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Applied and Environmental Microbiology, December 2004, p. 7511-7519, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7511-7519.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

Tamara M. Maier,1 Andrea Havig,1 Monika Casey,1 Francis E. Nano,2 Dara W. Frank,1 and Thomas C. Zahrt1*

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin,1 Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada2

Received 14 May 2004/ Accepted 3 August 2004

Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 x 107 CFU µg of DNA–1 in F. tularensis LVS, Francisella novicida U112, and E. coli DH5{alpha}. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, The Medical College of Wisconsin, 8701 Watertown Plank Rd., P.O. Box 26509, Milwaukee, WI 53226-0509. Phone: (414) 456-7429. Fax: (414) 456-6535. E-mail: tzahrt{at}mcw.edu.

Applied and Environmental Microbiology, December 2004, p. 7511-7519, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7511-7519.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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