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Applied and Environmental Microbiology, November 2004, p. 6901-6904, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6901-6904.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Thomas H. Kawula,* Joshua D. Hall, James R. Fuller, and Robin R. Craven

Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Received 29 March 2004/ Accepted 28 June 2004

ABSTRACT

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 x 10–8 ± 0.87 x 10–8 per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, CB#7290, 804 MEJB, University of North Carolina, Chapel Hill, NC 27599-7290. Phone: (919) 966-9699. Fax: (919) 962-8103. E-mail: kawula{at}med.unc.edu.

Applied and Environmental Microbiology, November 2004, p. 6901-6904, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6901-6904.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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