Site-directed (IS30-FljA) transposon mutagenesis system to produce nonflagellated mutants of SalmonellaEnteritidis

Abstract

Site-directed integration/mutagenesis systems are used to carry out targeted transpositions on DNA. The well-characterized IS30-element and its transposase have numerous advantages that predestine it to be a good candidate for such applications. In order to generate nonflagellated mutants of Salmonella Enteritidis, a new site-directed mutagenesis system has been developed and applied. The system was constructed based on the assumption that the DNA-binding FljA component of the fusion transposase would bind to its target (the operator of fliC), and as a consequence, insertions could be concentrated in the flagellin operon. The system consists of two components: one expresses the fusion transposase and the other is an integration donor plasmid harbouring the (IS30) 2 reactive structure. The application of this site-directed mutagenesis system on a strain of S. Enteritidis 11 (SE11) resulted in several nonmotile mutants with fliD insertion that could serve as negatively markered vaccine candidates. Analysis of less motile mutants generated by the fusion transposase revealed further hot spot sequences preferred by the fusion construct. © 2011 Federation of European Microbiological Societies.