Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori

Helicobacter pylori strains demonstrate substantial variability in the effi ciency of transformation by plasmids from Escherichia coil, and many strain s are completely resistant to transformation. Among the barriers to transfo rmation are numerous strain-specific restriction-modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site-specific methylation using cell-free extracts of H. pylor i before transformation. In two cases, plasmid DNA treated with cell-free e xtracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the tr ansformation frequency by treated plasmid shuttle and suicide vectors was s ignificantly increased compared with mock-treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should signifi cantly enhance the ability to manipulate gene function in H. pylori and oth er organisms that have substantial restriction barriers to transformation.