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Applied and Environmental Microbiology, May 2008, p. 2619-2626, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02905-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Targeted Inactivation of Francisella tularensis Genes by Group II Introns{triangledown}

Stephen A. Rodriguez,1 Jieh-Juen Yu,1 Greg Davis,2 Bernard P. Arulanandam,1 and Karl E. Klose1*

South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas San Antonio, San Antonio, Texas 78249,1 Sigma-Aldrich Biotechnology, Genomics R&D, St. Louis, Missouri2

Received 21 December 2007/ Accepted 21 February 2008

Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.

* Corresponding author. Mailing address: Dept. of Biology, University of Texas San Antonio, One UTSA Circle, San Antonio, TX 78249. Phone: (210) 458-6140. Fax: (210) 458-4468. E-mail: kklose{at}utsa.edu

{triangledown} Published ahead of print on 29 February 2008.

Applied and Environmental Microbiology, May 2008, p. 2619-2626, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02905-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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