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

产乙酸微生物的定义是它们以H2作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，产乙酸菌如何保存能量一直是一个谜，但最近对模式产乙酸菌A的研究揭开了谜底。woodii。已发现该产乙酸菌具有由膜结合的Na+转运铁氧还蛋白：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。从基因组的角度来看，不含rnf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。间接的证据，主要是古细菌收集，连同它们的进化相关性的呼吸链的复合物I被视为指示Ech复合物是呼吸酶，耦合电子转移从供体，如还原铁氧还蛋白的质子（下释放氢）与离子的易位跨细胞质膜。因此，假设它们产生电化学离子梯度，然后由ATP合酶用于合成ATP。假设呼吸链中的rnf-免费的，ech-containing产乙酸菌组成的质子/钠离子转运，质子还原ech-hydrogenase将解决在嗜热产乙酸细菌Thermoanaerodikivui。我们将采用双管齐下的方法来获得T. kivui使用生理/生化研究和遗传分析相结合。有趣的是，T。kivui基因组编码两种Ech复合物。我们将开发一种从T. Kivui在严格厌氧条件下使用不同的色谱步骤。将确定酶复合物的亚基组成和生化性质。特别感兴趣的是电子转移的潜在钠离子依赖性。最终的目标是将酶的脂质体，证明Ech是一个电子转移驱动的H+ / Na+泵，并表征离子易位Ech在这些蛋白脂质体。此外，我们将应用遗传操作的方法对T。kivui基因组最近建立在我们的实验室，产生染色体缺失的基因编码Ech 1，Ech 2，亚基的两个Ech复合物或两个Ech复合物同时。将在不同的生长条件下研究缺失突变体的生理学。随后，将在倒置膜囊泡中研究能量守恒。这将回答关于Ech复合物在T. kivui。

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.