Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFLN10 based plasmids

Abstract

Francisella tularensis is a category A bioterror pathogen which in some cases can cause a severe and fatal human infection. Very few virulence factors are known in this species due to the difficulty in working with it as well as the lack of tools for genetic manipulation. This work describes the construction of a shuttle vector that can replicate in Escherichia coli and F. tularensis as well as two distinct promoter trap constructs based on the shuttle vector backbone. Replication in F. tularensis is based on the promiscuous origin of replication from the Staphylococcus aureus plasmid pC194. We demonstrate the novel plasmids can coexist with established F. tularensis vectors based on the pFNL10 plasmid, the current workhorse of F. tularensis genetics. Our promoter trap can identify promoters that are activated during intracellular growth and survival. These new vectors provide additional tools for the genetic manipulation of F. tularensis.

Fig. 1

pFNL10 based vectors for F. tularensis. One series of plasmids based on the combination of pFNL10 and pCR2.1-TOPO includes the shuttle vectors pFNLTP5-9 (Maier et al., 2004, 2006). The pFNLTP vectors are now being modified for other uses such as the examination the F. tularensis β lactamases which expand the range of useful antimicrobial markers (Bina et al., 2006). Other plasmids were used to study the replication mechanism of pFNL10 as well as a few shuttle vectors each of which has utilized origins of replication from other plasmids in combination with pFNL10 (Kuoppa et al., 2001; Norqvist et al., 1996; Pavlov et al., 1996; Pomerantsev et al., 2001a, 2001b). Examination of hygromycin resistance and a suicide vector system in F. tularensis was examined by combining pMV261 and pFNL10 (Lovullo et al., 2006). These plasmids represent the full arsenal of plasmids currently available for F. tularensis work.

Fig. 2

Construction of the novel shuttle vectors and promoter trap plasmid for F. tularensis. Descriptions of restriction enzymes utilized to create each of pCUG1, pCUG2, and pCU18 are described in the text. Relevant features are highlighted as follows: antibiotic cassettes are represented by black arrows; other open reading frames are grey arrows; relevant features of the origins of replication are either in hollow arrows or hollow boxes. Note: pCUG1 and pCUG2 is essentially the same vector with the pC194 segment cloned in opposite orientation. pCU18 is a shuttle vector that can coexist in E. coli and F. tularensis.

Fig. 3

(a) Schema of analysis of both plasmids within a single F. tularensis LVS isolate. pCUG1 was electroporated into F. tularensis LVS and stably maintained with selective pressure and subsequently re-isolated. Additionally, pKK214GFP was transformed into the F. tularensis LVS containing pCUG1 and also stably maintained with selective pressure. (b) Analysis of the plasmid profiles of F. tularensis containing pCUG1 and or pKK214 demonstrating that both plasmids can be stably maintained together.

Fig. 4

Examination of the plasmid stability in the absence of selective pressure. Over time each of the plasmids, pCUG1 (boxes) and pKK214GFP (triangles), are rapidly lost. When the rate of loss for the singly maintained plasmids is compared to the rate of the cells containing both plasmids (circles) there does not appear to be an increase in the rate of plasmid loss.

Fig. 5

Identification of F. tularensis promoters activated during intracellular growth in macrophage. (a) depicts the experimental method used in the identification of intracellular activated promoter regions. (b) An example of nine fragments obtained from the promoter screen assay, demonstrating a range of insert sizes, suggesting that the screen is functional.