Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori.
Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation 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. pylori before transformation. In two cases, plasmid DNA treated with cell-free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly 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 significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.
Donahue
J P
JP
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, A-3310, MCN, Nashville, TN 37232, USA. john.donahue@mcmail.vanderbilt.edu
Israel
D A
DA
Peek
R M
RM
Blaser
M J
MJ
Miller
G G
GG
D001426:Bacterial Proteins; D002474:Cell-Free System; D019175:DNA Methylation; D004269:DNA, Bacterial; D016480:Helicobacter pylori / Q000235:genetics*; D010957:Plasmids; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013045:Species Specificity; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C108842:DNA modification methylase HpyI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)