Wakao Fukuda

Agmatine is essential for the cell growth of Thermococcus kodakaraensis

TK0149 (designated as Tk-PdaD) of a hyperthermophilic archaeon, Thermococcus kodakaraensis, was annotated as pyruvoyl-dependent arginine decarboxylase, which catalyzes agmatine formation by the decarboxylation of arginine as the first step of polyamine biosynthesis. In order to investigate its physiological roles, Tk-PdaD was purified as a recombinant form, and its substrate dependency was examined using the candidate compounds arginine, ornithine and lysine. Tk-PdaD, expressed in Escherichia coli, was cleaved into alpha and beta subunits, as other pyruvoyl-dependent enzymes, and the resulting subunits formed an (alphabeta)6 complex. The Tk-PdaD complex catalyzed the decarboxylation of arginine but not that of ornithine and lysine. A gene disruptant lacking Tk-pdaD was constructed, showing that it grew only in the medium in the presence of agmatine but not in the absence of agmatine. The obtained results indicate that Tk-pdaD encodes a pyruvoyl-dependent arginine decarboxylase and that agmatine is essential for the cell growth of T. kodakaraensis.

First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (PckTk). PckTk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in PckTk. However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant PckTk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of PckTk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of PckTk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.

Effect of Growth Temperature and Growth Phase on the Lipid Composition of the Archaeal Membrane from Thermococcus kodakaraensis

Archaea have unique membrane lipids typified by ether linkages of the glycerol-to-isoprenoid chains with sn-2,3 stereochemistry that runs against the naturally occurring sn-1,2 stereochemistry of the glycerophospholipids of Bacteria and Eukarya. Membrane lipids were extracted and analyzed from the hyperthermophilic archaeon, Thermococcus kodakaraensis, cultivated at various temperatures. At all growth temperatures examined, both the diphytanylglycerol diether (archaeol, C20) and diphytanyldiglycerol tetraether (caldarchaeol, C40) were identified as saturated forms, and no other lipids could be identified. The ratio of caldarchaeol to archaeol increased with increasing growth temperature, particularly at 93 °C. A larger amount of archaeol was detected from cells in the logarithmic phase than from those in the stationary phase at all temperatures examined. These results indicate that T. kodakaraensis modulated the membrane lipid composition depending on both the growth phase and the growth temperature, and suggest that the membrane fluidity to environmental change was maintained by altering the length of the hydrocarbon chains, and not by side-chain saturation such as double-bond hydrogenation nor by such a modification as cyclopentane ring formation.

Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N4-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N4-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N1-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher kcat/Km value for N1-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N1-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher kcat/Km value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N1-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N1-aminopropylagmatine. Furthermore, spermine and N4-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.

Expression Profiles and Physiological Roles of Two Types of Molecular Chaperonins from the Hyperthermophilic Archaeon Thermococcus kodakarensis

ABSTRACT Thermococcus kodakarensis possesses two chaperonins, CpkA and CpkB, and their expression is induced by the downshift and upshift, respectively, of the cell cultivation temperature. The expression levels of the chaperonins were examined by using specific antibodies at various cell growth temperatures in the logarithmic and stationary phases. At 60°C, CpkA was highly expressed in both the logarithmic and stationary phases; however, CpkB was not expressed in either phase. At 85°C, CpkA and CpkB were expressed in both phases; however, the CpkA level was decreased in the stationary phase. At 93°C, CpkA was expressed only in the logarithmic phase and not in the stationary phase. In contrast, CpkB was highly expressed in both phases. The results of reverse transcription-PCR experiments showed the same growth phase- and temperature-dependent profiles as observed in immunoblot analyses, indicating that the expression of cpkA and cpkB is regulated at the mRNA level. The cpkA or cpkB gene disruptant was then constructed, and its growth profile was monitored. The cpkA disruptant showed poor cell growth at 60°C but no significant defects at 85°C and 93°C. On the other hand, cpkB disruption led to growth defects at 93°C but no significant defects at 60°C and 85°C. These data indicate that CpkA and CpkB are necessary for cell growth at lower and higher temperatures, respectively. The logarithmic-phase-dependent expression of CpkA at 93°C suggested that CpkA participates in initial cell growth in addition to lower-temperature adaptation. Promoter mapping and quantitative analyses using the Phr (Pyrococcus heat-shock regulator) gene disruptant revealed that temperature-dependent expression was achieved in a Phr-independent manner.

Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Mae). The amino acid sequence of Tk-Mae was 52-58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP(+) rather than NAD(+), but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (K(m)) of Tk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence.

Lysobacter oligotrophicus sp. nov., isolated from an Antarctic freshwater lake in Antarctica.

A Gram-stain-negative, non-spore-forming, rod-shaped, aerobic bacterium (strain 107-E2(T)) was isolated from freshwater samples containing microbial mats collected at a lake in Skarvsnes, Antarctica (temporary lake name, Lake Tanago Ike). Strain 107-E2(T) grew between 5 and 25 °C, with an optimum of 23 °C. Moreover, colony formation was observed on agar media even at -5 °C. The pH range for growth was between 6.0 and 9.0, with an optimum of pH 7.0-8.0. The range of NaCl concentration for growth was between 0.0 and 0.5% (w/v), with an optimum of 0.0%. No growth was observed in media containing organic compounds at high concentrations, which indicated that strain 107-E2(T) was an oligotroph. In the late stationary phase, strain 107-E2(T) produced a dark brown water-soluble pigment. Esterase, amylase and protease production was observed. Antimicrobial-lytic activities for Gram-negative bacteria and yeast were observed. Ubiquinone-8 was the major respiratory quinone. The major fatty acids were iso-C15:0, iso-C(17:1)ω9c and iso-C(15:1) at 5. The G+C content of genomic DNA was 66.1 mol%. Analysis of the 16S rRNA gene sequences revealed that strain 107-E2(T) belonged to the genus Lysobacter, and low DNA-DNA relatedness values with closely related species distinguished strain 107-E2(T) from recognized species of the genus Lysobacter. The phylogenetic situation and physiological characteristics indicated that strain 107-E2(T) should be classified as a representative of a novel species of the genus Lysobacter, for which the name Lysobacter oligotrophicus sp. nov. is proposed. The type strain is 107-E2(T) ( =JCM 18257(T) =ATCC BAA-2438(T)).

Property of cold inducible DEAD-box RNA helicase in hyperthermophilic archaea

TK0306 (Tk-DeaD) of hyperthermophilic archaeon Thermococcus kodakaraensis is annotated as the DEAD-box helicase gene; nevertheless, its ortholog has not been identified in closely related genera, Pyrococcus spp., which generally grow at higher temperature than T. kodakaraensis, suggesting that the cold-inducible RNA helicase of Tk-DeaD functions under cold stress conditions. Quantitative RT-PCR revealed that Tk-deaD was more dominantly transcribed at 60 degrees C than at 85 degrees C and 93 degrees C in both logarithmic and stationary phases. Immunoblot analyses revealed that Tk-DeaD was detected only in logarithmic-phase cells cultivated at 60 degrees C but hardly detected at 85 degrees C and 93 degrees C in both phases. Tk-DeaD expression is, hence, post-transcriptionally regulated and appears under vigorous growth conditions at 60 degrees C. Recombinant Tk-DeaD purified to homogeneity started to unfold at 20 degrees C, fully unfolded at 70 degrees C, and exhibited maximal ATPase activity and unwinding activity specific for single-strand paired RNA at 50 degrees C, which is lower than the growth limit of T. kodakaraensis.

Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

Expression profiles and physiological roles of two types of prefoldins from the hyperthermophilic archaeon Thermococcus kodakaraensis.

The hyperthermophilic archaeon Thermococcus kodakaraensis possesses four prefoldin genes encoding two alpha subunits (pfdA and pfdC) and two beta subunits (pfdB and pfdD) of prefoldins on the genome. pfdC and pfdD are unique genes whose orthologues are not found in Pyrococcus spp., whereas pfdA and pfdB are commonly found in both Thermococcus and Pyrococcus spp. The pfdA and pfdB are located at different loci, and pfdC and pfdD were tandemly arranged on the genome. Immunoprecipitation experiments using specific antisera, anti-PfdB and anti-PfdD, revealed that PfdB and PfdD make a complex only with PfdA and PfdC, respectively. Both PfdA/PfdB and PfdC/PfdD complexes obtained as recombinant forms showed inhibitory activity against the thermal aggregation of citrate synthase. Immunoblot experiments indicated that the PfdA/PfdB complex was expressed at all examined temperatures; however, the PfdC/PfdD complex was specifically expressed under heat-stress conditions at 93 degrees C. Transcriptional analyses showed that pfdA and pfdB were transcribed at equal levels at all examined temperatures but pfdC and pfdD were transcribed at higher levels at 93 degrees C. Furthermore, pfdA and pfdB were transcribed individually, but pfdD was cotranscribed with pfdC. A typical Pyrococcus heat-shock regulator (Phr) recognition sequence was identified at the upstream region of pfdC. The transcriptional level of pfdCD was measured in a phr disruptant, showing that the pfdCD transcript in the phr disruptant was drastically increased in comparison with that of the wild type. However, the pfdCD level was also elevated at higher temperature, indicating that heat induction of PfdC/PfdD is mainly achieved by Phr derepression but that a certain degree of induction is not under Phr control. The pfdB and pfdD disruptants were then constructed, and the growth profiles were compared. At 85 degrees C cultivation, no significant difference was observed between the wild type and the pfdD disruptant; however, less growth was observed in the pfdB disruptant. At 93 degrees C, the pfdD disruptant grew less than the wild type, and the pfdB disruptant grew the least. The results suggest that the PfdA/PfdB complex plays a crucial role at all growth temperatures and the PfdC/PfdD complex contributes to survival in a high-temperature environment.