Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system

Blake W Buchan; Molly K McLendon; Bradley D Jones

doi:10.1128/AEM.02882-07

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Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system

Journal article

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Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system

Blake W Buchan, Molly K McLendon and Bradley D Jones

Applied and environmental microbiology, Vol.74(9), pp.2637-2645

05/2008

DOI: 10.1128/AEM.02882-07

PMCID: PMC2394869

PMID: 18344342

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Abstract

Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn5-based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42 degrees C) is applied to a bacterial culture carrying the transposon delivery plasmid, approximately 0.1% of the population is recovered with Tn5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains.

Genes, Bacterial

Up-Regulation

Mutagenesis, Insertional - methods

Kanamycin Resistance - genetics

Recombination, Genetic

Gene Expression Profiling

Francisella tularensis - genetics

Hot Temperature

beta-Galactosidase - genetics

Artificial Gene Fusion - methods

DNA Transposable Elements

Luciferases - genetics

Details

Title: Subtitle: Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system
Creators: Blake W Buchan - Department of Microbiology, University of Iowa School of Medicine, 51 Newton Road, Iowa City, IA 52242-1109, USA
Molly K McLendon
Bradley D Jones
Resource Type: Journal article
Publication Details: Applied and environmental microbiology, Vol.74(9), pp.2637-2645
DOI: 10.1128/AEM.02882-07
PMID: 18344342
PMCID: PMC2394869
NLM abbreviation: Appl Environ Microbiol
ISSN: 0099-2240
eISSN: 1098-5336
Grant note: P01 AI044642 / NIAID NIH HHS
Language: English
Date published: 05/2008
Academic Unit: Microbiology and Immunology
Record Identifier: 9984083885802771

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