Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: Construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases
Document Type
Article
Publication Date
2011
Abstract
In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile. © 2011, American Society for Microbiology.
Publication Title
Applied and Environmental Microbiology
Volume
77
First Page
2232
Last Page
2238
Recommended Citation
Lipscomb, G. L.; Stirrett, K.; Schut, G. J.; Yang, F.; Jenney, Francis E. Jr.; Scott, R. A.; Adams, M. W.; and Westpheling, J., "Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: Construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases" (2011). PCOM Scholarly Works. 598.
https://digitalcommons.pcom.edu/scholarly_papers/598
Comments
This article was published in Applied and Environmental Microbiology, Volume 77, Issue 7, Pages 2232-2238.
The published version is available at http://dx.doi.org/10.1128/AEM.02624-10.Copyright © 2011 ASM.