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A novel method of extracting plasmid DNA from Helicobacter species
M C De Ungria1, D Tillett, B A Neilan
1School of Microbiology and Immunology, University of New South Wales, Australia.
Background:
Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms.
Materials And Methods:
Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effectiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories.
Results:
High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to >16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes.
Conclusion:
This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms.
Insights
A new, cost-effective method efficiently extracts plasmid DNA from Helicobacter species, aiding future research into their roles in microbial physiology and ecology. This technique simplifies plasmid isolation for key bacterial pathogens.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Plasmids are extrachromosomal DNA elements that can confer advantageous traits such as virulence and antibiotic resistance.
- While plasmids are known in some Helicobacter species, their functions remain largely uncharacterized.
- Efficient plasmid DNA extraction from Helicobacter species is challenging due to high DNase activity.
Purpose of the Study:
- To develop an efficient, rapid, and inexpensive method for extracting plasmid DNA from Helicobacter species.
- To overcome the difficulties associated with DNase activity during plasmid isolation.
- To facilitate future studies on the role of plasmids in Helicobacter.
Main Methods:
- A novel potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer was employed for plasmid DNA extraction.
- PCR targeting the conserved repA gene confirmed plasmid presence in H. pylori isolates.
- Plasmid DNA yield was compared to commercial kits and validated by restriction enzyme digestion.
Main Results:
- High and low molecular weight plasmids (3 kb to >16 kb) were successfully extracted from H. pylori and H. felis.
- The XSP buffer method yielded comparable amounts of plasmid DNA to commercial kits.
- Plasmid DNA from H. pylori SS1 was effectively digested by restriction enzymes, confirming its integrity.
Conclusions:
- An efficient, inexpensive, and rapid method for extracting plasmids from Helicobacter species has been developed.
- This novel method will enable further isolation and characterization of plasmids in diverse Helicobacter species.
- Understanding Helicobacter plasmids can enhance knowledge of microbial physiology and ecology.