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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 [N.sup.4]-aminopropylspermine [3(3)43] were identified as major polyamines at 60[degrees]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[degrees]C and 93[degrees]C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and [N.sup.1]-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher [k.sub.cat]/[K.sub.m] value for [N.sup.1]-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as [N.sup.1]- 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[degrees]C and 93[degrees]C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher [k.sub.cat]/[K.sub.m] value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce [N.sup.1]-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via [N.sup.1]-aminopropylagmatine. Furthermore, spermine and [N.sup.4]-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines. doi: 10.1128/JB.00279-10

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the [alpha]-1,4 linkage and its subsequent transfer to the [alpha]-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus koda-karaensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain [alpha]-glucans, indicating that the reaction product contained many [alpha]-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70[degrees]C, and thermostability up to 90[degrees]C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436[DELTA]H protein) was used. The average molecular weight of branched glucan after reaction with the TK1436[DELTA]H protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.

Four orthologous genes (TK1108, TK1404, TK1777, and TK2185) that can be annotated as phosphomannomutase (PMM) genes (COG1109) have been identified in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. We previously found that TK1777 actually encodes a phosphopentomutase. In order to determine which of the remaining three orthologues encodes a phosphoglucomutase (PGM), we examined the PGM activity in T. kodakaraensis cells and identified the gene responsible for this activity. Heterologous gene expression and purification and characterization of the recombinant protein indicated that TK1108 encoded a protein with high levels of PGM activity (690 U [mg.sup.-l]), along with high levels of PMM activity (401 U [mg.sup.-1]). Similar analyses of the remaining two orthologues revealed that their protein products exhibited neither PGM nor PMM activity. PGM activity and transcription of TK1108 in T. kodakaraensis were found to be higher in cells grown on starch than in cells grown on pyruvate. Our results clearly indicate that, among the four PMM gene orthologues in T. kodakaraensis, only one gene, TK1108, actually encodes a protein with PGM and PMM activities.

Results demonstrate an upstream activation sequence which induces transcription in response to n-alkanes. Data suggest that induction of peroxisomal thiolase enzyme in Candida tropicalis is different from that of oleate-response element-mediated induction mechanism.

A research was conducted to study the expression of a truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis and its purification, characterization and production by S. cerevisiae. A protein sequencer was utilized to determine the N-terminal amino acid sequence for each molecule. Results suggested that CFTase production can be increased by optimizing fermentation conditions.

Research describes the cytoplasmic localization of an NiFe-hydrogenase in Thermococcus kodakaraensis KOD1, which is encoded by a gene cluster consisting of four open reading frames. The purified enzymes shows four subunits, each subunit containing 0.8 mol of nickel and 22.3 mol of iron, 21.1 mol of sulfide, and 1.01 mol of flavin adenine dinucleotide. Maximum hydrogen uptake is at 95 C and 90 C for hydrogen production.