New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori

Background. Because of limited genetic tools for use in Helicobacter pylori , tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to dev elop such tools for H. pylori. Materials and Methods. We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no via ble mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permi t the gene's disruption. For genes that yielded mutants with selectable phe notypes, a strategy was developed for reversion whereby an intact copy of t he gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. Results. After failure to attain transformants upon attempted mutation of g enes HP0166 and HP1365, we inserted a second copy of each gene within the H , pylori chromosome. In each case the second copy relieved the block of tra nsformation. Mutation of genes HP0703 and HP1021 gave non-motile and small- colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had rev erted following phenotypic selections. Conclusions. The methods used in this study provide new approaches for conf irming suspected genotype/phenotype associations and should be widely appli cable in the study of H, pylori.