Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis.
OBJECTIVE
To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its disrupted vacA gene.


RESULTS
A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript II SK, and then transformed into vacA+ H pylori by electroporation. Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction, and the allelic exchange was determined by polymerase chain reactions and sequencing. Loss of vacuolating activity of the vacA-knockout strain was confirmed by examining the gastric cells co-cultured with cell-free supernatants from H pylori wild strain or the mutant.


CONCLUSIONS
We constructed a vacA-knockout strain of H pylori through direct mutagenesis, which creates an important precondition for the future research on virulence comparison with gene expression analysis.
Yuan
Jian-Ping
JP
Department of Microbiology and Parasitology, Shanghai Second Medical University, Shanghai 200025, China.
Li
Tao
T
Shi
Xiao-Dong
XD
Hu
Bao-Yu
BY
Yang
Gui-Zhen
GZ
Tong
Shan-Qing
SQ
Guo
Xiao-Kui
XK
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D017353:Gene Deletion; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013274:Stomach Neoplasms; D014774:Virulence
D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori