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

*Department of Genome Sciences, University of Washington, Campus Box 355065, 1705 NE Pacific Street, Seattle, WA 98195; and
^†Department of Medicine, University of Washington, Campus Box 352145, 1705 NE Pacific Street, Seattle, WA 98195

Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved November 22, 2006 (received for review August 3, 2006)

Abstract

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is a category A select agent. We created a sequence-defined, near-saturation transposon mutant library of F. tularensis novicida, a subspecies that causes a tularemia-like disease in rodents. The library consists of 16,508 unique insertions, an average of >9 insertions per gene, which is a coverage nearly twice that of the greatest previously achieved for any bacterial species. Insertions were recovered in 84% (1,490) of the predicted genes. To achieve high coverage, it was necessary to construct transposons carrying an endogenous Francisella promoter to drive expression of antibiotic resistance. An analysis of genes lacking (or with few) insertions identified nearly 400 candidate essential genes, most of which are likely to be required for growth on rich medium and which represent potential therapeutic targets. To facilitate genome-scale screening using the mutant collection, we assembled a sublibrary made up of two purified mutants per gene. The library provides a resource for virtually complete identification of genes involved in virulence and other nonessential processes.

Footnotes

^‡To whom correspondence should be addressed. E-mail: manoil{at}u.washington.edu
Author contributions: L.A.G. and C.M. designed research; L.A.G., E.R., M.A.J., and R.K. performed research; L.A.G. and M.B. contributed new reagents/analytic tools; L.A.G. and M.B. analyzed data; and L.A.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0606713104/DC1.
Abbreviations:

TSAC,

trypticase soy agar supplemented with 0.1% l-cysteine HCl and 0.2% dextrose;

TSBC,

trypticase soy broth supplemented with 0.1% l-cysteine HCl and 0.2% dextrose