Helicobacter pylori mutagenesis by mariner in vitro transposition.
We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
B P
BP
Guo
J J
JJ
Mekalanos
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity; D016296:Mutagenesis; D019895:Transposases; D014774:Virulence / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C121269:HopZ protein, Helicobacter pylori; C074689:VacA protein, Helicobacter pylori; C111652:mariner transposases; D019895:Transposases