title: Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain.
abstract: Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria.
authors.affiliation: Bacterial Vaccines and Immune Sera, Department of Veterinary Medicine, Paul Ehrlich Institute, Langen, 63225, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Roos
authors.forename: Esther
authors.initials: E
authors.lastname: Werner
authors.forename: Holger
authors.initials: H
authors.lastname: Loessner
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D018628:Gene Dosage; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D022562:Salmonella Vaccines / Q000235:genetics* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D022562:Salmonella Vaccines