Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker.
Research on Helicobacter pylori has been hindered by the lack of useful genetic tools. Using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows introduction of unmarked mutations in H. pylori. A kan-sacB cassette, consisting of the sacB gene expressed from the H. pylori flagellin promoter and the kanamycin resistance module, was introduced by homologous recombination into a target H. pylori gene. The resultant strains were sucrose sensitive and kanamycin resistant. Following transformation with a mutated allele, growth in sucrose-containing medium allowed the selection of strains that had lost the kan-sacB module and had integrated the unmarked allele. We have used this cassette to perform a site-directed modification of two histidine residues encoded by the vacA gene in a two-step procedure. This system should prove useful in the site-directed mutagenesis of H. pylori genes.
Copass
M
M
IRIS, Chiron-Vaccines Immunobiological Research Institute in Siena, Italy.
Grandi
G
G
Rappuoli
R
R
D000483:Alleles; D001412:Bacillus subtilis / Q000235:genetics; D015153:Blotting, Western; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000235:genetics*; D005408:Flagellin / Q000235:genetics; D016480:Helicobacter pylori / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D007613:Kanamycin Resistance / Q000235:genetics; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013395:Sucrose / Q000494:pharmacology; D014170:Transformation, Genetic
D003603:Cytotoxins; D005408:Flagellin; D006639:Histidine; D013395:Sucrose