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Applied and Environmental Microbiology, April 2008, p. 2161-2170, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.02360-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis{triangledown}

Joseph Horzempa,1,{dagger} Deanna M. Tarwacki,1,{dagger} Paul E. Carlson Jr.,1 Cory M. Robinson,1 and Gerard J. Nau1,2,3*

Department of Microbiology and Molecular Genetics,1 Division of Infectious Diseases, Department of Medicine,2 Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152613

Received 19 October 2007/ Accepted 24 January 2008

Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, E1256 BSTWR, 200 Lothrop St., Pittsburgh, PA 15261. Phone: (412) 383-9986. Fax: (412) 624-1401. E-mail: gjnau{at}pitt.edu

{triangledown} Published ahead of print on 1 February 2008.

{dagger} J.H. and D.M.T. contributed equally to this paper.

Applied and Environmental Microbiology, April 2008, p. 2161-2170, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.02360-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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