Title: Engineering of bacteriophages Y2::dpoL1-C and Y2::luxAB for efficient control and rapid detection of the fire blight pathogen, Erwinia amylovora
Authors : Born, Yannick
Fieseler, Lars
Thöny, Valentin
Leimer, Nadja
Duffy, Brion
Loessner, Martin J.
Published in : Applied and Environmental Microbiology
Volume(Issue) : 83
Issue : 12
Pages : e00341-17
Publisher / Ed. Institution : American Society for Microbiology
Issue Date: Jun-2017
License (according to publishing contract) : Licence according to publishing contract
Type of review: Not specified
Language : English
Subjects : Bacteriophage; Depolymerase; Fire blight; Luciferase; Recombinant phage; Reporter; Reporter phage; Bacteriophages; Erwinia amylovora; Viral gene expression regulation; Genetic engineering; Malus; Plant diseases; Viral proteins; Virulence
Subject (DDC) : 570: Biology
Abstract: 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.
Departement: Life Sciences and Facility Management
Organisational Unit: Institute of Food and Beverage Innovation (ILGI)
Publication type: Article in scientific Journal
DOI : 10.1128/AEM.00341-17
ISSN: 0099-2240
1098-5336
URI: https://digitalcollection.zhaw.ch/handle/11475/12255
Appears in Collections:Publikationen Life Sciences und Facility Management

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