title: Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases.
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.
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA 30602, USA.
authors.forename: Gina L
authors.initials: GL
authors.lastname: Lipscomb
authors.forename: Karen
authors.initials: K
authors.lastname: Stirrett
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Schut
authors.forename: Fei
authors.initials: F
authors.lastname: Yang
authors.forename: Francis E
authors.initials: FE
authors.lastname: Jenney
authors.forename: Robert A
authors.initials: RA
authors.lastname: Scott
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
authors.forename: Janet
authors.initials: J
authors.lastname: Westpheling
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics; D017353:Gene Deletion; D005827:Genetics, Microbial / Q000379:methods*; D006864:Hydrogenase / Q000235:genetics; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic
chemical_list: D019843:Archaeal Proteins; C407406:pyrF protein, Archaeal; D006864:Hydrogenase