Single-copy chromosomal integration systems for Francisella tularensis

Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a beta-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel D-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.