title: New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence.
abstract: Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.
authors.affiliation: Microbiology Research Group, Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto 619-0292, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Suzuki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nonaka
authors.forename: Yota
authors.initials: Y
authors.lastname: Tsuge
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inui
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yukawa
mesh_terms: D001483:Base Sequence; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000254:growth & development; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D017353:Gene Deletion; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial