Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

Francisella tularensis; is the causative agent of the life-threatening disease tularemia. However, the molecular tools to study; Francisella; are limited. Especially, expression plasmids are sparse and difficult to use, as they are unstable and prone to spontaneous loss. Most; Francisella; expression plasmids lack inducible promoters making it difficult to control gene expression levels. In addition, available expression plasmids are mainly designed for; F. tularensis; , however, genetic differences including restriction-modification systems impede the use of these plasmids in; F. novicida; , which is often used as a model organism to study; Francisella; pathogenesis. Here we report construction and characterization of two mobilizable plasmids (pFNMB1 and pFNMB2) designed for regulated gene expression in; F. novicida; . pFNMB plasmids contain a tetracycline inducible promoter to control gene expression levels and; oriT; for RP4 mediated mobilization. We show that both plasmids are stably maintained in bacteria for more than 40 generations over 4 days of culturing in the absence of selection against plasmid loss. Expression levels are dependent on anhydrotetracycline concentration and homogeneous in a bacterial population. pFNMB1 and pFNMB2 plasmids differ in the sequence between promoter and translation start site and thus allow to reach different maximum levels of protein expression. We used pFNMB1 and pFNMB2 for complementation of; Francisella; Pathogenicity Island mutants Δ; iglF; , Δ; iglI; , and Δ; iglC in-vitro; and pFNMB1 to complement Δ; iglI; mutant in bone marrow derived macrophages