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Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori
1Department of Microbiology, Gyeongsang National University College of Medicine, Kyung-Nam, Republic of Korea.
Abstract:
In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.
Insights
A new Helicobacter pylori-Escherichia coli shuttle vector, pBHP489K, was developed for DNA transfer. This vector successfully restored urease activity in H. pylori mutants, proving its utility for research.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Helicobacter pylori is a significant human pathogen.
- Efficient genetic manipulation tools are crucial for studying H. pylori.
- Existing vectors may have limitations for H. pylori research.
Purpose of the Study:
- To construct and characterize a novel shuttle vector for H. pylori and E. coli.
- To enable efficient DNA transfer and gene expression in H. pylori.
- To facilitate research into H. pylori pathogenicity and genetic systems.
Main Methods:
- Selection of the smallest cryptic plasmid (pHP489) from H. pylori isolates.
- Ligation of a kanamycin resistance gene [aph(3')-III] to pHP489, creating pHP489K.
- Construction of the shuttle vector pBHP489K by combining pHP489, ColE1, and aph(3')-III sequences.
- Transformation of pBHP489K into H. pylori and E. coli.
- Introduction of H. pylori ureA and ureB genes into urease-negative H. pylori mutants using the shuttle vector.
Main Results:
- The recombinant plasmid pHP489K was efficiently transformed and stably maintained in H. pylori.
- The shuttle vector pBHP489K was successfully transformed and maintained in both H. pylori and E. coli.
- Introduction of ureA and ureB genes restored urease activity in H. pylori mutants.
- Transformants confirmed the presence of the introduced plasmid DNA.
Conclusions:
- pBHP489K functions effectively as an H. pylori-E. coli shuttle vector.
- This vector is a valuable tool for studying H. pylori pathogenicity.
- The shuttle vector may aid in investigating H. pylori restriction-modification systems.