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

The researchers developed new methods to demonstrate a gene’s role in viability in Helicobacter pylori by experimenting with mutational studies of response regulator genes. These methods have culminated in new ways for confirming genotype/phenotype associations, which could be widely used in the study of this organism.

Research Approach

• The researchers started by attempting to mutate five response regulator genes through a process known as allelic exchange insertional mutagenesis. This involves replacing one allele with another. The goal was to disrupt the genes and observe the result.
• When no viable mutants resulted, they took the approach of inserting a second copy of the gene into the chromosome. This was achieved using a suicide vector, which is a type of plasmid or virus that carries material into a cell causing it to change.
• They wanted to see if providing this second copy would allow the gene’s disruption to take place.
• For genes where mutants were successfully created with selectable phenotypes, a reversion strategy was developed. This involved introducing an intact copy of the gene to the organism through transformation with PCR (polymerase chain reaction) products.
• Following this, any potential revertants were selected through phenotypic tests and then tested for genetic reversion.

Research Findings

• Two genes, HP0166 and HP1365, failed to produce viable mutants. However, when a second copy of each gene was introduced into the H. pylori chromosome, it allowed the block of transformation to be overcome.
• Two other genes, HP0703 and HP1021, when mutated, yielded non-motile and small-colony phenotypes, respectively.
• Introduction of PCR products containing intact copies of the mutated genes resulted in the reversal of both phenotype and genotype after phenomenological selections.

Conclusions and Implications

• The techniques utilised in this study open up new possibilities for confirming suspected genotype/phenotype associations.
• These methods developed via mutational studies could be widely applicable in the study of Helicobacter pylori, an organism that previously had limited genetic tools available.