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 usefu l genetic tools. Using the sacB gene of Bacillus subtilis, we develope d a sucrose-based counterselection system that allows introduction of unmarked mutations in H. pylori. A kan-sacB cassette, consisting of th e sacB gene expressed from the H. pylori flagellin promoter and the ka namycin resistance module, was introduced by homologous recombination into a target H. pylori gene. The resultant strains were sucrose sensi tive and kanamycin resistant. Following transformation with a mutated allele, growth in sucrose-containing medium allowed the selection of s trains that had lost the kan-sacB module and had integrated the unmark ed allele. We have used this cassette to perform a site directed modif ication of two histidine residues encoded by the vacA gene in a two-st ep procedure. This system should prove useful in the site-directed mut agenesis of H. pylori genes.