R. Yatsunami, M. Sato, K. Orishimo, Y. Hatori, Y. Zhang, T. Takashina, T. Fukui, Satoshi Nakamura
An open reading frame encoding a chitinase homolog (ChiN1) was found in the genome of extremely halophilic archaeon Halobacterium salinarum NRC-1. ChiN1 is a multidomain enzyme consisting of a chitin-binding domain, a polycystic kidney disease domain and a catalytic domain belonging to glycoside hydrolase family 18. chiN1 gene was successfully expressed in extremely halophilic archaeon Haloarcula japonica TR-1 by employing the promoter sequence of its cell surface glycoprotein gene. A large amount of recombinant ChiN1 was secreted into the culture supernatant. The Ha. japonica-produced ChiN1 was purified and characterized. The optimal pH and temperature of ChiN1 are pH 4.5 and 55°C, respectively. ChiN1 was most active at 1.0 M NaCl and stable over a wide range of NaCl concentration from 1.0 to 4.5 M. This is the first report on a chitinase from extremely halophilic archaeon.
在极端嗜盐古细菌盐盐菌NRC-1基因组中发现了一个编码几丁质酶同源物(ChiN1)的开放阅读框。ChiN1是一种多结构域酶，由几丁质结合结构域、多囊肾病结构域和催化结构域组成，属于糖苷水解酶家族18。利用细胞表面糖蛋白基因启动子序列成功表达了chiN1基因在极嗜盐古菌(Haloarcula japonica TR-1)中的表达。大量重组ChiN1被分泌到培养上清液中。Ha。对日本产的ChiN1进行了纯化和鉴定。ChiN1的最佳pH和温度分别为pH 4.5和55℃。ChiN1在1.0 M NaCl条件下活性最强，在1.0 ~ 4.5 M NaCl浓度范围内稳定。这是对极嗜盐古菌中几丁质酶的首次报道。
{"title":"Gene expression and characterization of a novel GH family 18 chitinase from extremely halophilic archaeon Halobacterium salinarum NRC-1","authors":"R. Yatsunami, M. Sato, K. Orishimo, Y. Hatori, Y. Zhang, T. Takashina, T. Fukui, Satoshi Nakamura","doi":"10.3118/JJSE.9.19","DOIUrl":"https://doi.org/10.3118/JJSE.9.19","url":null,"abstract":"An open reading frame encoding a chitinase homolog (ChiN1) was found in the genome of extremely halophilic archaeon Halobacterium salinarum NRC-1. ChiN1 is a multidomain enzyme consisting of a chitin-binding domain, a polycystic kidney disease domain and a catalytic domain belonging to glycoside hydrolase family 18. chiN1 gene was successfully expressed in extremely halophilic archaeon Haloarcula japonica TR-1 by employing the promoter sequence of its cell surface glycoprotein gene. A large amount of recombinant ChiN1 was secreted into the culture supernatant. The Ha. japonica-produced ChiN1 was purified and characterized. The optimal pH and temperature of ChiN1 are pH 4.5 and 55°C, respectively. ChiN1 was most active at 1.0 M NaCl and stable over a wide range of NaCl concentration from 1.0 to 4.5 M. This is the first report on a chitinase from extremely halophilic archaeon.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"113 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124718730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Toru Mizuki, M. Kamekura, M. Ishibashi, R. Usami, Yasuhiko Yoshida, M. Tokunaga, K. Horikoshi
Nucleotide diphosphate kinase (NDK) was purified from 12 strains of halobacteria using ATP-agarose chromatography and their N-terminal amino acid sequences were determined. The electrophoretic mobilities of these enzymes differed significantly on the native-PAGE or the SDS-PAGE when the samples were not heat treated. Comparison of seven complete amino acid sequences from seven species of Haloarcula_and those from Har. californiae and Har. japonica, whose N-terminal 3 amino acids have not been determined yet, revealed that they are very similar and differed at only 1 to 4 residues in 153 residues. NDKs from Haloarcula quadrata and Har. sinaiiensis differed only at the 30th amino acid (arginine vs. cysteine), yet they showed a remarkable difference in their salt-response patterns, suggesting that a single amino acid substitution can cause a one molar shift in the optimal NaCl concentration.
{"title":"Nucleoside diphosphate kinase of halobacteria","authors":"Toru Mizuki, M. Kamekura, M. Ishibashi, R. Usami, Yasuhiko Yoshida, M. Tokunaga, K. Horikoshi","doi":"10.3118/JJSE.3.1_18","DOIUrl":"https://doi.org/10.3118/JJSE.3.1_18","url":null,"abstract":"Nucleotide diphosphate kinase (NDK) was purified from 12 strains of halobacteria using ATP-agarose chromatography and their N-terminal amino acid sequences were determined. The electrophoretic mobilities of these enzymes differed significantly on the native-PAGE or the SDS-PAGE when the samples were not heat treated. Comparison of seven complete amino acid sequences from seven species of Haloarcula_and those from Har. californiae and Har. japonica, whose N-terminal 3 amino acids have not been determined yet, revealed that they are very similar and differed at only 1 to 4 residues in 153 residues. NDKs from Haloarcula quadrata and Har. sinaiiensis differed only at the 30th amino acid (arginine vs. cysteine), yet they showed a remarkable difference in their salt-response patterns, suggesting that a single amino acid substitution can cause a one molar shift in the optimal NaCl concentration.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"170 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122422983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
N. Hirota, T. Matsuo, A. Ikeda, R. Yatsunami, T. Fukui, Satoshi Nakamura
The ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe-2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich in acidic amino acids, as well as a common core domain that contains the Fe-S cluster. The N-terminally HAT-tagged intact HjFd (HAT/HjFd) and spinach/Ha. japonica chimeric Fd (HAT/Sp/HjFd) were prepared and characterized. Escherichia coli-produced HAT/Sp/HjFd and Ha. japonica-produced HAT/HjFd were produced as holoproteins. On the other hand, E. coli-produced HAT/HjFd did not incorporate the Fe-S cluster. These results suggested that the N-terminal domain of HjFd contributed to the polypeptide folding and successive Fe-S cluster incorporation under high salt conditions. Both Ha. japonica-produced HAT/HjFd and E. coli-produced HAT/Sp/HjFd were stable at high salt concentrations (≥1.5 M NaCl), although a reduction in stability was observed at lower concentrations. Lack of the N-terminal domain did not affect the stability of HjFd, indicating that the core domain mainly contributed to the stability of HjFd at high salt concentrations. Solubility of E. coli-produced HAT/Sp/HjFd under high salt conditions was significantly lower than that of Ha. japonica-produced HAT/HjFd. It was revealed that substitution of the N-terminal domain of HjFd to that of spinach Fd injured the solubility of HjFd. Thus, it was concluded that the N-terminal domain of HjFd should perform the essential functions for halophilic adaptation from the folding process through the folded state.
来自Haloarcula japonica的铁氧还蛋白(Fd)具有植物型[2Fe-2S]簇，在高盐浓度下稳定。哈哈。japonica Fd (HjFd)包含一个富含酸性氨基酸的n端附加结构域，以及一个包含Fe-S簇的共同核心结构域。n端HAT标记的完整HjFd (HAT/HjFd)和菠菜/Ha。制备了粳稻嵌合Fd (HAT/Sp/HjFd)并对其进行了表征。大肠杆菌产生的HAT/Sp/HjFd和Ha。以全蛋白的形式制备HAT/HjFd。另一方面，大肠杆菌产生的HAT/HjFd不包含Fe-S簇。这些结果表明，在高盐条件下，HjFd的n端结构域参与了多肽折叠和Fe-S簇的连续结合。两公顷。日本产的HAT/HjFd和大肠杆菌产的HAT/Sp/HjFd在高盐浓度(≥1.5 M NaCl)下稳定，但在低盐浓度下稳定性降低。缺少n端结构域不影响HjFd的稳定性，表明核心结构域主要影响HjFd在高盐浓度下的稳定性。大肠杆菌产生的HAT/Sp/HjFd在高盐条件下的溶解度显著低于Ha。japonica-produced帽子/ HjFd。结果表明，HjFd的n端结构域被菠菜Fd取代会损害HjFd的溶解度。综上所述，HjFd的n端结构域从折叠过程到折叠态都发挥着亲盐适应的重要作用。
{"title":"Role of an N-terminal domain found in the ferredoxin from extremely halophilic archaeon Haloarcula japonica","authors":"N. Hirota, T. Matsuo, A. Ikeda, R. Yatsunami, T. Fukui, Satoshi Nakamura","doi":"10.3118/JJSE.4.14","DOIUrl":"https://doi.org/10.3118/JJSE.4.14","url":null,"abstract":"The ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe-2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich in acidic amino acids, as well as a common core domain that contains the Fe-S cluster. The N-terminally HAT-tagged intact HjFd (HAT/HjFd) and spinach/Ha. japonica chimeric Fd (HAT/Sp/HjFd) were prepared and characterized. Escherichia coli-produced HAT/Sp/HjFd and Ha. japonica-produced HAT/HjFd were produced as holoproteins. On the other hand, E. coli-produced HAT/HjFd did not incorporate the Fe-S cluster. These results suggested that the N-terminal domain of HjFd contributed to the polypeptide folding and successive Fe-S cluster incorporation under high salt conditions. Both Ha. japonica-produced HAT/HjFd and E. coli-produced HAT/Sp/HjFd were stable at high salt concentrations (≥1.5 M NaCl), although a reduction in stability was observed at lower concentrations. Lack of the N-terminal domain did not affect the stability of HjFd, indicating that the core domain mainly contributed to the stability of HjFd at high salt concentrations. Solubility of E. coli-produced HAT/Sp/HjFd under high salt conditions was significantly lower than that of Ha. japonica-produced HAT/HjFd. It was revealed that substitution of the N-terminal domain of HjFd to that of spinach Fd injured the solubility of HjFd. Thus, it was concluded that the N-terminal domain of HjFd should perform the essential functions for halophilic adaptation from the folding process through the folded state.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"9 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125150143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The product YkoN of the gene of unknown function, ykoN, of Bacillus subtilis Marburg has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and thus YkoN is expected to have a lipase or esterase activity. To characterize the expected enzyme activity the plasmid having a modified ykoN that include the sequence for His(x6) tag at its C-terminus of YkoN, which has 373 amino acid residues, was constructed. His-tagged YkoN protein of 39 kDa was induced in Escherichia coli BL21 (DE3) cells harboring chaperon plasmid pGro7 and purified to near homogeneity by using gel filtration and Ni-agarose. When p-nitrophenyl-esters of different fatty acid chain length were examined, the purified YkoN hydrolyzed the esters of fatty acid with short chain length (4-6 carbon atoms) preferentially. The esters of fatty acid with longer chain (C ≥ 10) were hydrolyzed inefficiently. The activity required no divalent cations and was not affected by addition of EDTA. The optimal pH for the activity was from pH 7.4 to pH 8.6. These results indicate that YkoN is a novel esterase which hydrolyzes the esters of fatty acid with short chain length.
{"title":"Characterization of a Novel Esterase YkoN from Bacillus subtilis Marburg","authors":"T. Matsuura, R. Usami, H. Hara, K. Matsumoto","doi":"10.3118/JJSE.6.32","DOIUrl":"https://doi.org/10.3118/JJSE.6.32","url":null,"abstract":"The product YkoN of the gene of unknown function, ykoN, of Bacillus subtilis Marburg has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and thus YkoN is expected to have a lipase or esterase activity. To characterize the expected enzyme activity the plasmid having a modified ykoN that include the sequence for His(x6) tag at its C-terminus of YkoN, which has 373 amino acid residues, was constructed. His-tagged YkoN protein of 39 kDa was induced in Escherichia coli BL21 (DE3) cells harboring chaperon plasmid pGro7 and purified to near homogeneity by using gel filtration and Ni-agarose. When p-nitrophenyl-esters of different fatty acid chain length were examined, the purified YkoN hydrolyzed the esters of fatty acid with short chain length (4-6 carbon atoms) preferentially. The esters of fatty acid with longer chain (C ≥ 10) were hydrolyzed inefficiently. The activity required no divalent cations and was not affected by addition of EDTA. The optimal pH for the activity was from pH 7.4 to pH 8.6. These results indicate that YkoN is a novel esterase which hydrolyzes the esters of fatty acid with short chain length.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"46 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125010692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shizuka Arakawa, M. Mori, L. Li, Y. Nogi, Takako Sato, Yasuhiko Yoshida, R. Usami, C. Kato
We have analyzed the microbial communities in the cold-seep sediment samples obtained from different depths (5800∼7500 m) of the Japan Trench land slope. The results indicated that the typical cold-seep microbial communities of bacteria and archaea were basically similar in different depth environments and consisted of the delta-Proteobacteria (including sulfate reducing bacterial group) as well as methanogenic archaea, which played an important role in sulfur circulation in the seep environment. More abundant microbes were also identified in deeper cold-seep environments. These observations suggested that the cold-seep activity at the deepest depths of the Japan Trench might be more dynamic than in the shallower land slope. This is the first suggestion describing the relationship between microbial mass and cold-seep activity.
{"title":"Cold-seep microbial communities are more abundant at deeper depths in the Japan Trench land slope.","authors":"Shizuka Arakawa, M. Mori, L. Li, Y. Nogi, Takako Sato, Yasuhiko Yoshida, R. Usami, C. Kato","doi":"10.3118/JJSE.4.50","DOIUrl":"https://doi.org/10.3118/JJSE.4.50","url":null,"abstract":"We have analyzed the microbial communities in the cold-seep sediment samples obtained from different depths (5800∼7500 m) of the Japan Trench land slope. The results indicated that the typical cold-seep microbial communities of bacteria and archaea were basically similar in different depth environments and consisted of the delta-Proteobacteria (including sulfate reducing bacterial group) as well as methanogenic archaea, which played an important role in sulfur circulation in the seep environment. More abundant microbes were also identified in deeper cold-seep environments. These observations suggested that the cold-seep activity at the deepest depths of the Japan Trench might be more dynamic than in the shallower land slope. This is the first suggestion describing the relationship between microbial mass and cold-seep activity.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"20 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"126679116","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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