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

Abstract

ABSTRACT 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 g lucose- r epressible p romoter, 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.