Genome Integration and Excision by a New Streptomyces Bacteriophage, ÏJoe.
Appl Environ Microbiol
; 83(5)2017 03 01.
Article
en En
| MEDLINE
| ID: mdl-28003200
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, ÏJoe, and investigate the conditions for integration and excision of the ÏJoe genome. ÏJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ÏJoe int-attP locus. The attB site for ÏJoe integrase was occupied in several Streptomyces genomes, including that of 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 ÏJoe RDF was identified, and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ÏJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox.IMPORTANCEStreptomyces 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, ÏJoe. ÏJoe 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Streptomyces
/
Bacteriófagos
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Integración Viral
/
Genoma Viral
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Appl Environ Microbiol
Año:
2017
Tipo del documento:
Article
País de afiliación:
Reino Unido