Novel virulent and broad-host-range Erwinia amylovora bacteriophages reveal a high degree of mosaicism and a relationship to Enterobacteriaceae phages.
ABSTRACT: A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ?Ea21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ?EcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.
Project description:Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting members of the Rosaceae Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range E. amylovora virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of E. amylovora virus L1 (dpoL1-C) or a bacterial luxAB fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein orf68, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::dpoL1-C demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of E. amylovora to colonize the surface of detached flowers. The reporter phage Y2::luxAB transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both in vitro and in plant material.IMPORTANCE Fire blight, caused by Erwinia amylovora, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce E. amylovora on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of E. amylovora cells.
Project description:Phages vB_EamP-S2 (S2) and vB_EamM-Bue1 (Bue1) infect the plant pathogen Erwinia amylovora. S2 has a genome size of 45,495?bp and belongs to the genus SP6virus. The genome size of Bue1, related to Salmonella phage Vil, is 164,037?bp. Both phages possess a depolymerase enzyme, a frequent feature of E. amylovora phages.
Project description:Newly discovered <i>Erwinia amylovora</i> phages PEar1, PEar2, PEar4 and PEar6 were isolated from three different orchards in North Tunisia to study their potential as biocontrol agents. Illumina sequencing revealed that the PEar viruses carry a single-strand DNA genome between 6608 and 6801 nucleotides and belong to the Inoviridae, making them the first described filamentous phages of <i>E. amylovora</i>. Interestingly, phage-infected cells show a decreased swimming and swarming motility and a cocktail of the four phages can significantly reduce infection of <i>E. amylovora</i> in a pear bioassay, potentially making them suitable candidates for phage biocontrol.
Project description:Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.
Project description:Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670?bp double-stranded DNA genome encodes 592 proteins and presents the longest (66?kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.
Project description:Erwinia amylovora is a globally devastating pathogen of apple, pear, and other Rosaceous plants. The use of lytic bacteriophages for disease management continues to garner attention as a possible supplement or alternative to antibiotics. A quantitative productive host range was established for 10 Erwinia phages using 106 wild type global isolates of E. amylovora, and the closely related Erwinia pyrifoliae, to investigate the potential regional efficacy of these phages within a biopesticide. Each host was individually infected with each of the 10 Erwinia phages and phage production after 8 h incubation was measured using quantitative real time PCR (qPCR) in conjunction with a standardized plasmid. PCR amplicons for all phages used in the study were incorporated into a single plasmid, allowing standardized quantification of the phage genome copy number after the infection process. Nine of the tested phages exhibited a broad host range, replicating their genomes by at least one log in over 88% of tested hosts. Also, every Amygdaloideae infecting E. amylovora host was able to increase at least one phage by three logs. Bacterial hosts isolated in western North America were less susceptible to most phages, as the mean genomic titre produced dropped by nearly two logs, and this phenomenon was strongly correlated to the amount of exopolysaccharide produced by the host. This method of host range analysis is faster and requires less effort than traditional plaque assay techniques, and the resulting quantitative data highlight subtle differences in phage host preference not observable with typical plaque-based host range assays. These quantitative host range data will be useful to determine which phages should be incorporated into a phage-mediated biocontrol formulation to be tested for regional and universal control of E. amylovora.
Project description:Fire blight, a plant disease of economic importance caused by Erwinia amylovora, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three E. amylovora-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an Erwinia tasmaniensis strain that is antagonistic for E. amylovora. The American phages resemble two already-described viral genomes, whereas the E. tasmaniensis phage displays a singular genomic sequence in BLAST searches.
Project description:Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E.?amylovora and Pantoea agglomerans with real-time polymerase chain reaction primers developed to detect E.?amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage-mediated biological control agent. Screening of 161 E.?amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P.?agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ?Ea35-20. These laboratory observations suggested that while lysogeny is possible in E.?amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages.
Project description:The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38?45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50?55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin.
Project description:A novel giant phage of the family Myoviridae is described. Pseudomonas phage PA5oct was isolated from a sewage sample from an irrigated field near Wroclaw, Poland. The virion morphology indicates that PA5oct differs from known giant phages. The phage has a head of about 131 nm in diameter and a tail of 136 × 19 nm. Phage PA5oct contains a genome of approximately 375 kbp and differs in size from any tailed phages known. PA5oct was further characterized by determination of its latent period and burst size and its sensitivity to heating, chloroform, and pH.