Open AccessA method for allelic replacement in Francisella tularensis
Author(s) -
Golovliov Igor,
Sjöstedt Anders,
Mokrievich Alexander,
Pavlov Vitaly
Publication year - 2003
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(03)00313-6
Subject(s) - francisella tularensis , virulence , biology , plasmid , mutagenesis , mutant , bacillus subtilis , genetics , pathogenicity island , francisella , gene , microbiology and biotechnology , bacteria
A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis , oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri , a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23‐kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis . A plasmid designated pPV‐Δ iglC was developed that contained only the regions flanking the encoding gene, iglC . By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted Δ iglC 1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated Δ iglC 1+2, was obtained. The Δ iglC 1+2 strain, in contrast to Δ iglC 1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23‐kDa protein, iglC, for the virulence of F. tularensis LVS.