Electron Transport in Archaeoglobus fulgidus

Hartzell古生物界的成员在极端环境中茁壮成长，包括热液和厌氧生态位，这些环境可能代表着生命起源时地球上的条件。古生菌属的成员是古生界中唯一的硫酸盐还原细菌，也是唯一的高温硫酸盐还原细菌。硫酸盐还原菌是一种严格的厌氧菌，它利用硫酸盐作为异化硫酸盐还原的末端电子受体来产生质子动力。该项目采用生化和微生物遗传学相结合的方法，了解黄褐古球藻如何利用D-乳酸作为生长的唯一碳源，并利用电子将硫酸盐还原为硫化氢。黄曲霉基因组的序列包括一个名为DLD的基因，该基因被预测为编码D-乳酸脱氢酶，D-乳酸脱氢酶是一种与膜相关的酶，可以将D-乳酸氧化成丙酮酸，并将电子传递到厌氧呼吸链。DLD基因是五个可能包含操纵子的黄曲霉基因的中心基因，将通过定位DLD mRNA的起始点来直接检测操纵子。其上游(noxA2；NADH氧化酶)和下游(ABC、ABC转运蛋白)基因也可能编码与D-乳酸分解代谢有关的产物。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的，具有热稳定性和D-乳酸专一性脱氢酶活性。纯化的NoxA2也很稳定，需要NAD才能发挥活性。纯化的DLD和NoxA2将进行生化表征，以确定可能参与电子转移的辅因子和金属。基因技术将被用来确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体，并鉴定与DLD相互作用的其他蛋白质，将使用亲和层析和酵母双杂交方法。黄曲霉是第一个可以获得完整基因组序列的硫酸盐还原剂。从这个序列开始，生化和遗传方法将提供强大的工具来重建其以D-乳酸开始的不寻常的电子流动的呼吸途径。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酶产生能量的相同蛋白质。与包括哺乳动物在内的生物体不同，在产生能量的过程中，哺乳动物将O2用作电子的接收器，而硫酸盐还原菌将SO4用作电子的接收器。该项目的目的是确定参与硫酸盐还原物质--黄褐古迹中的能量产生的蛋白质，并确定这些蛋白质如何相互作用以允许电子流向硫酸盐。黄曲霉是一个古老的微生物群体的成员，只在非常高的温度(83摄氏度，181华氏度)下生长。该项目最初将专注于乳酸脱氢酶和NADH氧化酶这两种蛋白质的活性和性质。这些蛋白质与其他单细胞生物体以及包括人类在内的复杂生物体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来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.