Characterization and application of a glucose-repressible promoter in Francisella tularensis

Department of Microbiology and Molecular Genetics, Department of Medicine – Division of Infectious Diseases, and Center for Vaccine Research University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

^* To whom correspondence should be addressed. Email: gjnau{at}pitt.edu.

	Abstract

Francisella tularensis, the causative agent of tularemia, is a Category A biodefense agent. 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, FGRp), allowing management of downstream gene expression. In bacteria cultured in media lacking glucose, this promoter induced 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 media restored IglC levels, indicating the usefulness of this promoter for controlling expression of both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during infection of human macrophages using the fluorescence reporter. In this environment, FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. Analysis of genomic sequence indicated that this promoter region controls a gene encoding a hypothetical protein, FTL_0580. Deletion analysis localized critical sites essential for FGRp activity to be within a 44 base pair 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.