Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Abstract

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 ؋ 10 ؊8 ؎ 0.87 ؋ 10 ؊8 per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.

A comprehensive transposon mutant library of Francisella novicida , a bioweapon surrogate

“…Southern analysis of ϽKAN-2Ͼ insertions in subsp. holarctica strain LVS, however, has shown identically sized bands before and after serial passage, suggesting that Tn5 insertions are stable in LVS (24). To examine transposon stability in F. novicida, eight auxotrophic mutants from the collection (three ϽKAN-2Ͼ, one T18, and four T20 insertions) were serially passaged in rich media without antibiotic selection for Ϸ50 generations and then plated on minimal media.…”

Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence

“…Tools for the genetic manipulation of F. tularensis have only recently become available (23)(24)(25)(26)(27)(28). We used the allelic replacement method of Golovliov et al (23) to construct deletion mutations of tolC and ftlC in the LVS.…”

Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

“…We used the commercially available EZ : : TN transposome system from Epicentre Technologies, which was previously shown to work well for mutagenesis of LVS (Kawula et al, 2004). The transposome consists of a purified transposase enzyme bound to the EZ : : TN transposon, a hyperactive version of the transposon Tn5 characterized by two 19 bp mosaic ends (ME) flanking an antibiotic resistance marker (Goryshin et al, 2000).…”

“…holarctica (Eigelsbach & Downs, 1961;Golovliov et al, 2003;Lauriano et al, 2003;Nano et al, 2004;Sjostedt et al, 1996). So far, three Escherichia coli-Francisella shuttle plasmids have been described for use in these two subspecies (Maier et al, 2004;Norqvist et al, 1996;Pavlov et al, 1996); allelic exchange methods were developed for novicida and LVS (Golovliov et al, 2003;Lauriano et al, 2003), shuttle transposon mutagenesis has been used for novicida (Cowley et al, 2000), while the EZ : : TN transposome system (Kawula et al, 2004) and a transposon based upon the Himar1 element (Maier et al, 2006) have been used for mutagenesis of LVS. However, few genetic tools have been tested with highly pathogenic, select agent strains of F. tularensis subsp.…”