title: Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes.
abstract: Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10(1-3) in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.
authors.affiliation: Department of Chemical Biology & Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan. nhirano@chem.ce.nihon-u.ac.jp
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Hirano
authors.forename: Tetsurou
authors.initials: T
authors.lastname: Muroi
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Kihara
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Hideo
authors.initials: H
authors.lastname: Takahashi
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
mesh_terms: D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005827:Genetics, Microbial / Q000379:methods*; D016680:Genome, Bacterial; D019426:Integrases / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D019426:Integrases