
6.
Environ Microbiol ; 17(11): 4730-44, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26271942

RESUMO

Integrative and conjugative elements (ICEs) contribute to the rapid evolution of bacterial pathogens via horizontal gene transfer of virulence determinants. ICEs have common mechanisms for transmission, yet the cues triggering this process under natural environmental or physiological conditions are largely unknown. In this study, mobilization of the putative ICE horizontally acquired island 2 (HAI2), present in the chromosome of the phytopathogen Pectobacterium atrosepticum SCRI1043, was examined during infection of the host plant potato. Under these conditions, mobilization of HAI2 increased markedly compared with in vitro cultures. In planta-induced mobilization of HAI2 was regulated by quorum sensing and involved the putative ICE-encoded relaxase ECA0613. Disruption of ECA0613 also reduced transcription of genes involved in production of coronafacic acid (Cfa), the major virulence factor harboured on HAI2, whereas their expression was unaffected in the quorum-sensing (expI) mutant. Thus, suppression of cfa gene expression was not regulated by the mobilization of the ICE per se, but was due directly to inactivation of the relaxase. The identification of genetic factors associated solely with in planta mobilization of an ICE demonstrates that this process is highly adapted to the natural environment of the bacterial host and can influence the expression of virulence determinants.


Assuntos
Pectobacterium/genética , Pectobacterium/patogenicidade , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Fatores de Virulência/genética , Sequência de Aminoácidos , Transferência Genética Horizontal , Indenos/metabolismo , Ilhas , Dados de Sequência Molecular , Pectobacterium/metabolismo , Percepção de Quorum/genética , Alinhamento de Sequência , Fatores de Virulência/metabolismo
7.
BMC Genomics ; 15: 163, 2014 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-24571088

RESUMO

BACKGROUND: The Type VI secretion system (T6SS) has been identified in several different bacteria, including the plant pathogenPantoea ananatis. Previous in silico analyses described three different T6SS loci present in the pathogenic strain of P. ananatis LMG 20103. This initial investigation has been extended to include an additional seven sequenced strains of P. ananatis together with 39 strains from different ecological niches. Comparative and phylogenetic analyses were used to investigate the distribution, evolution, intra-strain variability and operon structure of the T6SS in the sequenced strains. RESULTS: Three different T6SS loci were identified in P. ananatis strain LMG 20103 and designated PA T6SS 1-3. PA T6SS-1 was present in all sequenced strains of P. ananatis and in all 39 additional strains examined in this study. In addition, PA T6SS-1 included all 13 core T6SS genes required for synthesis of a functional T6SS. The plasmid-borne PA T6SS-2 also included all 13 core T6SS genes but was restricted to only 33% (15/46) of the strains examined. In addition, PA T6SS-2 was restricted to strains of P. ananatis isolated from symptomatic plant material. This finding raises the possibility of an association between PA T6SS-2 and either pathogenicity or host specificity. The third cluster PA T6SS-3 was present in all strains analyzed in this study but lacked 11 of the 13 core T6SS genes suggesting it may not encoded a functional T6SS. Inter-strain variability was also associated with hcp and vgrG islands, which are associated with the T6SS and encode a variable number of proteins usually of unknown function. These proteins may play a role in the fitness of different strains in a variety of ecological niches or as candidate T6SS effectors. Phylogenetic analysis indicated that PA T6SS-1 and PA T6SS-2 are evolutionarily distinct. CONCLUSION: Our analysis indicates that the three T6SSs of P. ananatis appear to have been independently acquired and may play different roles relating to pathogenicity, host range determination and/or niche adaptation. Future work will be directed toward understanding the roles that these T6SSs play in the biology of P. ananatis.


Assuntos
Sistemas de Secreção Bacterianos/genética , Meio Ambiente , Interação Gene-Ambiente , Genômica , Pantoea/genética , Ordem dos Genes , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Óperon
8.
BMC Genomics ; 15: 404, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24884520

RESUMO

BACKGROUND: Pantoea ananatis is found in a wide range of natural environments, including water, soil, as part of the epi- and endophytic flora of various plant hosts, and in the insect gut. Some strains have proven effective as biological control agents and plant-growth promoters, while other strains have been implicated in diseases of a broad range of plant hosts and humans. By analysing the pan-genome of eight sequenced P. ananatis strains isolated from different sources we identified factors potentially underlying its ability to colonize and interact with hosts in both the plant and animal Kingdoms. RESULTS: The pan-genome of the eight compared P. ananatis strains consisted of a core genome comprised of 3,876 protein coding sequences (CDSs) and a sizeable accessory genome consisting of 1,690 CDSs. We estimate that ~106 unique CDSs would be added to the pan-genome with each additional P. ananatis genome sequenced in the future. The accessory fraction is derived mainly from integrated prophages and codes mostly for proteins of unknown function. Comparison of the translated CDSs on the P. ananatis pan-genome with the proteins encoded on all sequenced bacterial genomes currently available revealed that P. ananatis carries a number of CDSs with orthologs restricted to bacteria associated with distinct hosts, namely plant-, animal- and insect-associated bacteria. These CDSs encode proteins with putative roles in transport and metabolism of carbohydrate and amino acid substrates, adherence to host tissues, protection against plant and animal defense mechanisms and the biosynthesis of potential pathogenicity determinants including insecticidal peptides, phytotoxins and type VI secretion system effectors. CONCLUSIONS: P. ananatis has an 'open' pan-genome typical of bacterial species that colonize several different environments. The pan-genome incorporates a large number of genes encoding proteins that may enable P. ananatis to colonize, persist in and potentially cause disease symptoms in a wide range of plant and animal hosts.


Assuntos
Infecções por Enterobacteriaceae/veterinária , Genoma Bacteriano , Pantoea/genética , Pantoea/fisiologia , Doenças das Plantas/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Interação Gene-Ambiente , Humanos , Insetos/microbiologia , Pantoea/classificação , Filogenia , Plantas/microbiologia , Vertebrados/microbiologia
9.
Environ Microbiol ; 16(7): 2181-95, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24148193

RESUMO

Bacterial attachment to plant and animal surfaces is generally thought to constitute the initial step in colonization, requiring adherence factors such as flagella and fimbriae. We describe the molecular mechanism underpinning flagella-mediated adherence to plant tissue for the foodborne pathogen, enterohaemorrhagic Escherichia coli. Escherichia coli H7 flagella interacted with a sulphated carbohydrate (carrageenan) on a glycan array, which occurred in a dose-dependent manner. Adherence of E. coli O157 : H-expressing flagella of serotype H7, H6 or H48 to plants associated with outbreaks from fresh produce and to Arabidopsis thaliana, was dependent on flagella interactions with phospholipids and sulpholipids in plasma membranes. Adherence of purified H7 and H48 flagella to carrageenan was reduced at higher concentrations of KH2 PO4 or KCl, showing an ionic basis to the interactions. Purified H7 flagella were observed to physically interact with plasma membranes in spinach plants and in A.thaliana. The results show a specific interaction between E. coli H7, H6 and H48 flagella and ionic lipids in plant plasma membranes. The work extends our understanding of the molecular mechanisms underpinning E.coli flagella targeting of plant hosts and suggests a generic mechanism of recognition common in eukaryotic hosts belonging to different biological kingdoms.


Assuntos
Arabidopsis/microbiologia , Membrana Celular/microbiologia , Escherichia coli O157/metabolismo , Flagelos/metabolismo , Lipídeos de Membrana/metabolismo , Spinacia oleracea/microbiologia , Arabidopsis/química , Aderência Bacteriana , Carragenina/metabolismo , Membrana Celular/química , Contagem de Colônia Microbiana , Escherichia coli O157/química , Flagelos/química , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Lipídeos de Membrana/química , Concentração Osmolar , Spinacia oleracea/química
10.
Int J Syst Evol Microbiol ; 64(Pt 3): 768-774, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24225027

RESUMO

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.


Assuntos
Enterobacteriaceae/classificação , Pectinas/metabolismo , Filogenia , Solanum tuberosum/microbiologia , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Europa (Continente) , Ácidos Graxos/química , Genes Bacterianos , Indóis/metabolismo , Israel , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Mol Plant Microbe Interact ; 26(3): 356-66, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23113713

RESUMO

Plant cell wall-degrading enzymes (PCWDE) are key virulence determinants in the pathogenesis of the potato pathogen Pectobacterium atrosepticum. In this study, we report the impact on virulence of a transposon insertion mutation in the metJ gene that codes for the repressor of the methionine biosynthesis regulon. In a mutant strain defective for the small regulatory RNA rsmB, PCWDE are not produced and virulence in potato tubers is almost totally abolished. However, when the metJ gene is disrupted in this background, the rsmB(-) phenotype is suppressed and virulence and PCWDE production are restored. Additionally, when metJ is disrupted, production of the quorum-sensing signal, N-(3-oxohexanoyl)-homoserine lactone, is increased. The metJ mutant strains showed pleiotropic transcriptional impacts affecting approximately a quarter of the genome. Genes involved in methionine biosynthesis were most highly upregulated but many virulence-associated transcripts were also upregulated. This is the first report of the impact of the MetJ repressor on virulence in bacteria.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Pectobacterium/genética , Percepção de Quorum/genética , Proteínas Repressoras/genética , Solanum tuberosum/microbiologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Metionina/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Motivos de Nucleotídeos , Análise de Sequência com Séries de Oligonucleotídeos , Pectobacterium/enzimologia , Pectobacterium/patogenicidade , Pectobacterium/fisiologia , Peptídeo Hidrolases/metabolismo , Fenótipo , Tubérculos/microbiologia , Polissacarídeo-Liases/metabolismo , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Virulência
12.
Mol Microbiol ; 84(4): 648-63, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22524709

RESUMO

Topoisomerase III enzymes are present only in a limited set of bacteria and their physiological role remains unclear. Here, we show that PbTopo IIIß, a homologue of topoisomerase III encoded on the chromosome of Pectobacterium atrosepticum strain SCRI1043 (Pba SCRI1043), is involved in excision of HAI2, a discrete ~100 kb region, from the Pba SCRI1043 chromosome. HAI2 is a Pathogenicity Island (PAI) that encodes coronafacic acid (Cfa), a major virulence determinant required for infection of potato. PAIs are horizontally acquired genetic elements that in some instances are able to excise from the chromosome of their host cell to form a circular episome prior to transfer to a recipient bacterium. We demonstrate excision of HAI2 from the chromosome, a process that is independent of growth phase and that results in the production of a circular intermediate. Inactivation of PbTopo IIIß causes a 10(3) - to 10(4) -fold increase in excision, leading to reduced fitness in vitro and a decrease in the virulence of Pba SCRI1043 on potato. These results suggest that PbTopo IIIß is required for stable maintenance of HAI2 in the chromosome of Pba SCRI1043 and may control as yet unidentified genes involved in viability and virulence of Pba SCRI1043 on potato.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/genética , Ilhas Genômicas , Pectobacterium/enzimologia , Pectobacterium/patogenicidade , Recombinação Genética , DNA Topoisomerases Tipo I/genética , DNA Bacteriano/metabolismo , Pectobacterium/genética , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Virulência
13.
Environ Microbiol ; 15(3): 687-701, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22788996

RESUMO

In the economically important phytopathogen, Pectobacterium atrosepticum, expression of plant cell wall degrading enzymes and other virulence determinants is controlled in a cell density-dependent fashion, termed quorum sensing (QS). Canonical QS systems in Gram-negative bacteria contain a LuxI-type protein, synthesizing a signalling molecule, and a LuxR-type regulator, responding to the signalling molecule above threshold concentrations. In P. atrosepticum, the central LuxR-type repressor of virulence, VirR, has been identified and its impacts on virulence characterized. Here we define the broader VirR regulon using chromatin immunoprecipitation (ChIP) and in planta microarrays. Ninety-four direct VirR targets were identified by ChIP microarrays and a consensus VirR binding site was determined. Purified VirR was used in DNA gel shift assays on target promoters and VirR : promoter binding was disrupted by exogenous addition of the signalling molecule, N-(3-oxohexanoyl)-l-homoserine lactone (OHHL). VirR autorepressed, and directly activated the transcription of rsmA in the absence of OHHL. Finally, we showed that VirR directly regulated the production of siderophores and controlled swimming motility. This is the first report characterizing the direct targets of VirR and provides clear evidence that this LuxR-type protein can act in vivo as both an activator and repressor of transcription in the absence of its cognate signalling molecule.


Assuntos
Pectobacterium/genética , Pectobacterium/patogenicidade , Percepção de Quorum/genética , Regulon/genética , Virulência/genética , 4-Butirolactona/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mutação , Pectobacterium/metabolismo , Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sideróforos/metabolismo , Transcriptoma
14.
Nature ; 450(7166): 115-8, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17914356

RESUMO

Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.


Assuntos
Proteínas de Algas/química , Proteínas de Algas/metabolismo , Nicotiana/metabolismo , Phytophthora/metabolismo , Sinais Direcionadores de Proteínas , Solanum tuberosum/metabolismo , Alanina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Biologia Computacional , Pectobacterium/genética , Phytophthora/química , Transporte Proteico , Pseudomonas syringae/genética , Solanum tuberosum/microbiologia , Nicotiana/microbiologia
15.
Phytopathology ; 103(4): 333-40, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23506361

RESUMO

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.


Assuntos
Escherichia coli O157/fisiologia , Escherichia coli/fisiologia , Lactuca/microbiologia , Raízes de Plantas/microbiologia , Spinacia oleracea/microbiologia , Verduras/microbiologia , Contagem de Colônia Microbiana , Endófitos , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli O157/citologia , Escherichia coli O157/crescimento & desenvolvimento , Contaminação de Alimentos , Microbiologia de Alimentos , Interações Hospedeiro-Patógeno , Humanos , Lactuca/citologia , Microscopia Eletrônica de Transmissão , Pectobacterium/citologia , Pectobacterium/crescimento & desenvolvimento , Pectobacterium/fisiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Rizosfera , Microbiologia do Solo , Solanum tuberosum/citologia , Solanum tuberosum/microbiologia , Spinacia oleracea/citologia , Nicotiana/citologia , Nicotiana/microbiologia
16.
Mol Plant Microbe Interact ; 25(4): 523-33, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22204647

RESUMO

Antimicrobial peptides constitute an important factor in the defense of plants against pathogens, and bacterial resistance to these peptides have previously been shown to be an important virulence factor in Dickeya dadantii, the causal agent of soft-rot disease of vegetables. In order to understand the bacterial response to antimicrobial peptides, a transcriptional microarray analysis was performed upon treatment with sub-lethal concentration of thionins, a widespread plant peptide. In all, 36 genes were found to be overexpressed, and were classified according to their deduced function as i) transcriptional regulators, ii) transport, and iii) modification of the bacterial membrane. One gene encoding a uricase was found to be repressed. The majority of these genes are known to be under the control of the PhoP/PhoQ system. Five genes representing the different functions induced were selected for further analysis. The results obtained indicate that the presence of antimicrobial peptides induces a complex response which includes peptide-specific elements and general stress-response elements contributing differentially to the virulence in different hosts.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Enterobacteriaceae/patogenicidade , Perfilação da Expressão Gênica , Genoma Bacteriano , Mutação , Peptídeos Cíclicos , Plantas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Virulência
17.
Mol Plant Microbe Interact ; 25(1): 6-17, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21936662

RESUMO

The exopolysaccharide amylovoran is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo but negatively controls expression in vitro. Besides amylovoran biosynthesis and regulatory genes, cell-wall and cell-envelope (membrane) as well as regulatory genes were identified as the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC, and rcsD genes but not the rcsB gene were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared with those grown in Luria Bertani medium. Furthermore, using the genome of E. amylovora ATCC 49946, a hidden Markov model predicted 60 genes with a candidate RcsB binding site in the intergenic region, 28 of which were identified in the microarray assay. Based on these findings as well as previous reported data, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.


Assuntos
Proteínas de Bactérias/genética , Erwinia amylovora/genética , Doenças das Plantas/microbiologia , Polissacarídeos Bacterianos/metabolismo , Regulon/genética , Proteínas de Bactérias/metabolismo , Erwinia amylovora/patogenicidade , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Genoma Bacteriano , Malus/microbiologia , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Óperon/genética , Polissacarídeos Bacterianos/genética , Regiões Promotoras Genéticas/genética , Pyrus/microbiologia , RNA Bacteriano/genética , Deleção de Sequência , Transdução de Sinais/genética , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
18.
Mol Microbiol ; 82(3): 719-33, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21992096

RESUMO

Cyclic diguanylate (c-di-GMP) is a second messenger controlling many important bacterial processes. The phytopathogen Pectobacterium atrosepticum SCRI1043 (Pba1043) possesses a Type I secretion system (T1SS) essential for the secretion of a proteinaceous multi-repeat adhesin (MRP) required for binding to the host plant. The genes encoding the MRP and the T1SS are tightly linked to genes encoding several putative c-di-GMP regulatory components. We show that c-di-GMP regulates secreted MRP levels in Pba1043 through the action of two genes encoding predicted diguanylate cyclase (DGC) and phosphodiesterase proteins (ECA3270 and ECA3271). Phenotypic analyses and quantification of c-di-GMP levels demonstrated that ECA3270 and ECA3271 regulate secreted MRP levels by increasing and decreasing, respectively, the intracellular levels of c-di-GMP. Moreover, ECA3270 represents the first active DGC reported to have an alternative active-site motif from the 'canonical' GG[D/E]EF. ECA3270 has an A-site motif of SGDEF and analysis of single amino acid replacements demonstrated that the first position of this motif can tolerate functional substitution. Serine in position one of the A-site is also observed in many other DGCs. Finally, another T1SS-linked regulator (ECA3265) also plays an important role in regulating secreted MRP, with an altered localization of MRP observed in an ECA3265 mutant background. Mutants defective in these three T1SS-linked regulators exhibit a reduction in root binding and virulence, confirming that this complex, finely tuned regulation system is crucial in the interaction with host plants.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Pectobacterium/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Pectobacterium/patogenicidade , Diester Fosfórico Hidrolases/genética , Fósforo-Oxigênio Liases/genética , Raízes de Plantas/microbiologia , Solanum tuberosum/microbiologia , Virulência
19.
Adv Appl Microbiol ; 81: 89-132, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22958528

RESUMO

Most reviews of climate change are epidemiological, focusing on impact assessment and risk mapping. However, there are many reports of the effects of environmental stress factors on defense mechanisms in plants against pathogens. We review those representative of key climate change-related stresses to determine whether there are any patterns or trends in adaptation responses. We recognize the complexity of climate change itself and the multitrophic nature of the complex biological interactions of plants, microbes, soil, and the environment and, therefore, the difficulty of reductionist dissection approaches to resolving the problems. We review host defense genes, germplasm, and environmental interactions in different types of organisms but find no significant group-specific trends. Similarly, we review by host defense mechanism type and by host-pathogen trophic relationship but identify no dominating mechanism for stress response. However, we do identify core stress response mechanisms playing key roles in multiple response pathways whether to biotic or abiotic stress. We suggest that these should be central to mechanistic climate change plant defense research. We also recognize biodiversity, heterogeneity, and the need for understanding stress in a true systems biology approach as being essential components of progressing our understanding of and response to climate change.


Assuntos
Mudança Climática , Plantas , Biodiversidade , Clima , Ecossistema , Plantas/metabolismo , Solo
20.
J Bacteriol ; 193(8): 2076-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21217001

RESUMO

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


Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Enterobacteriaceae/genética , Genoma Bacteriano , Enterobacteriaceae/isolamento & purificação , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Plantas/microbiologia , Análise de Sequência de DNA
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