Shewanella livingstonensis Ac10, a psychrotrophic bacterium isolated from Antarctic seawater, grows well at low temperatures close to 0 degrees 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 Hiss-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. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.