Allelic exchange in Francisella tularensis using PCR products

Crystal M. Lauriano, Jeffrey R. Barker, Francis E. Nano, Bernard P. Arulanandam, Karl E. Klose

Research output: Contribution to journalArticle

78 Scopus citations

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 Erm R 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.

Original languageEnglish (US)
Pages (from-to)195-202
Number of pages8
JournalFEMS Microbiology Letters
Volume229
Issue number2
DOIs
StatePublished - Dec 12 2003

Keywords

  • Francisella tularensis
  • Mutagenesis
  • Tularemia

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'Allelic exchange in Francisella tularensis using PCR products'. Together they form a unique fingerprint.

  • Cite this