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アイテム
大腸菌におけるオペロン欠失技術を活用したヒドロゲナーゼ機能の調査解明
https://doi.org/10.18997/00008988
https://doi.org/10.18997/0000898838a275b3-bff2-49da-b8f9-9873ab480f26
| 名前 / ファイル | ライセンス | アクション |
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sei_k_395.pdf (7.3 MB)
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| アイテムタイプ | 学位論文 = Thesis or Dissertation(1) | |||||||
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| 公開日 | 2022-10-24 | |||||||
| 資源タイプ | ||||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||||
| 資源タイプ | doctoral thesis | |||||||
| タイトル | ||||||||
| タイトル | Investigation of bacterial hydrogenase functions using an operon deletion technology in Escherichia coli | |||||||
| 言語 | en | |||||||
| タイトル | ||||||||
| タイトル | 大腸菌におけるオペロン欠失技術を活用したヒドロゲナーゼ機能の調査解明 | |||||||
| 言語 | ja | |||||||
| 言語 | ||||||||
| 言語 | eng | |||||||
| 著者 |
Shekhar, Chandra
× Shekhar, Chandra
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| 抄録 | ||||||||
| 内容記述タイプ | Abstract | |||||||
| 内容記述 | Hydrogen metabolism is among the most ancient and widespread metabolic trait of microbial life. Escherichia coli （E. coli） is one of the most studied microorganisms for hydrogen metabolism as well as other metabolic phenomena. The four hydrogenase isozymes, hydrogenase 1 （Hyd-1）, hydrogenase 2 （Hyd-2）, hydrogenase 3 （Hyd-3）, and hydrogenase 4 （Hyd-4）, play key roles in hydrogen metabolism by catalyzing the reversible reaction of hydrogen to protons and electrons （2H+ + 2e− H2 （g））. A comparison of the identity between large hydrogenase subunits indicates that either Hyd-1 and Hyd-2 or Hyd-3 and Hyd-4 have a similar homology that was supposed to be a potent reason for inconsistent reports regarding the functions of hydrogenases. Hydrogenases are important integral membrane proteins; therefore, they directly influence microbial membrane physiology. Apart from hydrogen metabolism, hydrogenases were hypothesized to have an essential role in other metabolic activities related to survival in the stressed condition. Hence, the purpose of my Ph. D. study is to investigate the absolute as well as other parallel roles of hydrogenases in hydrogen and associated metabolisms of E. coli. Two types of hydrogenase operon mutants were mainly constructed and employed in the study: （1） operon mutants devoid of only one hydrogenase operon and containing other three in the genome and （2） operon mutants devoid of three hydrogenase operons and possessing solely one hydrogenase operon in the genome. During the study, all single genes encoding large subunit gene deletion mutants along with a quadruple operon mutant devoid of all four hydrogenase operons were employed for their comparative analysis. Using single operon deleted mutants, along with the hydrogen production and uptake activities, hydrogenases were found to have crucial roles in growth, glucose metabolism, energy, redox balance, and pH homeostasis of the cell. The two hydrogenases, Hyd-2 and Hyd-3 were specifically confirmed to possess solely hydrogen productive capabilities, whereas all four have uptake activity. The four single hydrogenase operon mutants were further employed to investigate their roles in persistence and acid resistance mechanisms under oxygen-deprived （micro-aerobic） states along with anaerobic conditions. All the hydrogenases were identified as having remarkable regulating functions in cellular viabilities. However, the roles of energy （ATP） and metabolism （NADH） were found crucial in hydrogenase-mediated cell survival and glucose metabolic phenomenon. Moreover, persistence and acid resistance studies demonstrated the ROS-scavenging and intracellular pH homeostasis activities, respectively, of hydrogenases. Hydrogenases are pivotal in the maintenance of proton gradient across the membrane and quinine pool, therefore, in NAD+ reduction activity and ATP synthesis, hydrogenases are found to play significant roles in the cell. Global transcriptomic analysis demonstrated possible regulating roles of the hydrogenases in diverse and critical metabolic activities. The comprehensive genomic connectivity of the hydrogenases further indicates their roles in bacterial healthy metabolism and survival. Apart from the work with hydrogenases, hydrogen metabolism was further investigated by the random genomic deletion approach, where we designed a new genetic engineering approach by which genomic insertion-deletions can be performed randomly and easily using NNN-extension primer-based PCR products. The approach not only showed efficient and accurate but also successfully demonstrates causing multiple deletions at the same time in the bacterial genome. The study demonstrated that hydrogen metabolism in the bacteria is not an essential metabolic activity since growth and productivity were not affected in random and multiple genomic deletion clones. Taken together, all the findings of this study contribute to understanding microbial hydrogen metabolism and its various roles in bacterial physiology, survival, and adaptation to adverse environmental conditions. | |||||||
| 言語 | en | |||||||
| 目次 | ||||||||
| 内容記述タイプ | TableOfContents | |||||||
| 内容記述 | 1 Introduction and Literature Review||2 Investigation of The Glucose Metabolism by The Deletion of The Operons of Hydrogenase in Escherichia Coli||3 Investigation of The Absolute Role of Each Hydrogenase Operon in Hydrogen Metabolism by Constructing the Escherichia Coli Strains Possessing Only A Single Hydrogenase Operon in The Genome||4 Investigation of The Role of Hydrogenases in The Persistence of Escherichia Coli||5 Elucidating the Significance of Hydrogenases in Cellular Viability and Acid Resistance of Escherichia coli||6 Investigation of The Hydrogen Metabolism by A New Approach of Random Genomic Deletions Using Ambiguous Sequences in The Escherichia Coli Genome||7 Concluding Remarks and Suggestions for The Future | |||||||
| 備考 | ||||||||
| 内容記述タイプ | Other | |||||||
| 内容記述 | 九州工業大学博士学位論文 学位記番号：生工博甲第395号 学位授与年月日：令和3年3月25日 | |||||||
| キーワード | ||||||||
| 主題Scheme | Other | |||||||
| 主題 | Operon deletion | |||||||
| キーワード | ||||||||
| 主題Scheme | Other | |||||||
| 主題 | Hydrogenase | |||||||
| キーワード | ||||||||
| 主題Scheme | Other | |||||||
| 主題 | Escherichia coli | |||||||
| キーワード | ||||||||
| 主題Scheme | Other | |||||||
| 主題 | Hydrogen metabolism | |||||||
| キーワード | ||||||||
| 主題Scheme | Other | |||||||
| 主題 | Acid resistance | |||||||
| キーワード | ||||||||
| 主題Scheme | Other | |||||||
| 主題 | Persister | |||||||
| アドバイザー | ||||||||
| 前田, 憲成 | ||||||||
| 学位授与番号 | ||||||||
| 学位授与番号 | 甲第395号 | |||||||
| 学位名 | ||||||||
| 学位名 | 博士（工学） | |||||||
| 学位授与年月日 | ||||||||
| 学位授与年月日 | 2021-03-25 | |||||||
| 学位授与機関 | ||||||||
| 学位授与機関識別子Scheme | kakenhi | |||||||
| 学位授与機関識別子 | 17104 | |||||||
| 学位授与機関名 | 九州工業大学 | |||||||
| 学位授与年度 | ||||||||
| 内容記述タイプ | Other | |||||||
| 内容記述 | 令和2年度 | |||||||
| 出版タイプ | ||||||||
| 出版タイプ | VoR | |||||||
| 出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||
| アクセス権 | ||||||||
| アクセス権 | open access | |||||||
| アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||
| ID登録 | ||||||||
| ID登録 | 10.18997/00008988 | |||||||
| ID登録タイプ | JaLC | |||||||
