Introduction

Oxygen is primary element that is indispensable for life to constitute its body, as well as carbon, hydrogen, and nitrogen, and is the most abundant oxidant in a surface of the earth. The relationship between molecular oxygen (O2) and life is interesting. Organisms efficiently obtain energy by aerobic respiration of O2. On the other hand, reactive oxygen species (ROS), byproducts of the respiration, have high reactivity towards cellular molecules leading to irreversible damage and even cell death. In this way, the relationship with oxygen is always associated with both benefits and risks, although numerous organisms today cope well with oxygen. Then, how have the organisms handled the risks derived from ROS? Small amount of O2 is presumed to exist in a surface of the primitive earth. The pollution of ROS derived from gradually-increased O2 might be menace for ancient organisms.

The common strategy for respiration and oxidative-stress defense is the utilization of electron. O2 is reduced with four electrons at the terminus of the respiratory chain to produce H2O, while ROS can be detoxified by reduction catalyzed by oxidative-stress defense enzymes. Therefore, to investigate the electron utilization of the aerobic organism leads to know how the organism deals with oxygen. In this study, elucidation of the relationship between oxygen, electron, and life was primarily intended, through the investigation of energy metabolisms of an evolutionary-ancient bacterium Hydrogenobacter thermophilus.

Chapter 1. Electron acquisition

In Chapter 1, electron acquisition manner of H. thermophilus was investigated. This bacterium is chemolithoautotroph that utilize not only hydrogen (H2) but also thiosulfate as a sole source of energy and assimilates carbon dioxide via the reductive tricarboxylic acid (RTCA) cycle. Here, transcriptome analysis of metabolic enzymes in both H2- and thiosulfate-grown H. thermophilus cells was carried out.

The results indicated that the expression of hydrogenase genes is significantly repressed under thiosulfate-oxidation conditions, whereas some genes for sulfur metabolisms, including sox genes, showed almost the same expression levels under both H2- and thiosulfate-oxidation conditions. In addition, the genes for the RTCA cycle and several central metabolic pathways showed high expression levels under both conditions. It was suggested that such central metabolisms and sulfur metabolism function as forms of basal metabolism and H2-oxidation is inducible. Utilization of sulfur compounds may be advantageous for H. thermophilus to survive in nature, as the habitat of this bacterium is hot spring in which abundant sulfur compounds are available.

Chapter 2. Electron carriers

In Chapter 2, ferredoxin (Fd) and its redox partner are focused. Fd is a vital electron carrier protein in this bacterium, as it is an electron donor for several key enzymes catalyzing the reactions of the RTCA cycle. Further, in recent years a novel Fd1-dependent type glutamine:2-oxoglutarate amidotransferase was found from this bacterium. Hence, Fd appears to be an important hub for the electron flow in H. thermophilus, and novel Fd1-related enzymes are expected to be identified. By detecting protein-protein interactions (PPI), Fd-related proteins were screened.

Although many approaches to detect PPI have been developed in the last decade, majority of them are not suitable to screen Fd-related proteins, because almost all of them are meant to detect strong PPI, whereas Fd and its redox partner are predicted to interact weakly. To identify the proteins interacting with Fd weakly, I developed a novel method by modifying far-western blotting technique. In the developed method, Fd and Fd-interacting protein are covalently cross-linked in order to increase detectivity for Fd-interacting proteins. The effectiveness of the developed method to detect weak interactions was confirmed by the detection of the enzymes reported to be Fd-dependent. Finally, the screening of Fd-related proteins using the developed method was carried out, and resulted in the identification of bacterioferritin comigratory protein (BCP) as a candidate for Fd-interacting protein. BCP has been reported to function as a peroxidase that is a member of oxidative-stress defense enzymes. It was suggested that electrons from Fd may be utilized in the oxidative-stress defense system of H. thermophilus, indicating the importance of the defense system.

Chapter 3. Electron-mediated relationships between oxygen and H. thermophilus

In Chapter 3, aerobic respiration and oxidative-stress defense systems are focused.

Respiration

3-1. Respiration

Four genes for heme/copper type cytochrome c oxidases (cox1, cox2, cox3, and cyo), which catalyze the four-electron reduction of O2 at the terminus of respiratory chain, were found from H. thermophilus genome. Transcriptome analyses in the past showed different expression patterns for each enzyme gene, suggesting the appropriate usage depending on the environments.

In order to investigate the function of the oxidases in vivo, each gene disruptant was tried to be constructed, and three of them (Δcox2, Δcox3, Δcyo) were obtained, although cox1 disruptant has not been obtained yet. Observation of the growth profiles of the three mutants suggested the roles of each enzyme. Δcox2 showed high growth rate compared with wild-type (WT) strain, implying low proton pumping efficiency of Cox2. Maximum optical density at 540 nm of cultures of Δcox3 and Δcyo was lower than that of WT, suggesting Cox3 and Cyo may function as high affinity enzymes that can be mainly used in low O2 concentration conditions such as stationary growth phase.

Cytochrome c oxidases have been proposed to be classified into three groups (Type A, B, and C) according to the phylogeny, and to conserved amino acids in the conserved domains. To our surprise, phylogenetic analysis revealed that Cyo does not belong to any of the three groups, and compose another group in the phylogenetic tree with its homologous proteins. Biochemical analysis of Cyo, predicted to be a novel member of respiratory enzyme, is now being carried out.

Oxidative-stress defense systems

3-2. Bacterioferritin comigratory protein

BCP, identified as a candidate for Fd-interacting protein in Chapter 2, is focused. To investigate the function in vivo, bcp gene defect mutants (Δbcp) was constructed, and cultivated under several conditions. As BCP has been reported to function as a peroxidase, which is a member of ROS detoxifier, the growth profiles under various concentrations of O2 and peroxide were observed. As a result, Δbcp showed higher sensitivity toward peroxide than WT, implying that BCP functions as antioxidant in vivo. BCP was heterologously expressed in Escherichia coli, and was purified. Purified BCP exhibited peroxide reductase activity in the presence of dithiothreitol as an electron donor, indicating the function as a thiol peroxidase. Although BCP was predicted to show Fd-dependent peroxidase activity, the activity has not been detected yet.

One to three cysteine (Cys) residues are conserved in the amino acid sequences of BCPs reported so far. In contrast, H. thermophilus BCP has four Cys residues. To clarify the roles of these Cys, amino acid substitution variants (C12A, C26A, C48A, and C53A) were constructed. Interestingly, only C48A lost peroxidase activity, although the others remained the activity. Taken together with the high conservativeness of Cys48 among other BCP sequences, at least Cys48 appears to be crucial for the activity. By homology modeling, three-dimensional structure of BCP was predicted. In the modeled structure, locations of Cys12 and Cys26 are predicted to be quite displaced from that of Cys48 and Cys53, thus, Cys12 and Cys26 are presumed to be not involved in the direct peroxide reducing reaction. Several BCPs are reported to reduce peroxides by the redox of intra- or inter-molecular two Cys residues, however, the counterpart of Cys48 has not been revealed in H. thermophilus BCP. Unusual peroxidase mechanism may be employed in H. thermophilus BCP. Further characterization of BCP is being carried out.

3-3. Ferriperoxin

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in H. thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H2O2) and organic hydroperoxides in the presence of NADPH and FNR as electron donors. The calculated Km and Vmax values of Fpx for organic hydroperoxides were comparable to that for H2O2, demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, which clearly shows the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree constructed with the sequences of Rbr and Rbr-like proteins. This finding, together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system.

3-4. Physiological partner of ferriperoxin

Since the gene for FNR was not found in genomes of some of organisms possessing fpx gene, FNR may not be a physiological partner of Fpx. By comparing the genomes, approximately 13 kb gene was found to be located next to fpx gene in many organisms possessing fpx gene, although annotations are diverse. In the genome of H. thermophilus, the gene (HTH_1527) was quite displaced from the locus of fpx. I tried to heterologously express HTH_1527 under various conditions using several plasmids, hosts, additive to the medium, temperatures, however almost all of the proteins formed inclusion bodies, and the appropriate protein has not been obtained.

3-5. Suppressor mutant of fpx gene defect mutant

By continually culturing Δfpx under aerobic conditions, suppressor mutant that is capable of growing under aerobic conditions was obtained. To reveal what recovers O2-sensitive phenotype, transcriptome analysis was carried out. It was demonstrated that expression levels of alkyl hydroperoxide reductase gene (ahpC) in suppressor mutant cells was 18-fold higher than that in WT strain cells under aerobic conditions. AhpC is reported to function as a peroxidase, thus, large expression of ahpC gene may compensate the lack of Fpx. To clarify the trigger of high expression of ahpC, whole genome mutation analysis was carried out, although significant mutation was not found.

Conclusion

This study elucidated a part of energy metabolism of H. thermophilus, including several new insights into ROS detoxification systems and respiratory enzymes. The manner of acquisition and consumption of electrons in this bacterium implies the "conservative" relationship of H. thermophilus with O2, namely this bacterium favors safety more than efficiency. H. thermophilus appears to obtain electrons in a reliable manner, and to keep cells highly reduced using a series of antioxidant enzymes by consuming abundant reducing power. Even respiration can be considered as a system to reductively detoxify molecular oxygen. Recently, one of hydrogenase of this bacterium was clarified to be necessary to survive under high O2 conditions nevertheless it is not ROS detoxifier. Interestingly, that hydrogenase, Fpx and Cyo are absent in the genome of Aquifex aeolicus, which is a closely related species of H. thermophilus but is O2-sensitive. Like these, H. thermophilus might have evolved against aerobic environments through obtaining such enzymatic equipments to handle oxidative-stress. Further, like H. thermophilus, numerous aerobic organisms living in the earth today might have evolved through continual struggle against oxygen, and finally obtained sophisticated mechanisms to get the most benefit from oxygen utilization.

多くの生物にとって最も重要な生命活動として呼吸が挙げられる。呼吸では酸素を水に還元することで効率的にエネルギーを獲得するが、それに伴い「活性酸素種」という有害な副産物が生じてしまう。活性酸素種は生体に深刻なダメージを与えるため、ほとんど全ての生物は活性酸素種からの防御酵素を装備している。一方、太古の地球には酸素はほとんど存在せず、当時の生物は酸素に非常に弱かったと考えられている。本研究では古い進化的起源を有する細菌、Hydrogenobacter thermophilusを研究対象に用いた。興味深いことに、本菌は進化的起源を色濃く残す「酸素に弱い代謝系」を持つにも関わらず、酸素存在下で旺盛に生育する。本研究ではこの矛盾に着目した。また、酸素からの利益の享受を担う「呼吸」と、危機からの保身を担う「防御系」は全く異質であるが、いずれも電子の供給に依存しており、電子なしでは機能しえない。このように電子の流れ、すなわちエネルギー代謝系は酸素と生物との関係に密接にリンクしており、本研究ではH. thermophilusのエネルギー代謝を理解することで生物と酸素との関係を明らかにすることを目的に掲げた。

第一章では電子の獲得系の特徴づけがなされた。本菌は水素またはチオ硫酸を酸化することによって電子(還元力)を得ているが、水素酸化による生育の方が格段に速いことが分かった。しかし興味深いことに、チオ硫酸代謝酵素はチオ硫酸の有無に関わらず常に高い発現量を示した。反対に水素代謝酵素であるhydrogenaseは水素が無いと発現しなかった。チオ硫酸代謝系を構成的に利用することで高い生存率を維持し、環境に応じて水素酸化系のON-OFFを切り替えていることが示唆された。すなわち本菌では、効率的なエネルギー獲得系より、低効率でも生存に有利なものが選択されているという結論を得た。

第二章では本菌の電子伝達体に着目した。本菌の中央代謝系ではferredoxin (Fd)という電子伝達タンパク質が電子供与体として活躍しており、これまでに炭素・窒素代謝系で新規のFd利用酵素が見つかってきた。そこでタンパク質間相互作用(PPI ; Protein-Protein Interaction)を指標に、Fdを利用する新規酵素の探索を行った。FdとそのパートナーのPPIは非常に弱く、既存の手法では検出が非常に困難であった。そこで本研究ではそのような弱いPPIを検出する新規手法を開発した。開発した手法を用いた研究によってFdがbacterioferritin comigratory protein (BCP)という酸化ストレス防御酵素に電子を供給することが示された。Fdが酸化ストレス防御に関与するという報告はなく、新規の電子ネットワークの可能性を示す重要な知見である。

第三章では電子が介する酸素と本菌との関係について、本菌の呼吸系および酸化ストレス防御機構について研究を行った。まず、系統解析の結果、本菌は新規の呼吸酵素持つことが示唆された。また、本菌の持つ4種類の呼吸酵素は酸素濃度に応じた使い分けがされていることが示唆され、本菌の様々な酸素濃度下での生育を支えていると考えられた。

酸化ストレス防御酵素としてはBCPに着目した。本菌BCPはperoxidaseとして働き、生体内では過酸化物解毒に寄与することが示された。アミノ酸置換体の解析から、本菌BCPが新規の反応機構を有することが示唆された。

また本菌の近縁でありながら酸素感受性を示す細菌との比較ゲノム解析を行った結果、本菌の酸化ストレス防御系の中核を成すことが予期される酵素遺伝子を見出した。この遺伝子の破壊株は好気的に生育することができず、この酵素が好気生育に必須であることが示された。この酵素を精製し解析したところ、過酸化物還元(peroxidase)活性を示した。またこの酵素は他のホモログには見られない基質への広い反応性を示した。すなわち本酵素は新規の酸化ストレス防御酵素とされ、Ferriperoxin (Fpx)と命名した。この酵素は、他の多くの微生物が持つ防御酵素の祖先型に当たることが示唆された。

本菌の好気生育を支えている酵素は酸化ストレス防御酵素のみではなく、他の酵素も独自に酸素耐性型に進化し、細胞全体で酸素が存在する環境に適応していったと考えられる。その詳細の理解と、各防御酵素を統率する転写制御因子を理解することが、本菌と酸素との関係をより明らかにするために、今後より重要であると考える。

以上本研究は、生物学上最も重要なテーマのひとつである生物と酸素との関係を、Hydrogenobacter thermophilusを研究対象として新規の酸化ストレス防御酵素を発見、本酵素は酸素存在下で生育するために必要不可欠であること、さらには新しいタイプの呼吸酵素を発見することにより、明らかにしてきたもので、審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。