Insertion-duplication mutagenesis of Salmonella enterica and related species using a novel thermosensitive vector.
We constructed a novel temperature-sensitive vector as a tool for gene disruption by insertion-duplication mutagenesis (IDM) in Salmonella enterica and related species. A phoN insertion mutant was proven highly stable during growth in LB medium and during infection of macrophage cells in the absence of selection pressure. By progressive shortening of a phoN fragment, the minimal length for effective insertional mutagenesis driven by homologous recombination was determined to be 50 bp, allowing to disrupt even short genes that could not yet be subjected to site-specific IDM. We also showed that plasmid excision from the chromosome restores the wild-type genotype with a reliability of 98%. Intracellular recovery of the excised vector provides the option to switch between two genotypes and thus to rapidly attribute the observed mutant phenotype to the targeted gene. In addition, a fragment library was used to measure the integration rate at various chromosomal sites that varies greatly by at least 2.5 magnitudes, independently from the length of the cloned fragment.
Fuchs
Thilo M
TM
Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, Weihenstephaner Berg 3, D-85350 Freising, Germany. thilo.fuchs@wzw.tum.de
Klumpp
Jochen
J
Przybilla
Karin
K
D000818:Animals; D002478:Cells, Cultured; D002876:Chromosomes, Bacterial / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020440:Gene Duplication; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008264:Macrophages / Q000502:physiology; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D011995:Recombination, Genetic; D019779:Salmonella enterica / Q000235:genetics*; D013696:Temperature; D014169:Transformation, Bacterial
D017931:DNA Primers; D004269:DNA, Bacterial