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Rachel A Mann, Theo H M Smits,
Andreas Bühlmann,
Jochen Blom,
Alexander Goesmann,
Jürg E Frey,
Kim M Plummer,
Steven V Beer,
Joanne Luck,
Brion Duffy,
Brendan Rodoni
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ABSTRACT: The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1(Ea) and a putative secondary metabolite pathway only present in Rubus-infecting strains.
PLoS ONE 01/2013; 8(2):e55644. · 4.09 Impact Factor
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ABSTRACT: Several molecular methods have been developed for detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomics pipeline. The target coding sequences (CDS) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with Optigene GenieII chemistry and instrumentation proved to be an exceptionally rapid (under 15 minutes) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing.
Journal of microbiological methods 12/2012; · 2.43 Impact Factor
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ABSTRACT: Fibrisoma limi strain BUZ 3(T), a Gram-negative bacterium, was isolated from coastal mud from the North Sea (Fedderwardersiel, Germany) and characterized using a polyphasic approach in 2011. The genome consists of a chromosome of about 7.5 Mb and three plasmids.
Journal of bacteriology 08/2012; 194(16):4445. · 3.94 Impact Factor
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ABSTRACT: Fibrisoma limi strain BUZ3T, a Gram-negative bacterium, was isolated from coastal mud from the North Sea (Fedderwardersiel, Germany) and characterized using a polyphasic approach in 2011. The genome consists of a chromosome of about 7.5 Mb and three plasmids.
Journal of Bacteriology 08/2012; 194(16). · 3.83 Impact Factor
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ABSTRACT: Fibrella aestuarina BUZ 2(T) is the type strain of the recently characterized genus Fibrella. Here we report the draft genome sequence of this strain, which consists of a single scaffold representing the chromosome (with 11 gaps) and a 161-kb circular plasmid.
Journal of bacteriology 07/2012; 194(13):3555. · 3.94 Impact Factor
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ABSTRACT: Pantoea agglomerans is a cosmopolitan plant epiphytic bacterium that includes some of the most effective biological antagonists against the fire blight pathogen Erwinia amylovora, a major threat to pome fruit production worldwide. Strain E325 is commercially available as Bloomtime Biological™ in the USA and Canada. New quantitative PCR (qPCR) assays were developed for species- and strain -specific detection in the environment, and for detection of indigenous strains carrying the biocontrol antibacterial peptide biosynthesis gene paaA. The qPCR assays were highly specific, efficient and sensitive, detecting fewer than three cells per reaction or 700 colony forming units per flower, respectively. The qPCR assays were tested on field samples, giving first indications to the incidence of P. agglomerans E325 related strains, total P. agglomerans and pantocin A producing bacteria in commercial orchards. These assays will facilitate monitoring the environmental behavior of biocontrol P. agglomerans after orchard application for disease protection, proprietary strain-tracking, and streamlined screening for discovery of new biocontrol strains.
Journal of microbiological methods 06/2012; 90(3):315-20. · 2.43 Impact Factor
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ABSTRACT: Comparative genomic analysis revealed differences in the lipopolysaccharide (LPS) biosynthesis gene cluster between the Rubus-infecting strain ATCC BAA-2158 and the Spiraeoideae-infecting strain CFBP 1430 of Erwinia amylovora. These differences corroborate rpoB-based phylogenetic clustering of E. amylovora into four different groups and enable the discrimination of Spiraeoideae- and Rubus-infecting strains. The structure of the differences between the two groups supports the hypothesis that adaptation to Rubus spp. took place after species separation of E. amylovora and E. pyrifoliae that contrasts with a recently proposed scenario, based on CRISPR data, in which the shift to domesticated apple would have caused an evolutionary bottleneck in the Spiraeoideae-infecting strains of E. amylovora which would be a much earlier event. In the core region of the LPS biosynthetic gene cluster, Spiraeoideae-infecting strains encode three glycosyltransferases and an LPS ligase (Spiraeoideae-type waaL), whereas Rubus-infecting strains encode two glycosyltransferases and a different LPS ligase (Rubus-type waaL). These coding domains share little to no homology at the amino acid level between Rubus- and Spiraeoideae-infecting strains, and this genotypic difference was confirmed by polymerase chain reaction analysis of the associated DNA region in 31 Rubus- and Spiraeoideae-infecting strains. The LPS biosynthesis gene cluster may thus be used as a molecular marker to distinguish between Rubus- and Spiraeoideae-infecting strains of E. amylovora using primers designed in this study.
Molecular Plant Pathology 05/2012; 13(8):975-84. · 3.90 Impact Factor
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Rachel A Mann,
Jochen Blom,
Andreas Bühlmann,
Kim M Plummer,
Steven V Beer,
Joanne E Luck,
Alexander Goesmann,
Jürg E Frey,
Brendan C Rodoni,
Brion Duffy, Theo H M Smits
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ABSTRACT: The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora not only encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICEs in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation.
Gene 05/2012; 504(1):6-12. · 2.34 Impact Factor
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ABSTRACT: The bacterial plant pathogen Erwinia amylovora causes fire blight, a major disease threat to pome fruit production worldwide with further impact on a wide-range of Rosaceae species. Important factors contributing to the development of the disease were discovered in the last decades. Comparative
genomics of the genera Erwinia and Pantoea is coming into focus with the recent availability of complete genome sequences. Insights from comparative genomics now position
us to answer fundamental questions regarding the evolution of E. amylovora as a successful pathogen and the critical elements for biocontrol activity of Pantoea spp. This trove of new data promises to reveal novel determinants and to understand interactive pathways for virulence, host
range and ecological fitness. The ultimate aim is now to apply genomics and identify the pathogen Achilles heels and antagonist
mechanisms of action as targets for designing innovative control strategies for fire blight.
KeywordsComparative genomics–Type III secretion systems–Exopolysaccharides–Virulence factor–Biocontrol
Trees 04/2012; 26(1):227-238. · 1.68 Impact Factor
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ABSTRACT: Pantoea vagans C9-1 is a biocontrol strain that produces at least two antibiotics inhibiting the growth of Erwinia amylovora, the causal agent of fire blight disease of pear and apple. One antibiotic, herbicolin I, was purified from culture filtrates of P. vagans C9-1 and determined to be 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine, also known as N(ß)-epoxysuccinamoyl-DAP-valine. A plasposon library was screened for mutants that had lost the ability to produce herbicolin I. It was shown that mutants had reduced biocontrol efficacy in immature pear assays. The biosynthetic gene cluster in P. vagans C9-1 was identified by sequencing the flanking regions of the plasposon insertion sites. The herbicolin I biosynthetic gene cluster consists of 10 coding sequences (CDS) and is located on the 166-kb plasmid pPag2. Sequence comparisons identified orthologous gene clusters in Pantoea agglomerans CU0119 and Serratia proteamaculans 568. A low incidence of detection of the biosynthetic cluster in a collection of 45 Pantoea spp. from biocontrol, environmental, and clinical origins showed that this is a rare trait among the tested strains.
Applied and environmental microbiology 04/2012; 78(12):4412-9. · 3.69 Impact Factor
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Pieter De Maayer,
Wai Yin Chan,
Fabio Rezzonico,
Andreas Bühlmann,
Stephanus N Venter,
Jochen Blom,
Alexander Goesmann,
Jürg E Frey, Theo H M Smits,
Brion Duffy,
Teresa A Coutinho
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ABSTRACT: The enterobacterium Pantoea ananatis is an ecologically versatile species. It has been found in the environment, as plant epiphyte and endophyte, as an emerging phytopathogen, and as a presumptive, opportunistic human pathogen. Here, we report the complete genome sequence of P. ananatis LMG 5342, isolated from a human wound.
Journal of bacteriology 03/2012; 194(6):1615-6. · 3.94 Impact Factor
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ABSTRACT: The LuxS enzyme, an S-ribosyl-homocysteine lyase, catalyzes the production of the signal precursor for autoinducer-2 mediated quorum sensing (QS-2) in Vibrio. Its widespread occurrence among bacteria is often considered the evidence for a universal language for interspecies communication. Presence of the luxS gene and production of the autoinducer-2 (AI-2) signal have repeatedly been the only evidences presented to assign a functional QS-2 to the most diverse species. In fact, LuxS has a primary metabolic role as part of the activated methyl cycle. In this review we have analyzed the distribution of QS-2 related genes in Enterobacteriaceae by moving the focus of the investigation from AI-2 production to the detection of potential AI-2 receptors. The latter are common in pathogens or endosymbionts of animals, but were also found in a limited number of Enterobacteriaceae of the genera Enterobacter, Klebsiella, and Pantoea that live in close association with plants or fungi. Although a precise function of QS-2 in these species has not been identified, they all show an endophytic or endosymbiontic lifestyle that suggests a role of type-2 quorum sensing in the adaptation to closed ecosystems.
Sensors 01/2012; 12(5):6645-65. · 1.74 Impact Factor
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ABSTRACT: Genomics has clarified the biosynthetic pathway for desferrioxamine E critical for iron acquisition in the enterobacterial fire blight pathogen Erwinia amylovora. Evidence for each of the individual steps and the role of desferrioxamine E biosynthesis in pathogen virulence and cell protection from host defenses is presented. Using comparative genomics, it can be concluded that desferrioxamine biosynthesis is ancestral within the genera Erwinia and Pantoea.
Archives of Microbiology 08/2011; 193(10):693-9. · 1.43 Impact Factor
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ABSTRACT: The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated. Comparative analysis with 15 Xanthomonas plasmids and 19 complete genomes revealed that nearly one-fourth of this plasmid has high sequence identity to plasmid pXAC64 and an 8.8-kb chromosomal region of Xanthomonas axonopodis pv. citri strain 306 carrying genes that encode type III effectors and helper proteins. The presence of pXap41 in all X. arboricola pv. pruni genotypes was confirmed for eight strains by plasmid profiling and for 35 X. arboricola pv. pruni isolates with a new plasmid multiplex PCR assay. This plasmid was not detected in any other X. arboricola pathovars (n=12), indicating the potential for the application of the pXap41 PCR method as a pathovar-level detection and identification tool.
FEMS Microbiology Letters 07/2011; 323(1):52-60. · 2.04 Impact Factor
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ABSTRACT: The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system confers acquired heritable immunity against mobile nucleic acid elements in prokaryotes, limiting phage infection and horizontal gene transfer of plasmids. In CRISPR arrays, characteristic repeats are interspersed with similarly sized nonrepetitive spacers derived from transmissible genetic elements and acquired when the cell is challenged with foreign DNA. New spacers are added sequentially and the number and type of CRISPR units can differ among strains, providing a record of phage/plasmid exposure within a species and giving a valuable typing tool. The aim of this work was to investigate CRISPR diversity in the highly homogeneous species Erwinia amylovora, the causal agent of fire blight. A total of 18 CRISPR genotypes were defined within a collection of 37 cosmopolitan strains. Strains from Spiraeoideae plants clustered in three major groups: groups II and III were composed exclusively of bacteria originating from the United States, whereas group I generally contained strains of more recent dissemination obtained in Europe, New Zealand, and the Middle East. Strains from Rosoideae and Indian hawthorn (Rhaphiolepis indica) clustered separately and displayed a higher intrinsic diversity than that of isolates from Spiraeoideae plants. Reciprocal exclusion was generally observed between plasmid content and cognate spacer sequences, supporting the role of the CRISPR/Cas system in protecting against foreign DNA elements. However, in several group III strains, retention of plasmid pEU30 is inconsistent with a functional CRISPR/Cas system.
Applied and environmental microbiology 04/2011; 77(11):3819-29. · 3.69 Impact Factor
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Rachel Powney, Theo H M Smits,
Tim Sawbridge,
Beatrice Frey,
Jochen Blom,
Jürg E Frey,
Kim M Plummer,
Steven V Beer,
Joanne Luck,
Brion Duffy,
Brendan Rodoni
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ABSTRACT: Here, we present the genome of a strain of Erwinia amylovora, the fire blight pathogen, with pathogenicity restricted to Rubus spp. Comparative genomics of ATCC BAA-2158 with E. amylovora strains from non-Rubus hosts identified significant genetic differences but support the inclusion of this strain within the species E. amylovora.
Journal of bacteriology 02/2011; 193(3):785-6. · 3.94 Impact Factor
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ABSTRACT: The genome of Salmonella enterica subsp. enterica serovar Weltevreden strain 2007-60-3289-1 was sequenced. The genome sequence of this fresh-vegetable isolate from Scandinavia will be useful for the elucidation of plant host factors in comparison to other serovars of S. enterica subsp. enterica.
Journal of bacteriology 02/2011; 193(8):2066. · 3.94 Impact Factor
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Pablo Llop,
Jordi Cabrefiga, Theo H M Smits,
Tanja Dreo,
Silvia Barbé,
Joanna Pulawska,
Alain Bultreys,
Jochen Blom,
Brion Duffy,
Emilio Montesinos,
María M López
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ABSTRACT: Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.
PLoS ONE 01/2011; 6(12):e28651. · 4.09 Impact Factor
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ABSTRACT: Pantoea vagans is a commercialized biological control agent used against the pome fruit bacterial disease fire blight, caused by Erwinia amylovora. Compared to other biocontrol agents, relatively little is currently known regarding Pantoea genetics. Better understanding of antagonist mechanisms of action and ecological fitness is critical to improving efficacy.
Genome analysis indicated two major factors Contribute to biocontrol activity: competition for limiting substrates and antibacterial metabolite production. Pathways for utilization of a broad diversity of sugars and acquisition of iron were identified. Metabolism of sorbitol by P. vagans C9-1 may be a major metabolic feature in biocontrol of fire blight. Biosynthetic genes for the antibacterial peptide pantocin A were found on a chromosomal 28-kb genomic island, and for dapdiamide E on the plasmid pPag2. There was no evidence of potential virulence factors that could enable an animal or phytopathogenic lifestyle and no indication of any genetic-based biosafety risk in the antagonist.
Identifying key determinants contributing to disease suppression allows the development of procedures to follow their expression in planta and the genome sequence contributes to rationale risk assessment regarding the use of the biocontrol strain in agricultural systems.
PLoS ONE 01/2011; 6(7):e22247. · 4.09 Impact Factor
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ABSTRACT: Pantoea vagans is a Gram-negative enterobacterial plant epiphyte of a broad range of plants. Here we report the 4.89-Mb genome sequence of P. vagans strain C9-1 (formerly Pantoea agglomerans), which is commercially registered for biological control of fire blight, a disease of pear and apple trees caused by Erwinia amylovora.
Journal of bacteriology 10/2010; 192(24):6486-7. · 3.94 Impact Factor
