Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants: usi ng mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into t he purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia Loll. purified mutagenized plasmid was introduc ed into Helicobacter pylori by natural transformation. Transformants were s elected by plating on kanamycin. Mutants were predominantly the result of d ouble homologous recombination, and multiple mutants (with insertions in di stinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulen ce genes, including urcA, hopZ. and vacA, using kanamycin- and kanamycin/la cZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appear ed blue on medium containing the chromogenic agent X-gal, allowing discrimi nation of mutant and wild-type H. pylori in mixed competition experiments. (C) 2001 Federation of European Microbiological Societies. Published by Els evier Science B.V. All rights reserved.