Constant Enthalpy Change Value during Pyrophosphate Hydrolysis within the Physiological Limits of NaCl

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. However, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at ∼4 m NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 m NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of −35 kJ/mol was obtained for the halophile and non-halophiles at 1.5–4.0 and 0.1–2.0 m NaCl, respectively. These results show that solvation changes caused by up to 4.0 m NaCl (water activity of ∼0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.This work was supported by Grant-in-aid for Scientific Research on Innovative Areas 20118005 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan

Difference in NaCl tolerance of membrane-bound 5′-nucleotidases purified from deep-sea and brackish water Shewanella species

Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5′-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance in order to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl2 for their activities, and they exhibited the same pH dependency and substrate specificity for 5′-nucleotides. However, S. violacea 5′-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5′-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5′-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [No. 26240045], a grant from the Japan Society for the Promotion of Science [No. 25-1446], and The Salt Science Research Foundation [No. 1655]

16S-23S rDNA スペーサー領域の制限断片長多型解析によるAcidithiobacillus 属3種、A.ferroxidans、A.thiooxidans およびA.caldus の識別

The PCR-amplified 16S-23S rDNA intergenic spacer regions (ISRs) of Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus strains were scquenced and evaluated for differentiation and identification of these bacteria. The total length of the 16S-23S ISRs of A. ferrooxidans and A. thiooxidans strains and A.caldus GO-1 were 441, 456, nd 379bp, respectively. Two genes. encoding tRNA and tRNA, and the box A-like sequences were highly conserved in the ISRs of all Acidithiobacillus species. The restriction fragment length polymorphism (RFLP) profiles of the PCR-amplified 16S-23S rDNA ISRs digested by HaeIII and AluI could clearly discriminate A. ferrooxidans from A. thiooxidans and A. caldus. The results indicated that RFLP analysis of the 16S-23S ISRs is an easy and rapid method for discrimination and identification of Acidithiobacillus species.Acidithiobacillus ferrooxidans，A.thiooxidansおよびA.caldusの16S-23S rDNAスペーサー領域の塩基配列を決定し，これらのバクテリアの識別・同定への有効性を評価した．A.ferrooxidans，A.thiooxidansおよびA.caldusのスペーサー領域の長さは，それぞれ441，456および379bpであった．３種のAcidithiobacillusスペーサー領域では，tRNA，tRNAをコードする遺伝子の塩基配列が高度に保存されていた．PCR増幅した16S-23S rDNAスペーサー領域をHaeおよびAluで酵素処理することによって得られた断片の解析によって，A.ferrooxidansをA.thiooxidansとA.caldusから識別できた．Acidithiobacillus種の16S-23S rDNAスペーサー領域の制限断片長多型解析は，Acidithiobacillus属の種の同定およびA.ferrooxidansに属する株の同定のための迅速で，技術的に簡便な方法であることが明らかとなった

好酸性鉄酸化細菌 Acidithiobacillus ferrooxidans の染色体凝集・分配タンパク質（ScpB）の性質

　Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide. This bacterium uses ferrous iron and reduced inorganic sulfur compounds (RISCs) as energy sources. Transcriptions of genes thought to be involved in the oxidation of RISCs have been known to be highly activated in A. ferrooxidans cells grown on RISCs, while transcriptions of genes involved in the iron oxidation were repressed in the cells grown on RISCs. A gene encoding a putative chromosome segregation and condensation protein (ScpB) with a helix-turn-helix motif was found in the upstream region of sulfide : quinone oxidoreductase gene, whose expression was up-regulated in cells grown in sulfur and tetrathionate. A semi-quantitative PCR analysis using cDNA prepared from iron-, sulfur-, or tetrathionate-grown cells revealed that the transcription of scpB was up-regulated in cells grown on sulfur or tetrathionate as the energy source. Electrophoretic mobility shift assays were employed to examine whether the ScpB functions as a transcription factor. The result indicated that the recombinant His-tagged ScpB protein was able to nonspecifically bind in vitro to DNA. This is the first report on a direct association of ScpB with DNA.　Acidithiobacillus ferrooxidans は，低品位の銅鉱石から銅を回収するバイオリーチングにおいて使用される微生物 の一つである．この細菌は，エネルギー源として二価鉄イオンや還元型無機硫黄化合物（RISC）を使用する．鉄の酸化に関与する遺伝子の転写は，A. ferrooxidans が RISC で生育したときには抑制されるが，RISC の酸化に関与すると考えられている遺伝子の転写は活性化されることが知られている．硫黄やテトラチオン酸で生育したときにその発現が上方制御される硫化水素：キノン酸化還元酵素のすぐ上流に，ヘリックスターンへリックスモティーフを持つ，ScpB と推定されるタンパク質をコードする遺伝子が存在していた．鉄，硫黄，テトラチオン酸生育細胞から調製した cDNA を用いた半定量的 PCR 分析の結果，硫黄やテトラチオン酸で生育した細胞内の scpB 遺伝子の転写は，鉄生育細胞と比較すると上方制御されていた．組換え ScpB タンパク質を用いたゲルシフトアッセイ法で，ScpB が転写制御因子として機能するかどうかを調べた．その結果，ScpB は DNA に結合したが，結合の特異性はなかった．ScpB が直接 DNA と相互作用する報告はこれまでになかった

好酸性鉄酸化細菌 Acidithiobacillus ferrooxidans の染色体凝集・分配タンパク質（ScpB）の性質

　Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide. This bacterium uses ferrous iron and reduced inorganic sulfur compounds (RISCs) as energy sources. Transcriptions of genes thought to be involved in the oxidation of RISCs have been known to be highly activated in A. ferrooxidans cells grown on RISCs, while transcriptions of genes involved in the iron oxidation were repressed in the cells grown on RISCs. A gene encoding a putative chromosome segregation and condensation protein (ScpB) with a helix-turn-helix motif was found in the upstream region of sulfide : quinone oxidoreductase gene, whose expression was up-regulated in cells grown in sulfur and tetrathionate. A semi-quantitative PCR analysis using cDNA prepared from iron-, sulfur-, or tetrathionate-grown cells revealed that the transcription of scpB was up-regulated in cells grown on sulfur or tetrathionate as the energy source. Electrophoretic mobility shift assays were employed to examine whether the ScpB functions as a transcription factor. The result indicated that the recombinant His-tagged ScpB protein was able to nonspecifically bind in vitro to DNA. This is the first report on a direct association of ScpB with DNA.　Acidithiobacillus ferrooxidans は，低品位の銅鉱石から銅を回収するバイオリーチングにおいて使用される微生物 の一つである．この細菌は，エネルギー源として二価鉄イオンや還元型無機硫黄化合物（RISC）を使用する．鉄の酸化に関与する遺伝子の転写は，A. ferrooxidans が RISC で生育したときには抑制されるが，RISC の酸化に関与すると考えられている遺伝子の転写は活性化されることが知られている．硫黄やテトラチオン酸で生育したときにその発現が上方制御される硫化水素：キノン酸化還元酵素のすぐ上流に，ヘリックスターンへリックスモティーフを持つ，ScpB と推定されるタンパク質をコードする遺伝子が存在していた．鉄，硫黄，テトラチオン酸生育細胞から調製した cDNA を用いた半定量的 PCR 分析の結果，硫黄やテトラチオン酸で生育した細胞内の scpB 遺伝子の転写は，鉄生育細胞と比較すると上方制御されていた．組換え ScpB タンパク質を用いたゲルシフトアッセイ法で，ScpB が転写制御因子として機能するかどうかを調べた．その結果，ScpB は DNA に結合したが，結合の特異性はなかった．ScpB が直接 DNA と相互作用する報告はこれまでになかった

Structural and functional insights into thermally stable cytochrome c' from a thermophile

Thermophilic Hydrogenophilus thermoluteolus cytochrome c0 (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c0 (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 A ° . In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit–subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit–subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit–subunit interactions with conservation of the ligand-binding ability.This work was performed under the Cooperative Research Program of the “Network Joint Research Center for Materials and Devices”

DNA Damage Sensor γ

Background. Phosphorylated histone H2AX (γ-H2AX) is a potential regulator of DNA repair and is a useful tool for detecting DNA damage. To evaluate the clinical usefulness of γ-H2AX in hepatocellular carcinoma (HCC), we measured the level of γ-H2AX in HCC, dysplastic nodule, and nontumorous liver diseases. Methods. The level of γ-H2AX was measured by immunohistochemistry in fifty-eight HCC, 18 chronic hepatitis, 22 liver cirrhosis, and 19 dysplastic nodules. Appropriate cases were also examined by fluorescence analysis and western blotting. Results. All cases with chronic liver disease showed increased levels of γ-H2AX expression. In 40 (69.9%) of 58 cases with HCC, the labeling index (LI) of γ-H2AX was above 50% and was inversely correlated with the histological grade. Mean γ-H2AX LI was the highest in dysplastic nodule (74.1±22.1%), which was significantly higher than HCC (P<0.005). Moreover, γ-H2AX was significantly increased in nontumorous tissues of HCC as compared with liver cirrhosis without HCC (62.5±24.7%, from 5.1 to 96.0%, P<0.005). Conclusions. γ-H2AX was increased in the preneoplastic lesions of HCC and might be a useful biomarker for predicting the risk of HCC