Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker

作者：Michael K. Copass, G Grandi, Rino Rappuoli · 发表于：Infection and Immunity · 年份：1997 · DOI：10.1128/iai.65.5.1949-1952.1997 · 被引用次数：100 · 研究领域：Helicobacter pylori-related gastroenterology studies、RNA and protein synthesis mechanisms、Microbial Metabolites in Food Biotechnology

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.