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We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF -deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (Δ pyrF ), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (Δ pyrF Δ trpE :: pyrF ), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (Δ pyrF Δ trpE ), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE . Likewise, a triple deletion mutant, KUWH1 (Δ pyrF Δ trpE Δ hisD ), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.

Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism. In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected. Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism. As PPM activity was detected in T. kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence. The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs. Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM. Both enzyme activities could be observed in T. kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2′-deoxynucleosides in the medium did not lead to a significant induction of these activities. Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T. kodakaraensis , providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM.

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (Fbp Tk ) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the “missing” FBPase. For this study, using T. kodakaraensis , we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis , Imp Tk , was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Δ fbp and Δ imp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Δ fbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbp Tk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous imp Tk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that imp Tk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the Fbp Tk ortholog, not the IMPase/FBPase ortholog.

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5′-monophosphate decarboxylase gene ( pyrF ) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl 2 method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE . As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain,Archaea. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon fromThermococcus kodakaraensisKOD1 (LonTk) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA+superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that LonTkwas actually a membrane-bound protein. The recombinant LonTkpossessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70°C, respectively. Unlike the enzyme fromEscherichia coli, we found that LonTkshowed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that LonTkrequired ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, LonTkdegraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in LonTkdifferent from that of its bacterial counterpart.