Electron Transport in Archaeoglobus fulgidus

古细菌领域的成员在极端环境中茁壮成长，包括热液和厌氧生态位，这可能代表了地球上生命起源的条件。古藻属的成员是古菌中唯一的硫酸盐还原剂，也是唯一的超嗜热硫酸盐还原剂。硫酸盐还原剂是一种严格的厌氧菌，它使用硫酸盐作为异化硫酸盐还原的终端电子受体来产生质子动力。该项目采用生物化学和微生物遗传学相结合的方法来了解富氏古舌藻如何利用d -乳酸作为生长的唯一碳源和将硫酸盐还原为硫化氢的电子。a . fulgidus基因组序列包括一个单一基因dld，该基因被预测编码d -乳酸脱氢酶，这是一种膜相关酶，可将d -乳酸氧化为丙酮酸并将电子转移到厌氧呼吸链。dld基因是五个可能包含操纵子的黄颡鱼基因的中心基因，将通过绘制dld mRNA的起始位点直接进行测试。其上游（noxA2； NADH氧化酶）和下游（abc， abc转运蛋白）基因也可能编码d -乳酸分解代谢相关的产物。编码Dld和NoxA2的基因已在大肠杆菌中表达。Dld是从大肠杆菌中纯化出来的，具有耐热性和d -乳酸特异性脱氢酶活性。纯化后的NoxA2也是稳定的，需要NAD才能发挥活性。纯化后的Dld和NoxA2将进行生物化学表征，以确定可能参与电子转移的辅因子和金属。遗传技术将用于鉴定这些酶的关键氨基酸残基，这些氨基酸残基参与辅因子结合和氧化还原循环。为了确定Dld和NoxA2是否相互作用形成复合物，以及鉴定与Dld相互作用的其他蛋白，将使用亲和层析和酵母双杂交方法。a . fulgidus是第一个硫酸盐还原菌的完整基因组序列。从这个序列出发，生化和遗传方法将提供强大的工具来重建其不寻常的由d -乳酸引发的电子流呼吸途径。硫酸盐还原生物通过降解和解毒沉积物和水中的化合物在环境中发挥关键作用。用于消除这些化合物的细胞成分是在生长过程中为硫酸盐还原剂生产能量的相同蛋白质。与包括哺乳动物在内的生物在能量产生过程中使用O2作为电子的吸收源不同，硫酸盐还原剂使用SO4作为电子的吸收源。这个项目的目的是鉴定硫酸盐还原菌中参与能量产生的蛋白质，并确定这些蛋白质如何相互作用以允许电子流向硫酸盐。a . fulgidus是一种古老微生物群的成员，只能在非常高的温度下生长（83摄氏度，181华氏度）。该项目最初将侧重于两种蛋白质的活性和性质，乳酸脱氢酶和NADH氧化酶。这些蛋白质与其他单细胞生物以及包括人类在内的复杂生物产生能量的蛋白质有关。通过这项研究获得的信息将帮助科学家了解哪些蛋白质参与了能量产生的基本过程，以及这些蛋白质如何相互联系以控制这一普遍而复杂的过程。

HartzellMembers of the domain Archaea thrive in extreme environments, including hydrothermal and anaerobic niches, that may represent the conditions on the earth when life originated. Members of the genus Archaeoglobus are the only sulfate reducers in the Archaea and the only hyperthermophilic sulfate-reducers. Sulfate-reducers are strict anaerobes that use sulfate as the terminal electron acceptor of dissimilatory sulfate reduction to generate a proton motive force. This project uses a combination of biochemical and microbial genetic approaches to understand how Archaeoglobus fulgidus utilizes D-lactate as a sole source of carbon for growth and electrons for reduction of sulfate to hydrogen sulfide. The sequence of the A. fulgidus genome includes a single gene, dld, predicted to encode D-lactate dehydrogenase, the membrane-associated enzyme that oxidizes D-lactate to pyruvate and transfers electrons to the anaerobic respiratory chain. The dld gene is the central gene of five A. fulgidus genes that likely comprise an operon, which will be tested directly by mapping the start site of the dld mRNA. Its upstream (noxA2; NADH oxidase) and downstream (abc, ABC transporter) genes may also encode products involved in D-lactate catabolism. The genes encoding Dld and NoxA2 have been expressed in E. coli. Dld, purified from E. coli, is thermostable and has D-lactate -specific dehydrogenase activity. Purified NoxA2 also is stable and requires NAD for activity. Purified Dld and NoxA2 will be characterized biochemically to identify cofactors and metals that may be involved in electron transfer. Genetic techniques will be used to identify the critical amino acid residues of these enzymes that are involved in cofactor binding and oxidation-reduction cycles. To determine if Dld and NoxA2 interact to form a complex, and to identify other proteins that interact with Dld, affinity chromatography and the yeast two-hybrid approach will be used. A. fulgidus is the first sulfate reducer for which a complete genome sequence is available. Starting from this sequence, biochemical and genetic approaches will provide powerful tools to reconstitute its unusual respiratory pathway of electron flow that initiates with D-lactate.Sulfate-reducing organisms play critical roles in the environment by degrading and detoxifying compounds in sediments and waters. The cellular components used to eliminate these compounds are the same proteins that carry out energy production for the sulfate-reducer during growth. Unlike organisms, including mammals, which use O2 as a sink for electrons during energy production, sulfate-reducers use SO4 as a sink for electrons. This project is aimed at identifying the proteins that are involved in energy production in the sulfate-reducer, Archaeoglobus fulgidus and determining how these proteins interact with one another to permit the flow of electrons to sulfate. A. fulgidus, a member of an ancient group of microbes, grows only at very high temperatures (83 C, 181 F). The project will focus initially on the activity and properties of two proteins, lactate dehydrogenase and NADH oxidase. These proteins are related to proteins involved in energy production from other single-cells organisms as well as complex organisms, including humans. The information obtained through this research will help scientists understand which proteins are involved the essential process of energy production and how these proteins link to one another to control this universal, complex process.