title: Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu.
abstract: An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10(4) to 10(6) CFU/microg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.
authors.affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
authors.forename: Arja
authors.initials: A
authors.lastname: Lamberg
authors.forename: Sari
authors.initials: S
authors.lastname: Nieminen
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Qiao
authors.forename: Harri
authors.initials: H
authors.lastname: Savilahti
mesh_terms: D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D016680:Genome, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*
chemical_list: D004251:DNA Transposable Elements