Hiroki Higashibata

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.

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.

Heme environment in aldoxime dehydratase involved in carbon–nitrogen triple bond synthesis

Resonance Raman spectra have been measured to characterize the heme environment in aldoxime dehydratase (OxdA), a novel hemoprotein, which catalyzes the dehydration of aldoxime into nitrile. The spectra showed that the ferric heme in the enzyme is six‐coordinate low spin, whereas the ferrous heme is five‐coordinate high spin. We assign a prominent vibration that occurs at 226 cm−1 in the ferrous enzyme to the Fe‐proximal histidine stretching vibration. In the CO‐bound form of OxdA, the correlation between the Fe–CO stretching (512 cm−1) and C–O stretching (1950 cm−1) frequencies also supports our assignment of proximal histidine coordination.

Effect of polyamines on histone-induced DNA compaction of hyperthermophilic archaea

The effect of polyamines on histone-mediated DNA compaction was examined in vitro with archaeal histone HpkA from Pyrococcus kodakaraensis KOD1. An agarose gel mobility-shift experiment indicated that histone-bound DNA (compacted DNA) was further compacted by addition of a polyamine (putrescine, spermidine, or spermine) or its acetylated form (N-acetylputrescine, N1-acetylspermidine, N8-acetylspermidine, or N1-acetylspermine) when the mixture was incubated at above 75 degrees C. Spermine was most effective in compaction enhancement among all the polyamines tested. A high concentration of potassium ion (1.0 M) did not stabilize the compacted form of DNA even though double-stranded DNA was stably maintained against thermal denaturation at elevated temperatures under this condition. It appears likely that multivalent polyamines have a nucleosome maintenance function in hyperthermophilic archaea in high-temperature environments.

Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA

We have isolated TBP (TATA‐binding protein)‐interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP‐agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein (Pk‐TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk‐TIP is composed of 224 amino acid residues (molecular weight of 25 558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk‐TIP and recombinant Pk‐TBP indicated that they interact with each other with an equilibrium dissociation constant, K D, of 1.24–1.46 μM. A gel mobility shift assay indicated that Pk‐TIP inhibited the interaction between Pk‐TBP and a TATA‐DNA. Pk‐TIP may be one of the archaeal factors which negatively regulate transcription.

Stopped‐flow spectrophotometric and resonance Raman analyses of aldoxime dehydratase involved in carbon–nitrogen triple bond synthesis

On stopped‐flow analysis of aliphatic aldoxime dehydratase (OxdA), a novel hemoprotein, a spectrum derived from a reaction intermediate was detected on mixing ferrous OxdA with butyraldoxime; it gradually changed into that of ferrous OxdA with an isosbestic point at 421 nm. The spectral change on the addition of butyraldoxime to the ferrous H320A mutant showed the formation of a substrate‐coordinated mutant, the absorption spectrum of which closely resembled that of the above intermediate. These observations and the resonance Raman investigation revealed that the substrate actually binds to the heme in OxdA, forming a hexa‐coordinate low‐spin heme.

Optimum Culture Conditions for the Production of N-Substituted Formamide Deformylase by Arthrobacter pascens F164

We investigated the optimum culture conditions for the production of a novel enzyme, N-substituted formamide deformylase, which acts mainly on N-benzylformamide, in Arthrobacter pascens F164. The highest enzyme activity was obtained when this strain F164 was cultivated in a synthetic medium with N-benzylformamide as sole nitrogen source. This deformylase was found to be an inducible enzyme depending on N-benzylformamide.

Surface histidine residue of archaeal histone affects DNA compaction and thermostability

Archaeal histone, which possesses only the core domain part of eukaryal histone, induced DNA compaction by binding to DNA. Based on structural modeling, tetramer formation by dimer-dimer interaction is considered to require two intermolecular ion pairs formed between histidine and aspartate. To examine the role of the ion pairs on DNA compaction, mutant histones were constructed and analyzed using HpkB from Thermococcus kodakaraensis KOD1 as a model protein. The mutant histones, HpkB-H50A, HpkB-H50V, and HpkB-H50G were constructed by replacing conserved surface His50 with Ala, Val, and Gly, respectively. Circular dichroism analysis indicated no significant difference between wild-type and mutants in their structures. Gel mobility shift assays showed that all mutants possessed DNA binding ability, like wild-type HpkB, however all mutants compacted DNA less efficiently than the wild-type. Moreover, all mutants could not maintain the nucleosome-like structure (compacted form of DNA) above 80 degrees C. These results suggest that surface ion pairs between His and Asp play an important role in maintenance of nucleosome structure and DNA stabilization at high temperature.