Comparative genomics to examine the endophytic potential of Pantoea agglomerans DAPP-PG 734.

Pantoea agglomerans DAPP-PG 734 was isolated as endophyte from knots (tumors) caused by Pseudomonas savastanoi pv. savastanoi DAPP-PG 722 in olive trees. To understand the plant pathogen-endophyte interaction on a genomic level, the whole genome of P. agglomerans DAPP-PG 734 was sequenced and annotated. The complete genome had a total size of 5'396'424 bp, containing one circular chromosome and four large circular plasmids. The aim of this study was to identify genomic features that could play a potential role in the interaction between P. agglomerans DAPP-PG 734 and P. savastanoi pv. savastanoi DAPP-PG 722. For this purpose, a comparative genomic analysis between the genome of P. agglomerans DAPP-PG 734 and those of related Pantoea spp. was carried out. In P. agglomerans DAPP-PG 734, gene clusters for the synthesis of the Hrp-1 type III secretion system (T3SS), type VI secretion systems (T6SS) and autoinducer, which could play an important role in a plant-pathogenic community enhancing knot formation in olive trees, were identified. Additional gene clusters for the biosynthesis of two different antibiotics, namely dapdiamide E and antibiotic B025670, which were found in regions between integrative conjugative elements (ICE), were observed. The in-depth analysis of the whole genome suggested a characterization of the P. agglomerans DAPP-PG 734 isolate as endophytic bacterium with biocontrol activity rather than as a plant pathogen.

Resolving taxonomic confusion: establishing the genus Phytobacter on the list of clinically relevant Enterobacteriaceae.

Although many clinically significant strains belonging to the family Enterobacteriaceae fall into a restricted number of genera and species, there is still a substantial number of isolates that elude this classification and for which proper identification remains challenging. With the current improvements in the field of genomics, it is not only possible to generate high-quality data to accurately identify individual nosocomial isolates at the species level and understand their pathogenic potential but also to analyse retrospectively the genome sequence databases to identify past recurrences of a specific organism, particularly those originally published under an incorrect or outdated taxonomy. We propose a general use of this approach to classify further clinically relevant taxa, i.e., Phytobacter spp., that have so far gone unrecognised due to unsatisfactory identification procedures in clinical diagnostics. Here, we present a genomics and literature-based approach to establish the importance of the genus Phytobacter as a clinically relevant member of the Enterobacteriaceae family.

Environmental parameters and microbial community profiles as indication towards microbial activities and diversity in aquaponic system compartments.

BACKGROUND: An aquaponic system couples cultivation of plants and fish in the same aqueous medium. The system consists of interconnected compartments for fish rearing and plant production, as well as for water filtration, with all compartments hosting diverse microbial communities, which interact within the system. Due to the design, function and operation mode of the individual compartments, each of them exhibits unique biotic and abiotic conditions. Elucidating how these conditions shape microbial communities is useful in understanding how these compartments may affect the quality of the water, in which plants and fish are cultured. RESULTS: We investigated the possible relationships between microbial communities from biofilms and water quality parameters in different compartments of the aquaponic system. Biofilm samples were analyzed by total community profiling for bacterial and archaeal communities. The results implied that the oxygen levels could largely explain the main differences in abiotic parameters and microbial communities in each compartment of the system. Aerobic system compartments are highly biodiverse and work mostly as a nitrifying biofilter, whereas biofilms in the anaerobic compartments contain a less diverse community. Finally, the part of the system connecting the aerobic and anaerobic processes showed common conditions where both aerobic and anaerobic processes were observed. CONCLUSION: Different predicted microbial activities for each compartment were found to be supported by the abiotic parameters, of which the oxygen saturation, total organic carbon and total nitrogen differentiated clearly between samples from the main aerobic loop and the anaerobic compartments. The latter was also confirmed using microbial community profile analysis.

Broad diversity of bacteria degrading 17ß-estradiol-3-sulfate isolated from river sediment and biofilm at a wastewater treatment plant discharge.

Conjugated estrogens, such as 17ß-estradiol-3-sulfate (E2-3S), can be released into aquatic environments through wastewater treatment plants (WWTP). There, they are microbiologically degraded into free estrogens, which can have harmful effects on aquatic wildlife. Here, the degradation of E2-3S in environmental samples taken upstream, downstream and at the effluent of a WWTP was assessed. Sediment and biofilm samples were enriched for E2-3S-degrading microorganisms, yielding a broad diversity of bacterial isolates, including known and novel degraders of estrogens. Since E2-3S-degrading bacteria were also isolated in the sample upstream of the WWTP, the WWTP does not influence the ability of the microbial community to degrade E2-3S.

Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens.

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum. A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum. Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum. The genome sequences of the strains had average nucleotide identity values ranging from 94.35-95.19 % and in silico DNA-DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain.

Phytobacter palmae sp. nov., a novel endophytic, N2 fixing, plant growth promoting Gammaproteobacterium isolated from oil palm (Elaeis guineensis Jacq.).

A novel strain S29T with high nitrogen fixing ability was isolated from surface-sterilized leaf tissues of oil palm (Elaeis guineensis) growing in Science Park II, Singapore. On the basis of 16S rRNA gene analysis and multilocus sequence typing with the rpoB, gyrB, infB and atpD genes, strain S29T was a member of the genus Phytobacter, with Phytobacter ursingii ATCC 27989T and Phytobacter diazotrophicus LS 8T as its closest relatives. Unique biochemical features that differentiated strain S29T from its closest relatives were the ability to utilize melibiose, α-cyclodextrin, glycogen, adonital, d-arabitol, m-inositol and xylitol. The major fatty acids were C14 : 0, C16 : 0, C17 : 0, C16 : 1 ω5c and summed feature 2 containing C16 : 1 ω7c and/or C16 : 1 ω6c. The polar fatty acid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and aminolipids. The draft genome of strain S29T comprised 5, 284, 330 bp with a G + C content of 52.6 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain S29T and the phylogenetically related Enterobacterales species were lower than 95 % and 70 %, respectively. Thus, the polyphasic evidences generated through the phenotypic, chemotaxonomic and genomic methods confirmed that strain S29T represents a novel species of the genus Phytobacter, for which we propose the name Phytobacter palmae sp. nov. with the type strain of S29T (=DSM 27342T=KACC 17598T).

The importance of genome sequence quality to microbial comparative genomics.

The quality of microbial genome sequences has been a concern ever since the emergence of genome sequencing. The quality of the genome assemblies is dependent on the sequencing technology used and the aims for which the sequence was generated. Novel sequencing and bioinformatics technologies are not intrinsically better than the older technologies, although they are generally more efficient. In this correspondence, the importance for comparative genomics of additional manual assembly efforts over autoassembly and careful annotation is emphasized.

Comparative genomics and pathogenicity potential of members of the Pseudomonas syringae species complex on Prunus spp.

BACKGROUND: Diseases on Prunus spp. have been associated with a large number of phylogenetically different pathovars and species within the P. syringae species complex. Despite their economic significance, there is a severe lack of genomic information of these pathogens. The high phylogenetic diversity observed within strains causing disease on Prunus spp. in nature, raised the question whether other strains or species within the P. syringae species complex were potentially pathogenic on Prunus spp. RESULTS: To gain insight into the genomic potential of adaptation and virulence in Prunus spp., a total of twelve de novo whole genome sequences of P. syringae pathovars and species found in association with diseases on cherry (sweet, sour and ornamental-cherry) and peach were sequenced. Strains sequenced in this study covered three phylogroups and four clades. These strains were screened in vitro for pathogenicity on Prunus spp. together with additional genome sequenced strains thus covering nine out of thirteen of the currently defined P. syringae phylogroups. Pathogenicity tests revealed that most of the strains caused symptoms in vitro and no obvious link was found between presence of known virulence factors and the observed pathogenicity pattern based on comparative genomics. Non-pathogenic strains were displaying a two to three times higher generation time when grown in rich medium. CONCLUSION: In this study, the first set of complete genomes of cherry associated P. syringae strains as well as the draft genome of the quarantine peach pathogen P. syringae pv. persicae were generated. The obtained genomic data were matched with phenotypic data in order to determine factors related to pathogenicity to Prunus spp. Results of this study suggest that the inability to cause disease on Prunus spp. in vitro is not the result of host specialization but rather linked to metabolic impairments of individual strains.

Pantocin A, a peptide-derived antibiotic involved in biological control by plant-associated Pantoea species.

The genus Pantoea contains a broad range of plant-associated bacteria, including some economically important plant pathogens as well as some beneficial members effective as biological control agents of plant pathogens. The most well-characterized representatives of biological control agents from this genus generally produce one or more antimicrobial compounds adding to biocontrol efficacy. Some Pantoea species evaluated as biocontrol agents for fire blight disease of apple and pear produce a histidine-reversible antibiotic. Three commonly studied histidine-reversible antibiotics produced by Pantoea spp. are herbicolin O, MccEh252, and pantocin A. Pantocin A is a novel ribosomally encoded and post-translationally modified peptide natural product. Here, we review the current knowledge on the chemistry, genetics, biosynthesis, and incidence and environmental relevance of pantocin A and related histidine-reversible antibiotics produced by Pantoea.

Pseudomonas tructae sp. nov., novel species isolated from rainbow trout kidney.

This study describes the biochemical and phylogenetic characteristics of a Gram-negative strain, SNU WT1T, isolated from rainbow trout kidney. The 16S rRNA gene sequencing indicated that strain SNU WT1T was highly similar to Pseudomonas wadenswilerensis CCOS 864T and closely related to other Pseudomonas putida-related strains. Multilocus sequence analysis of concatenated partial gyrB, rpoB and rpoD sequences revealed that strain SNU WT1T was distinct from P. putida-related strains and formed a separate clade. The average nucleotide identity and Genome-to-Genome Distance Calculator values were 90.19 and 41.7 %with its closest relative P. wadenswilerensis CCOS 864T; however, it was phenotypically distinct from CCOS 864T with respect to arginine dihydrolase, glucose fermentation, aesculin hydrolysis and N-acetyl-glucosamine assimilation. The major polar lipid of the strain was phosphatidylethanolamine and the major quinone was Q-9. The genome of strain SNU WT1T had 5 685 196 bp with a G+C content of 61.83 mol%. We describe a novel species of genus Pseudomonas, for which the name Pseudomonastructae has been proposed, with SNU WT1T (=KCTC 72265=JCM 33436) as the type strain.

Emended description of the genus Phytobacter, its type species Phytobacter diazotrophicus (Zhang 2008) and description of Phytobacter ursingii sp. nov.

The species Phytobacter diazotrophicus and the associated genus Phytobacter were originally described by Zhanget al. [Arch Microbiol189 (2008), 431-439] on the basis of few endophytic nitrogen-fixing bacteria isolated from wild rice (Oryza rufipogon) in China. In this study, we demonstrate that a number of clinical isolates that were either described in the literature, preserved in culture collections, or obtained during a 2013 multi-state sepsis outbreak in Brazil also belong to the same genus. 16S rRNA gene sequencing, multilocus sequence analysis based on gyrB, rpoB, atpD and infB genes, as well as digital DNA-DNA hybridization support the existence of a second species within the genus Phytobacter. All isolates from the recent Brazilian outbreak, along with some older American clinical strains, were found to belong to the already described species Phytobacterdiazotrophicus, whereas three clinical strains retrieved in the USA over a time span of almost four decades, could be assigned to a new Phytobacter species. Implementation of an extended set of biochemical tests showed that the two Phytobacter species could phenotypically be discriminated from each other by the ability to utilize l-sorbose and d-serine. This feature was limited to the strains of the novel species described herein, for which the name Phytobacter ursingii sp. nov. is proposed, with ATCC 27989T (=CNCTC 5729T) as the designated type strain. An emended description of the species Phytobacter diazotrophicus and of the genus Phytobacter is also provided.

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil.

BACKGROUND: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. METHODS: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. RESULTS: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970's. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. CONCLUSION: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans.

Role of the type VI secretion systems during disease interactions of Erwinia amylovora with its plant host.

BACKGROUND: Type VI secretion systems (T6SS) are widespread among Gram-negative bacteria and have a potential role as essential virulence factors or to maintain symbiotic interactions. Three T6SS gene clusters were identified in the genome of E. amylovora CFBP 1430, of which T6SS-1 and T6SS-3 represent complete T6SS machineries, while T6SS-2 is reduced in its gene content. RESULTS: To assess the contribution of T6SSs to virulence and potential transcriptomic changes of E. amylovora CFBP 1430, single and double mutants in two structural genes were generated for T6SS-1 and T6SS-3. Plant assays showed that mutants in T6SS-3 were slightly more virulent in apple shoots while inducing less disease symptoms on apple flowers, indicating that T6SSs have only a minor effect on virulence of E. amylovora CFBP 1430. The mutations led under in vitro conditions to the differential expression of type III secretion systems, iron acquisition, chemotaxis, flagellar, and fimbrial genes. Comparison of the in planta and in vitro transcriptome data sets revealed a common differential expression of three processes and a set of chemotaxis and motility genes. Additional experiments proved that T6SS mutants are impaired in their motility. CONCLUSION: These results suggest that the deletion of T6SSs alters metabolic and motility processes. Nevertheless, the difference in lesion development in apple shoots and flower necrosis of T6SS mutants was indicative that T6SSs influences the disease progression and the establishment of the pathogen on host plants.

Microbial diversity in different compartments of an aquaponics system.

Aquaponics is a solution for sustainable production of fish and plants in a single semi-closed system, where nutrient-rich water from the aquaculture provides nutrients for plant growth. We examined the microbial communities within an experimental aquaponics system. Whereas the fish feces contained a separate community dominated by bacteria of the genus Cetobacterium, the samples from plant roots, biofilter, and periphyton were more similar to each other, while the communities were more diverse. Detailed examination of the data gave the first indications to functional groups of organisms in the different compartments of the aquaponic system. As other nitrifiers other than members of the genus Nitrospira were only present at low numbers, it was anticipated that Nitrospirae may perform the nitrification process in the biofilm.

Pseudomonas wadenswilerensis sp. nov. and Pseudomonas reidholzensis sp. nov., two novel species within the Pseudomonas putida group isolated from forest soil.

Within the frame of a biotechnological screening, we isolated two Pseudomonas strains from forest soil. 16S rRNA gene sequence analysis indicated that strain CCOS 864T shared 99.8 % similarity with Pseudomonas donghuensis HYST, while strain CCOS 865T shared 99.0 % similarity with Pseudomonas putida DSM 291T and lower similarity with other P. putida group type strains. Based on multilocus sequence analysis, the two strains were genotypically distinct from each other, each forming a separate clade. Strains CCOS 864T and CCOS 865T were Gram-stain-negative, motile and rod-shaped, growing at a temperature range of 4-37 °C. Strain CCOS 864T could be phenotypically distinguished from P. putida group species by the combination of gelatinase-positive reaction and positive growth on N-acetyl-d-glucosamine, p-hydroxyphenylacetic acid and inosine but lack of fluorescein production on King's B medium, while strain CCOS 865T could be distinguished from P. putida group species by the combination of positive growth with saccharic acid and negative growth with p-hydroxyphenylacetic acid and l-pyroglutamic acid. The major polar lipid for both strains was phosphatidylethanolamine; the major quinone was ubiquinone Q-9. DNA-DNA hybridization and average nucleotide identities confirmed the novel species status for the two strains. The DNA G+C contents of CCOS 864T and CCOS 865T were 62.1 and 63.8 mol%, respectively. The phenotypic, phylogenetic and DNA-DNA relatedness data support the suggestion that CCOS 864T and CCOS 865T represent two novel Pseudomonas species. The names Pseudomonas wadenswilerensis sp. nov. (type strain CCOS 864T=LMG 29327T) and Pseudomonas reidholzensis sp. nov. (type strain CCOS 865T=LMG 29328T) are proposed.

Phylogenomic resolution of the bacterial genus Pantoea and its relationship with Erwinia and Tatumella.

Investigation of the evolutionary relationships between related bacterial species and genera with a variety of lifestyles have gained popularity in recent years. For analysing the evolution of specific traits, however, a robust phylogeny is essential. In this study we examined the evolutionary relationships among the closely related genera Erwinia, Tatumella and Pantoea, and also attempted to resolve the species relationships within Pantoea. To accomplish this, we used the whole genome sequence data for 35 different strains belonging to these three genera, as well as nine outgroup taxa. Multigene datasets consisting of the 1039 genes shared by these 44 strains were then generated and subjected to maximum likelihood phylogenetic analyses, after which the results were compared to those using conventional multi-locus sequence analysis (MLSA) and ribosomal MLSA (rMLSA) approaches. The robustness of the respective phylogenies was then explored by considering the factors typically responsible for destabilizing phylogenetic trees. We found that the nucleotide datasets employed in the MLSA, rMLSA and 1039-gene datasets contained significant levels of homoplasy, substitution saturation and differential codon usage, all of which likely gave rise to the observed lineage specific rate heterogeneity. The effects of these factors were much less pronounced in the amino acid dataset for the 1039 genes, which allowed reconstruction of a fully supported and resolved phylogeny. The robustness of this amino acid tree was also supported by different subsets of the 1039 genes. In contrast to the smaller datasets (MLSA and rMLSA), the 1039 amino acid tree was also not as sensitive to long-branch attraction. The robust and well-supported evolutionary hypothesis for the three genera, which confidently resolved their various inter- and intrageneric relationships, represents a valuable resource for future studies. It will form the basis for studies aiming to understand the forces driving the divergence and maintenance of lineages, species and biological traits in this important group of bacteria.

Specific surface glycan decorations enable antimicrobial peptide resistance in plant-beneficial pseudomonads with insect-pathogenic properties.

Some plant-beneficial pseudomonads can invade and kill pest insects in addition to their ability to protect plants from phytopathogens. We explored the genetic basis of O-polysaccharide (O-PS, O-antigen) biosynthesis in the representative insecticidal strains Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 and investigated its role in insect pathogenicity. Both strains produce two distinct forms of O-PS, but differ in the organization of their O-PS biosynthesis clusters. Biosynthesis of the dominant O-PS in both strains depends on a gene cluster similar to the O-specific antigen (OSA) cluster of Pseudomonas aeruginosa. In CHA0 and other P. protegens strains, the OSA cluster is extensively reduced and new clusters were acquired, resulting in high diversity of O-PS structures, possibly reflecting adaptation to different hosts. CHA0 mutants lacking the short OSA form of O-PS were significantly impaired in insect virulence in Galleria injection and Plutella feeding assays. CHA0, PCL1391, and other insecticidal pseudomonads exhibited high resistance to antimicrobial peptides, including cecropins that are central to insect immune defense. Resistance of both model strains depended on the dominant OSA-type O-PS. Our results suggest that O-antigen is essential for successful insect infection and illustrate, for the first time, its importance in resistance of Pseudomonas to antimicrobial peptides.

Erwinia gerundensis sp. nov., a cosmopolitan epiphyte originally isolated from pome fruit trees.

A survey to obtain potential antagonists of pome fruit tree diseases yielded two yellow epiphytic bacterial isolates morphologically similar to Pantoea agglomerans, but showing no biocontrol activity. Whole-cell MALDI-TOF mass spectrometry and analysis of 16S rRNA gene and gyrB sequences suggested the possibility of a novel species with a phylogenetic position in either the genus Pantoea or the genus Erwinia. Multi-locus sequence analysis (MLSA) placed the two strains in the genus Erwinia and supported their classification as a novel species. The strains showed general phenotypic characteristics typical of this genus and results of DNA-DNA hybridizations confirmed that they represent a single novel species. Both strains showed a DNA G+C content, as determined by HPLC, of 54.5 mol% and could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, potassium 2-ketogluconate, maltose, melibiose and raffinose. Whole-genome sequencing of strain EM595T revealed the presence of a chromosomal carotenoid biosynthesis gene cluster similar to those found in species of the genera Cronobacter and Pantoea that explains the pigmentation of the strain, which is atypical for the genus Erwinia. Additional strains belonging to the same species were recovered from different plant hosts in three different continents, revealing the cosmopolitan nature of this epiphyte. The name Erwinia gerundensis sp. nov. is proposed, with EM595T ( = LMG 28990T = CCOS 903T) as the designated type strain.

Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites.

Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region.

A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.