title: In vivo Himar1-based transposon mutagenesis of Francisella tularensis.
abstract: Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
authors.forename: Tamara M
authors.initials: TM
authors.lastname: Maier
authors.forename: Roger
authors.initials: R
authors.lastname: Pechous
authors.forename: Monika
authors.initials: M
authors.lastname: Casey
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Zahrt
authors.forename: D W
authors.initials: DW
authors.lastname: Frank
mesh_terms: D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics; D005604:Francisella tularensis / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D005816:Genetic Complementation Test; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation; D010957:Plasmids; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements