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.Several reports addressed the initial establishment of genetic techniques for the Thermococcales, a major group of hyperthermophilic archaea including the genera Thermococcus and Pyrococcus. The first experiments described used the plasmid pGT5 from Pyrococcus abyssi. This plasmid is only 3,440 bp in size and replicates via a rolling circle mechanism (7). The archaeal plasmid was fused with a pUC19 vector to create a potential shuttle vector between Escherichia coli and Pyrococcus furiosus (1). This construct could be transformed in both organisms by CaCl 2 treatment. Later, this construct was modified by introducing the alcohol dehydrogenase gene from Sulfolobus solfataricus as a selectable marker (3). The resulting plasmids pAG1 and pAG2 were maintained for several generations in E. coli, in the euryarchaeote P. furiosus, and also in the crenarchaeote Sulfolobus acidocaldarius. The presence of these plasmids in the two archaea conferred resistance to butanol and benzyl alcohol.As the attempts to use this selection system for P. abyssi failed, a new shuttle vector, pYS2, was created (17). This construct is also based on the archaeal pGT5 plasmid and a bacterial vector, pLitmus38. It contains the pyrE gene of S. acidocaldarius, a key enzyme of the pyrimidine biosynthetic pathway, as a selectable marker. For the transformation procedure, a Pyrococcus strain was used containing a pyrE mutation which led to a uracil-auxotrophic phenotype. Using the shuttle vector pYS2 in combination with a polyethylene glycolspheroplast method, it was possible to transform the pyrE mutant of P. abyssi to uracil prototrophy. Although the transformation frequency was very low, the shuttle vector was stably maintained at high copy number under selective conditions in both E. coli and P. abyssi (17).A major breakthrough in the establishment of genetic tools for hyperthermophilic euryar...

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…furiosus and Tco. kodakarensis as well as those of several other members of the Thermococcales have been sequenced (446,447), and genetic systems have been established (27,(448)(449)(450)(451).…”

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection

“…In such a selection system, simvastatin, a competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was utilized to select for overexpression of the HMG-CoA reductase gene (hmgA) under the control of a strong constitutive promoter of glutamate dehydrogenase (Matsumi et al, 2007;Santangelo et al, 2008). This selection strategy has now been exploited in another hyperthermophilc euryarchaeon, Pyrococcus furious (optimal temperature 100 u C), with great success (Lipscomb et al, 2011;Waege et al, 2010). Recently, simvastatin has been used to rescue lethal deletion mutants in S. islandicus by transformation with a shuttle vector overexpressing HMG-CoA reductase from Sulfolobus tokodaii under the control of a constitutive promoter from S. acidocaldarius 7d (Zheng et al, 2012).…”

Natural Competence in the Hyperthermophilic ArchaeonPyrococcus furiosusFacilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases

“…Antibiotic selection strategies used in mesophilic bacteria are typically ineffective because the molecular machineries of archaea are not affected by the antibiotic (9,29) or, in the case of hyperthermophiles, because of the instability of either the drug or the heterologously expressed resistance protein at high temperatures (2,25). One exception is the drug simvastatin (or mevinolin), first utilized in the haloarchaea (18,28), which is sufficiently thermostable to inhibit growth of both T. kodakarensis (85°C) (23) and P. furiosus (45). Simvastatin competitively inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, the rate-limiting step in the biosynthesis of isoprenoids, the major component of archaeal membrane lipids.…”