Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1994 Mar;60(3):903–907. doi: 10.1128/aem.60.3.903-907.1994

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts †

Girishchandra B Patel 1,*, John H E Nash 1, Brian J Agnew 1, G Dennis Sprott 1
PMCID: PMC201408  PMID: 16349218

Abstract

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.

Full text

PDF
903

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bertani G., Baresi L. Genetic transformation in the methanogen Methanococcus voltae PS. J Bacteriol. 1987 Jun;169(6):2730–2738. doi: 10.1128/jb.169.6.2730-2738.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gernhardt P., Possot O., Foglino M., Sibold L., Klein A. Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol Gen Genet. 1990 Apr;221(2):273–279. doi: 10.1007/BF00261731. [DOI] [PubMed] [Google Scholar]
  3. Hopwood D. A. Genetic studies with bacterial protoplasts. Annu Rev Microbiol. 1981;35:237–272. doi: 10.1146/annurev.mi.35.100181.001321. [DOI] [PubMed] [Google Scholar]
  4. Jarrell K. F., Koval S. F. Ultrastructure and biochemistry of Methanococcus voltae. Crit Rev Microbiol. 1989;17(1):53–87. doi: 10.3109/10408418909105722. [DOI] [PubMed] [Google Scholar]
  5. Jones W. J., Nagle D. P., Jr, Whitman W. B. Methanogens and the diversity of archaebacteria. Microbiol Rev. 1987 Mar;51(1):135–177. doi: 10.1128/mr.51.1.135-177.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jones W. J., Whitman W. B., Fields R. D., Wolfe R. S. Growth and plating efficiency of methanococci on agar media. Appl Environ Microbiol. 1983 Jul;46(1):220–226. doi: 10.1128/aem.46.1.220-226.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Koval S. F., Jarrell K. F. Ultrastructure and biochemistry of the cell wall of Methanococcus voltae. J Bacteriol. 1987 Mar;169(3):1298–1306. doi: 10.1128/jb.169.3.1298-1306.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mercenier A., Chassy B. M. Strategies for the development of bacterial transformation systems. Biochimie. 1988 Apr;70(4):503–517. doi: 10.1016/0300-9084(88)90086-7. [DOI] [PubMed] [Google Scholar]
  9. Micheletti P. A., Sment K. A., Konisky J. Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae. J Bacteriol. 1991 Jun;173(11):3414–3418. doi: 10.1128/jb.173.11.3414-3418.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Patel G. B., Choquet C. G., Nash J. H., Sprott G. D. Formation and Regeneration of Methanococcus voltae Protoplasts. Appl Environ Microbiol. 1993 Jan;59(1):27–33. doi: 10.1128/aem.59.1.27-33.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Possot O., Gernhardt P., Klein A., Sibold L. Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering. Appl Environ Microbiol. 1988 Mar;54(3):734–740. doi: 10.1128/aem.54.3.734-740.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Reeve J. N. Molecular biology of methanogens. Annu Rev Microbiol. 1992;46:165–191. doi: 10.1146/annurev.mi.46.100192.001121. [DOI] [PubMed] [Google Scholar]
  13. Sandbeck K. A., Leigh J. A. Recovery of an integration shuttle vector from tandem repeats in Methanococcus maripaludis. Appl Environ Microbiol. 1991 Sep;57(9):2762–2763. doi: 10.1128/aem.57.9.2762-2763.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Worrell V. E., Nagle D. P., Jr, McCarthy D., Eisenbraun A. Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg. J Bacteriol. 1988 Feb;170(2):653–656. doi: 10.1128/jb.170.2.653-656.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES