Cultivation of Piezotolerant and Piezophilic Hyperthermophiles with a Newly Developed Constant High Pressure and Temperature Culturing and Monitoring System.

The Earth's microbial biosphere extends from ambient to extreme environments, including deep-sea hydrothermal vents and subseafloor habitats. Despite efforts to understand the physiological adaptations of these microbes, our knowledge is limited due to the technological challenges associated with reproducing in situ high temperature (HT)-high hydrostatic pressure (HHP) conditions and sampling HT-HHP cultures. In the present study, we developed a new high temperature and pressure (HTP) incubation system that enabled the maintenance of HT-HHP conditions while sampling incubation medium and mostly eliminated non-biological reactions, including hydrogen generation or the leakage of small gaseous molecules. The main characteristics of our system are (1) a chamber made of gold with gold-etched lid parts that suppress the majority of non-biological reactions, (2) the exceptional containment of dissolved gas, even small molecules, such as hydrogen, and (3) the sampling capacity of intra-chamber liquid without depressurization and the isobaric transfer of a culture to inoculate new medium. We initially confirmed the retention of dissolved hydrogen in the incubation container at 82°C and 20| |MPa for 9 days. Cultivation tests with an obligate hyperthermophilic piezophile (Pyrococcus yayanosii), hydrogenotrophic hyperthermophile (Archaeoglobus profundus), and heterotrophic hyperthermophile (Pyrococcus horikoshii) were successful based on growth monitoring and chemical ana-lyses. During HTP cultivation, we observed a difference in the duration of the lag phase of P. horikoshii, which indicated the potential effect of a pressure change on the physiology of piezophiles. The present results suggest the importance of a cultivation system designed and developed explicitly for HTP conditions with the capacity for sampling without depressurization of the entire system.

Simple In-liquid Staining of Microbial Cells for Flow Cytometry Quantification of the Microbial Population in Marine Subseafloor Sediments.

Microbial cell counting provides essential information for the study of cell abundance profiles and biogeochemical interactions with the surrounding environments. However, it often requires labor-intensive and time-consuming processes, particularly for subseafloor sediment samples, in which non-cell particles are abundant. We developed a rapid and straightforward method for staining microbial intracellular DNA by SYBR Green I (SYBR-I) to enumerate cells by flow cytometry (FCM). We initially examined the efficiency of microbial cell staining at various dye/sediment ratios (volume ratio of SYBR-I/sediment [vSYBR/vSed]). Non-cell particles in sediment strongly and preferentially adsorbed SYBR-I dye, resulting in the unsuccessful staining of microbial cells when an insufficient ratio (<1.63 vSYBR/vSed) of SYBR-I dye was present per volume of sediment. SYBR-I dye at an abundance of 10 vSYBR/vSed successfully and stably stained microbial cells in green fluorescence, while the fluorescent color of non-cell particles red-shifted to yellow-orange with the overaccumulation of SYBR-I dye. A low vSYBR/vSed ratio was quickly recognized by a colorless supernatant after centrifugation. At the appropriate vSYBR/vSed ratio, FCM-measured cell concentrations in subseafloor sediments were consistently similar to microscopy counts (>106 cells cm-3). Samples with low cell abundance (<105 cells cm-3) still require cell separation. This modified staining allows us to efficiently process and perform the microbial cell counting of sediment samples to a depth of a few hundred meters below the seafloor with a higher throughput and capability to scale up than procedures employing microscopy-based observations.

Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments.

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Biochemical characterization of mismatch-binding protein MutS1 and nicking endonuclease MutL from a euryarchaeon Methanosaeta thermophila.

In eukaryotes and most bacteria, the MutS1/MutL-dependent mismatch repair system (MMR) corrects DNA mismatches that arise as replication errors. MutS1 recognizes mismatched DNA and stimulates the nicking endonuclease activity of MutL to incise mismatch-containing DNA. In archaea, there has been no experimental evidence to support the existence of the MutS1/MutL-dependent MMR. Instead, it was revealed that a large part of archaea possess mismatch-specific endonuclease EndoMS, indicating that the EndoMS-dependent MMR is widely adopted in archaea. However, some archaeal genomes encode MutS1 and MutL homologs, and their molecular functions have not been revealed. In this study, we purified and characterized recombinant MutS1 and the C-terminal endonuclease domain of MutL from a methanogenic archaeon Methanosaeta thermophila (mtMutS1 and the mtMutL CTD, respectively). mtMutS1 bound to mismatched DNAs with a higher affinity than to perfectly-matched and other structured DNAs, which resembles the DNA-binding specificities of eukaryotic and bacterial MutS1 homologs. The mtMutL CTD showed a Mn2+/Ni2+/Co2+-dependent nicking endonuclease activity that introduces single-strand breaks into a circular double-stranded DNA. The nicking endonuclease activity of the mtMutL CTD was impaired by mutagenizing the metal-binding motif that is identical to those of eukaryotic and bacterial MutL endonucleases. These results raise the possibility that not only the EndoMS-dependent MMR but also the traditional MutS1/MutL-dependent MMR exist in archaea.

The GIY-YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures.

In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle-specific homolog of bacterial mismatch-binding protein MutS1. The C-terminal region of plant MSH1 contains the GIY-YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown. Here, we characterized the recombinant GIY-YIG domain of Arabidopsis thaliana MSH1, showing that the domain possesses branched DNA-specific DNA-binding activity. Interestingly, the domain exhibited no endonuclease activity, suggesting that the mismatch-binding domain is required for DNA incision. Based on these results, we propose a possible mechanism for MSH1-dependent suppression of homeologous recombination.

Archaeal MutS5 tightly binds to Holliday junction similarly to eukaryotic MutSγ.

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutSγ. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutSγ and bacterial MutS2, a Holliday junction-resolving MutS homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)+-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD+ Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD+/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the ß-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. IMPORTANCE: In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase.

Rapid purification and plasticization of D-glutamate-containing poly-γ-glutamate from Japanese fermented soybean food natto.

Poly-γ-glutamate (PGA) is a major component of mucilage derived from natto, a Japanese fermented food made from soybeans, and PGAs obtained under laboratory's conditions contain numerous d-glutamyl residues. Natto foods are thus promising as a source for nutritionally safe d-amino acids present in intact and digested polymers, although there is little information on the stereochemistry of PGA isolated directly from natto. Here, we describe the development of a new process for rapid purification of PGA using alum and determined the D-glutamate content of natto PGA by chiral high-performance liquid chromatographic analysis. Further, using hexadecylpyridinium cation (HDP(+)), which is a compound of toothpaste, we chemically transformed natto PGA into a new thermoplastic material, called DL-PGAIC. (1)H nuclear magnetic resonance and calorimetric measurements indicate that DL-PGAIC is a stoichiometric complex of natto PGA and HDP(+) with glass transition points of -16.8 °C and -3.1 °C. Then, DL-PGAIC began decomposing at 210°C, suggesting thermal stability suitable for use as a supramolecular soft plastic.

Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. l-Trp was the preferred substrate for deamination, though l-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of l-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of l-Trp, maximum enzymatic activity was observed at 45°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°C. Unlike l-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and l-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S (B-type) stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family.

Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species.

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 µmol·min(-1)·mg(-1), respectively, and those for d-allo-isoleucine were 13.2 mM and 286 µmol·min(-1)·mg(-1), respectively. Hydroxylamine and other inhibitors of pyridoxal 5'-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5'-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.

Crystal structure of the ligand-binding form of nanoRNase from Bacteroides fragilis, a member of the DHH/DHHA1 phosphoesterase family of proteins.

NanoRNase (Nrn) specifically degrades nucleoside 3',5'-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. Two Mn²âº ions are coordinated by conserved residues in the DHH motif of the N-terminal domain. GMP binds near the DHHA1 motif region in the C-terminal domain. Our structure enables us to predict the substrate-bound form of Nrn as well as other DHH/DHHA1 phosphoesterase family proteins.

Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis.

Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD(+), the other for NADP(+) and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81%), but was not high in the region recognizing the 2' position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties.

In vivo, in vitro, and x-ray crystallographic analyses suggest the involvement of an uncharacterized triose-phosphate isomerase (TIM) barrel protein in protection against oxidative stress.

Accumulating genome sequences have revealed the existence of a large number of conserved hypothetical proteins. Characterization of these proteins is considered essential in the elucidation of intracellular biological pathways. Our previous transcriptomic analysis suggested that, in Thermus thermophilus HB8, loss of an oxidized DNA-repairing activity leads to the up-regulation of a function-unknown gene, tthb071, which is conserved in a wide range of bacteria. Interestingly, the tthb071 gene product, TTHB071, showed a significant primary structure similarity to apurinic/apyrimidinic (AP) endonucleases, which are required for the repair of oxidized DNA. In the present study, we observed that disruption of tthb071 increases the H(2)O(2) sensitivity in T. thermophilus HB8, suggesting the involvement of tthb071 in a protection mechanism against oxidative stress. However, purified TTHB071 exhibited no AP endonuclease or DNA-binding activities, indicating that TTHB071 plays no major role in repairing oxidative DNA damage. Then we determined the three-dimensional structure of TTHB071 complexed with zinc ions by x-ray crystallography. In addition to the overall structural similarity, the zinc-binding fashion was almost identical to that of the phosphatase active site of an AP endonuclease, implying that TTHB071 possesses a phosphatase activity. Based on the structural information around the zinc-binding site, we investigated the binding of TTHB071 to 14 different compounds. As a result, TTHB071 favorably bound FMN and pyridoxal phosphate in a zinc ion-mediated manner. Our results suggest that TTHB071 protects the cell from oxidative stress, through controlling the metabolism of FMN, pyridoxal phosphate, or an analogous compound.

Inactivation of the DNA repair genes mutS, mutL or the anti-recombination gene mutS2 leads to activation of vitamin B1 biosynthesis genes.

Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B(1), which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Co-immunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system.

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation.

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.

Molecular mechanisms of the whole DNA repair system: a comparison of bacterial and eukaryotic systems.

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.

Structure of RecJ exonuclease defines its specificity for single-stranded DNA.

RecJ is a single-stranded DNA (ssDNA)-specific 5'-3' exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn(2+) or Mg(2+) by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure. One of two newly identified domains had structural similarities to an oligonucleotide/oligosaccharide-binding (OB) fold. The OB fold domain alone could bind to DNA, indicating that this domain is a novel member of the OB fold superfamily. The truncated RecJ containing only the core domain exhibited much lower affinity for the ssDNA substrate compared with intact RecJ. These results support the hypothesis that these structural features allow specific binding of RecJ to ssDNA. In addition, the structure of the RecJ-Mn(2+) complex suggests that the hydrolysis reaction catalyzed by RecJ proceeds through a two-metal ion mechanism.

Insights into different dependence of dNTP triphosphohydrolase on metal ion species from intracellular ion concentrations in Thermus thermophilus.

Deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) from Thermus thermophilus HB8 (TTHB8) hydrolyzes wide variety of dNTPs to deoxyribonucleoside and inorganic triphosphate in magnesium-dependent manner. In this paper, we assess the specificity for various metal ions and of the dNTP triphosphohydrolase activity of the dNTPase from TTHB8. Manganese and cobalt ions more effectively induced the activity for dNTPs than magnesium and, unexpectedly, brought about the degradation of single kind of dNTP. Manganese and cobalt concentrations of 10 nM were enough to induce the activity, while magnesium of about 1 mM was required for the induction of the activity. To further evaluate metal ions inherent to dNTPase in TTHB8 cells, we measured intracellular concentrations of major metal ions in TTHB8 cells by inductively coupled plasma emission spectroscopy and compared them with the dependence of metal ion concentration on dNTPase activity. Though cobalt ion was below detectable level, magnesium and manganese ions were detected at sufficient level to induce dNTPase activity. These results suggest that both manganese and magnesium ions are likely to be functional under intracellular condition. In addition, the proposed model of dNTPase activity induced by magnesium and multiple dNTPs was discussed based on the results obtained in this study.