Fogg, Paul C M orcid.org/0000-0001-5324-4293, Haley, Joshua A, Stark, W Marshall et al. (1 more author) (2017) Genome integration and excision by a new Streptomyces bacteriophage, ϕJoe. Applied and Environmental Microbiology. e02767-16. pp. 1-43. ISSN 0099-2240
Abstract
Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the φJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from S. venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int/attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE: Streptomyces spp. are prolific producers of secondary metabolites including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo The specific recognition site for integration is present in a wide range of different Actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2016 Fogg et al. |
Keywords: | Recombination directionality factor,Streptomyces coelicolor,Streptomyces venezuelae,Serine integrase,Integration vector,Bacteriophage genetics,Mobile genetic elements,R4-like phage,Bacteriophages/enzymology,Viral Proteins/genetics,Interspersed Repetitive Sequences/genetics,Attachment Sites, Microbiological/genetics,Recombination, Genetic,Base Sequence,Genes, Viral,Soil Microbiology,Escherichia coli/genetics,Virus Integration/genetics,Sequence Alignment,Streptomyces/genetics,Models, Biological,Plasmids,Genetic Engineering/methods,Genome, Viral/genetics,Genetic Vectors,Integrases/metabolism,DNA, Viral |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 17 Jan 2017 17:04 |
Last Modified: | 07 Dec 2024 00:12 |
Published Version: | https://doi.org/10.1128/AEM.02767-16 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1128/AEM.02767-16 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:110735 |
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