title: Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe.
abstract: Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. InStreptomyces, 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 newStreptomycesphage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largestStreptomycesphage cluster (R4-like) and encodes a serine integrase. TheattBsite fromStreptomyces venezuelaewas used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoeint-attPlocus. TheattBsite for ϕJoe integrase was occupied in severalStreptomycesgenomes, including that ofS. 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 confirmedin vivoBoth the integrase and RDF were active inin vitrorecombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox.IMPORTANCEStreptomycesspp. 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 theStreptomyceschromosome 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 isolatedStreptomycesphage, ϕJoe. ϕJoe integrase is activein vitroandin vivoThe specific recognition site for integration is present in a wide range of different actinobacteria, includingStreptomyces venezuelae, an emerging model bacterium inStreptomycesresearch.
authors.affiliation: Biology Department, University of York, York, United Kingdom paul.fogg@york.ac.uk.
authors.forename: Paul C M
authors.identifier: http://orcid.org/0000-0001-5324-4293
authors.initials: PCM
authors.lastname: Fogg
authors.affiliation: Biology Department, University of York, York, United Kingdom.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Haley
authors.affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, United Kingdom.
authors.forename: W Marshall
authors.initials: WM
authors.lastname: Stark
authors.affiliation: Biology Department, University of York, York, United Kingdom.
authors.forename: Margaret C M
authors.identifier: http://orcid.org/0000-0002-4150-0496
authors.initials: MCM
authors.lastname: Smith
mesh_terms: D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004279:DNA, Viral; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics*; D019426:Integrases / Q000378:metabolism; D020071:Interspersed Repetitive Sequences / Q000235:genetics; D008954:Models, Biological; D010957:Plasmids; D011995:Recombination, Genetic; D016415:Sequence Alignment; D012988:Soil Microbiology; D013302:Streptomyces / Q000235:genetics* / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics; D016662:Virus Integration / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; D019426:Integrases
keywords: R4-like phage; Streptomyces coelicolor; Streptomyces venezuelae; bacteriophage genetics; integration vector; mobile genetic elements; recombination directionality factor; serine integrase