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...

BackgroundBioinformatic analysis of the genes coding for the chitinase in Pyrococcus furiosus and Thermococcus kodakarensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase gene. This splits the enzyme into two separate genes, PF1233 and PF1234, in comparison to Thermococcus kodakarensis. Furthermore, our attempts to grow the wild type strain of Pyrococcus furiosus on chitin were negative. From these data we assume that Pyrococcus furiosus is most likely unable to use chitin as a carbon source. The aim of this study was to analyze in vivo if the one nucleotide insertion is responsible for the inability to grow on chitin, using a recently described genetic system for Pyrococcus furiosus.ResultsA marker-less genetic system for Pyrococcus furiosus was developed using simvastatin for positive selection and 6-methylpurine for negative selection. Resistance against simvastatin was achieved by overexpression of the hydroxymethylglutaryl coenzyme A reductase gene. For the resistance to 6-methylpurine the hypoxanthine-guanine phosphoribosyltransferase gene was deleted. This system was used to delete the additional nucleotide at position 1006 in PF1234. The resulting chitinase in the mutant strain was a single subunit enzyme and aligns perfectly to the enzyme from Thermococcus kodakarensis. A detailed analysis of the wild type and the mutant using counted cell numbers as well as ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. An additional mutant strain containing a reduced chitinase version containing just one catalytic and one chitin-binding domain showed diminished growth on chitin in comparison to the mutant containing the single large enzyme.ConclusionsWild type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. The overall high sequence identity of the two chitinases between P. furiosus and T. kodakarensis indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme using programmed +/−1 frameshifting.

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix-turn-helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2 bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°.While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent.This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein-DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA.

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analysed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterise the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 microM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the complex regulatory network that enables Pyrococcus to respond to increased copper concentrations.

Rpb5 is a general subunit of all eukaryotic RNA polymerases which consists of a N-terminal and a C-terminal domain. The corresponding archaeal subunit RpoH contains only the conserved C-terminal domain without any N-terminal extensions. A chimeric construct, termed rp5H, which encodes the N-terminal yeast domain and the C-terminal domain from Pyrococcus furiosus is unable to complement the lethal phenotype of a yeast rpb5 deletion strain (Δrpb5). By applying a random mutagenesis approach we found that the amino acid exchange E197K in the C-terminal domain of the chimeric Rp5H, either alone or with additional exchanges in the N-terminal domain, leads to heterospecific complementation of the growth deficiency of Δrpb5. Moreover, using a recently described genetic system for Pyrococcus we could demonstrate that the corresponding exchange E62K in the archaeal RpoH subunit alone without the eukaryotic N-terminal extension was stable, and growth experiments indicated no obvious impairment in vivo. In vitro transcription experiments with purified RNA polymerases showed an identical activity of the wild type and the mutant Pyrococcus RNA polymerase. A multiple alignment of RpoH sequences demonstrated that E62 is present in only a few archaeal species, whereas the great majority of sequences within archaea and eukarya contain a positively charged amino acid at this position. The crystal structures of the Sulfolobus and yeast RNA polymerases show that the positively charged arginine residues in subunits RpoH and Rpb5 most likely form salt bridges with negatively charged residues from subunit RpoK and Rpb1, respectively. A similar salt bridge might stabilize the interaction of Rp5H-E197K with a neighboring subunit of yeast RNA polymerase and thus lead to complementation of Δrpb5.