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FEMS Microbiol Lett. 2003 Dec 12;229(2):195-202.

Allelic exchange in Francisella tularensis using PCR products.

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1
Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.

Abstract

We describe here a technique for allelic exchange in Francisella tularensis subsp. novicida utilizing polymerase chain reaction (PCR) products. Linear PCR fragments containing gene deletions with an erythromycin resistance cassette insertion were transformed into F. tularensis. The subsequent ErmR progeny were found to have undergone allelic exchange at the correct location in the genome; the minimum flanking homology necessary was 500 bp. This technique was used to create mglA, iglC, bla, and tul4 mutants in F. tularensis subsp. novicida strains. The mglA and iglC mutants were defective for intramacrophage growth, and the tul4 mutant lacked detectable Tul4 by Western immunoblot, as expected. Interestingly, the bla mutant maintained resistance to ampicillin, indicating the presence of multiple ampicillin resistance genes in F. tularensis.

PMID:
14680699
DOI:
10.1016/S0378-1097(03)00820-6
[Indexed for MEDLINE]
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