ArticleApplied and environmental microbiology2002
Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.
Article in Applied and environmental microbiology, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Who cites it
31 citing papers in PubMed.
- The Thermococcales as a model system: historical perspectives and emerging tools.Journal of bacteriology · 2025Review
- Metabolic engineering of Caldicellulosiruptor bescii for hydrogen production.Applied microbiology and biotechnology · 2024Review
- Gene transfer to plants by electroporation: methods and applications.Molecular biology reports · 2020Review
- The global distribution and evolutionary history of the pT26-2 archaeal plasmid family.Environmental microbiology · 2019Article
- Biotechnology of extremely thermophilic archaea.FEMS microbiology reviews · 2018Review
- Development of a pyrE-based selective system for Thermotoga sp. strain RQ7.Extremophiles : life under extreme conditions · 2017Article
- Mechanisms of Evolutionary Innovation Point to Genetic Control Logic as the Key Difference Between Prokaryotes and Eukaryotes.Journal of molecular evolution · 2015Article
- Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.Extremophiles : life under extreme conditions · 2015Article
- Utilization of virus φCh1 elements to establish a shuttle vector system for Halo(alkali)philic Archaea via transformation of Natrialba magadii.Applied and environmental microbiology · 2013Article
- Article
- Overview of the genetic tools in the Archaea.Frontiers in microbiology · 2012Article
- Defining components of the chromosomal origin of replication of the hyperthermophilic archaeon Pyrococcus furiosus needed for construction of a stable replicating shuttle vector.Applied and environmental microbiology · 2011Article
- Genetic tool development underpins recent advances in thermophilic whole-cell biocatalysts.Microbial biotechnology · 2011Review
- Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases.Applied and environmental microbiology · 2011Article
- Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins.Nucleic acids research · 2010Article
- Shuttle vector-based transformation system for Pyrococcus furiosus.Applied and environmental microbiology · 2010Article
- Thermus thermophilus as biological model.Extremophiles : life under extreme conditions · 2009Review
- Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome.Archaea (Vancouver, B.C.) · 2008Article
- Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon.Applied and environmental microbiology · 2008Article
- Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.Journal of bacteriology · 2007Article
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8 authors.
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Abstract
Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.
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