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

产醋酸微生物的定义是它们以H2为电子供体生长的能力，通过Wood-Ljungdahl途径将CO2还原为乙酸，这可能是最古老的CO2固定途径。几十年来，它一直是一个谜，但最近解开了模型醋酸A.伍迪。已发现这种氧气具有由膜结合的Na+易位铁氧还蛋白、NAD氧化还原酶、Rnf复合物和钠离子依赖的ATP合成酶组成的呼吸链。然而，许多产醋细菌缺乏rnf基因。从基因组的角度来看，不含rnf的酵素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，膜积分，电子转移蛋白复合物。间接证据，主要是为古细菌收集的，以及它们与呼吸链复合体I的进化关系表明，Ech复合体是一种呼吸酶，通过离子在细胞质膜上的易位，将电子从一个供体（如还原铁氧还蛋白）转移到质子上（在氢的释放下）。因此，假设它们产生电化学离子梯度，然后被ATP合酶用来合成ATP。在无rnf、含Ech的醋酸菌中，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醋酸菌热厌氧菌kivui中得到解决。我们将采用双管齐下的方法，利用生理/生化研究和遗传分析相结合的方法来获得T. kivui能量代谢的完整图像。有趣的是，T. kivui基因组编码两个Ech复合物。我们将在严格的厌氧条件下使用不同的色谱步骤从T. kivui中纯化Ech复合物（es）。该酶复合物的亚基组成和生化性质将被确定。特别有趣的是钠离子对电子转移的潜在依赖性。最终目标是将酶纳入脂质体，以证明Ech是电子转移驱动的H+ / Na+泵，并表征Ech在这些蛋白脂质体中的离子易位。此外，我们将应用我们实验室最近建立的对T. kivui基因组进行遗传操作的方法，以产生编码Ech1， Ech2， Ech复合物亚基或同时编码Ech复合物的基因的染色体缺失。将在不同生长条件下研究缺失突变体的生理特性。随后，我们将研究倒膜囊泡中的能量守恒。这将回答有关Ech复合物在T. kivui生理和能量保存中的作用的问题。

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.