A novel method of extracting plasmid DNA from Helicobacter species.
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.


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.


CONCLUSIONS
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.
De Ungria
M C
MC
School of Microbiology and Immunology, University of New South Wales, Australia.
Tillett
D
D
Neilan
B A
BA
Cox
P T
PT
Lee
A
A
D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods*; D002021:Buffers; D004269:DNA, Bacterial / Q000302:isolation & purification*; D003851:Deoxyribonucleases / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism; D016998:Helicobacter / Q000737:chemistry / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics; D008828:Microbiological Techniques; D008969:Molecular Sequence Data; D019800:Phenol; D010957:Plasmids / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013871:Thiones / Q000737:chemistry
D002021:Buffers; D004269:DNA, Bacterial; D013871:Thiones; D019800:Phenol; D012967:Sodium Dodecyl Sulfate; C004918:ethylxanthate; D003851:Deoxyribonucleases; C027908:endodeoxyribonuclease DdeI; C040838:endodeoxyribonuclease MaeIII; C056436:endodeoxyribonuclease SpeI; D015252:Deoxyribonucleases, Type II Site-Specific