title: Shuttle vector-based transformation system for Pyrococcus furiosus.
abstract: Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furiosus, but the copy number was only one to two per chromosome. This system was used for overexpression of His(6)-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.
authors.affiliation: Universität Regensburg, Lehrstuhl für Mikrobiologie, Universitätsstr. 31, 93053 Regensburg, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Waege
authors.forename: Georg
authors.initials: G
authors.lastname: Schmid
authors.forename: Sybille
authors.initials: S
authors.lastname: Thumann
authors.forename: Michael
authors.initials: M
authors.lastname: Thomm
authors.forename: Winfried
authors.initials: W
authors.lastname: Hausner
mesh_terms: D000214:Acyl Coenzyme A; D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D020027:Pyrococcus furiosus / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D000214:Acyl Coenzyme A; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D012321:DNA-Directed RNA Polymerases