Energy conservation in the Ech-containing thermophilic acetogenic bacterium Thermoanaerobacter kivui

含 Ech 的嗜热产乙酸细菌 Thermoanaerobacter kivui 中的能量守恒

• 批准号: 394854436
• 负责人: Professor Dr. Mirko Basen
• 金额: --
• 依托单位: Lehrstuhl Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/394854436?language=en
• 关键词: Energy; conservation; Ech; containing; thermophilic

Acetogenic microorganisms are defined by their ability to grow with H2 as electron donor, reducing CO2 to acetic acid via the Wood-Ljungdahl pathway, the presumably oldest CO2 fixation pathway. It has been an enigma for decades how acetogens conserve energy, but was recently unravelled for the model acetogen A. woodii. This acetogen has been found to have a respiratory chain consisting of a membrane bound Na+ translocating ferredoxin:NAD oxidoreductase, the Rnf complex, and a sodium ion-dependent ATP synthase. However, many acetogenic bacteria are devoid of rnf genes. A genomic perspective revealed that rnf-free acetogens have genes encoding energy-converting hydrogenases (ech) instead (some may have both). The latter encode multi-subunit, membrane-integral, electron transfer protein complexes. Indirect evidence, mostly gathered for archaea, together with their evolutionary relatedness to complex I of the respiratory chain are taken as indication that Ech complexes are respiratory enzymes that couple electron transfer from a donor such as reduced ferredoxin to protons (under liberation of hydrogen) with the translocation of ions across the cytoplasmic membrane. Thus, it is assumed that they create an electrochemical ion gradient that is then used by an ATP synthase to synthesize ATP. The hypothesis that the respiratory chain in rnf-free, ech-containing acetogens consists of a proton/sodium ion-translocating, proton-reducing Ech hydrogenase will be addressed in the thermophilic acetogenic bacterium Thermoanaerobacter kivui. We will use a two-pronged approach to obtain a complete picture of the energy metabolism of T. kivui using a combination of physiological/biochemical studies and genetic analyses. Interestingly, the T. kivui genome encodes for two Ech complexes. We will develop a protocol for the purification of Ech complex(es) from T. kivui using different chromatographic steps under strictly anaerobic conditions. The subunit composition and biochemical properties of the enzyme complex will be determined. Of special interest is a potential sodium ion dependence of the electron transfer. The final goal is to incorporate the enzyme in liposomes, to proof that Ech is an electron transfer-driven H+ / Na+ pump and to characterize ion translocation by Ech in these proteoliposomes. Moreover, we will apply methods for genetic manipulations on the T. kivui genome recently established in our lab to generate chromosomal deletions of genes encoding Ech1, Ech2, subunits of both Ech complexes or both Ech complexes simultaneously. The physiology of the deletion mutants will be studied under different growth conditions. Subsequently, energy conservation will be studied in inverted membrane vesicles. This will answer the question about the role of Ech complexes in physiology and energy conservation in T. kivui.

产乙酸微生物的定义是它们能够以 H2 作为电子供体生长，通过 Wood-Ljungdahl 途径（可能是最古老的 CO2 固定途径）将 CO2 还原为乙酸。几十年来，产乙酸菌如何节省能量一直是个谜，但最近，产乙酸菌 A. woodii 模型被解开了。已发现这种产乙酸菌具有由膜结合的 Na+ 易位铁氧还蛋白：NAD 氧化还原酶、Rnf 复合物和钠离子依赖性 ATP 合酶组成的呼吸链。然而，许多产乙酸细菌缺乏 rnf 基因。基因组视角显示，不含核因子的产乙酸菌具有编码能量转换氢化酶（ech）的基因（有些可能两者都有）。后者编码多亚基、膜整合、电子传递蛋白复合物。主要为古细菌收集的间接证据，连同它们与呼吸链复合物 I 的进化相关性，被认为表明 Ech 复合物是一种呼吸酶，它将电子从供体（如还原铁氧还蛋白）转移到质子（在释放氢的情况下）与离子穿过细胞质膜的易位结合在一起。因此，假设它们产生电化学离子梯度，然后由 ATP 合成酶用来合成 ATP。无 rnf、含 ech 的产乙酸菌中的呼吸链由质子/钠离子易位、质子还原的 Ech 氢化酶组成的假设将在嗜热产乙酸细菌基伍热厌氧杆菌中得到解决。我们将采用双管齐下的方法，结合生理/生化研究和遗传分析，全面了解 T. kivui 的能量代谢。有趣的是，T. kivui 基因组编码两个 Ech 复合物。我们将开发一种在严格厌氧条件下使用不同色谱步骤从 T. kivui 中纯化 Ech 复合物的方案。将确定酶复合物的亚基组成和生化特性。特别令人感兴趣的是电子转移的潜在钠离子依赖性。最终目标是将酶合并到脂质体中，以证明 Ech 是电子转移驱动的 H+/Na+ 泵，并表征这些蛋白脂质体中 Ech 的离子易位。此外，我们将应用我们实验室最近建立的 T. kivui 基因组的遗传操作方法，以产生编码 Ech1、Ech2、两个 Ech 复合物的亚基或同时编码两个 Ech 复合物的基因的染色体缺失。将在不同的生长条件下研究缺失突变体的生理学。随后，将研究倒置膜囊泡中的能量守恒。这将回答有关 Ech 复合物在 T. kivui 生理学和能量守恒中的作用的问题。