title: Development of a versatile method for targeted gene deletion and insertion by using the pyrF gene in the psychrotrophic bacterium, Shewanella livingstonensis Ac10.
abstract: Shewanella livingstonensis Ac10, a psychrotrophic bacterium isolated from Antarctic seawater, grows well at low temperatures close to 0°C. The bacterium is useful as a host in a low-temperature protein expression system. It is also useful as a model microorganism to investigate the mechanisms of microbial cold-adaptation. Versatile genetic manipulation techniques would be useful to investigate the biology of this bacterium and to develop its applications. In this study, we developed a method for targeted gene deletion and insertion by using the gene coding for orotidine-5'-phosphate decarboxylase (pyrF), which is involved in pyrimidine synthesis. We found that S. livingstonensis Ac10 is sensitive to 5-fluoroorotic acid (5-FOA), which is converted to a highly toxic compound by the product of pyrF. A uracil-auxotrophic strain resistant to 5-FOA was constructed by deleting pyrF, thus allowing the use of a plasmid-borne copy of pyrF for selection of recombinants. We constructed the pyrF complementation suicide plasmid pKKP, which contains pyrF, the R6K replication origin, the mob site of RP4, an antibiotic marker gene, and a multiple cloning site. To demonstrate pyrF-based gene replacement, we deleted the internal region of orf5, the gene coding for an eicosapentaenoic acid (EPA) synthesis enzyme. We also successfully inserted a His6-tag-coding sequence into orf8, the gene coding for another EPA synthesis enzyme. This system allows the markerless deletion and insertion of desired sequences at specific sites in the genome, which remarkably facilitates genetic manipulation of this bacterium.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ito
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Chunjie
authors.initials: C
authors.lastname: Gong
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Kawamoto
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address: kurihara@scl.kyoto-u.ac.jp.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kurihara
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics; D001483:Base Sequence; D003080:Cold Temperature; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D030781:Organisms, Genetically Modified; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D009963:Orotic Acid; C001242:5-fluoroorotic acid; D009964:Orotidine-5'-Phosphate Decarboxylase
keywords: Gene deletion; Gene insertion; Homologous recombination; Shewanella; pyrF