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Applied and Environmental Microbiology, February 2002, p. 705-712, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.705-712.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

Arja Lamberg, Sari Nieminen, Mingqiang Qiao,^, and Harri Savilahti^*

Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, 00014 Helsinki, Finland

Received 17 July 2001/ Accepted 16 November 2001

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 10⁴ to 10⁶ CFU/µg 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.

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* Corresponding author. Mailing address: Institute of Biotechnology, Viikki Biocenter, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Finland. Phone: 358-9-191 59516. Fax: 358-9-191 59366. E-mail: harri.savilahti{at}helsinki.fi.

Present address: Institute for Molecular Biology, Nankai University, Tianjin 300071, People’s Republic of China.

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Applied and Environmental Microbiology, February 2002, p. 705-712, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.705-712.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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