ANAEROBIC EXPRESSION OF FUMARATE REDUCTASE IN E COLI

富马酸还原酶在大肠杆菌中的厌氧表达

• 批准号: 2413518
• 负责人: ROBERT P GUNSALUS
• 金额: $ 22.43万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2413518
• 关键词: DNA footprinting; Escherichia coli; anaerobic bacteria; bacterial genetics; bacterial proteins; dicarboxylate; electron transport; enzyme complex; enzyme induction /repression; gene expression; gene interaction; gene mutation; genetic manipulation; genetic promoter element; genetic regulation; genetic transcription; mutant; nitrate reductases; oxidoreductase; phosphorylation; polymerase chain reaction; protein purification; protein structure; respiratory enzyme; site directed mutagenesis

The aim of this research proposal is to elucidate the mechanisms responsible for regulation of the anaerobic respiratory pathway genes of includes the fumarate reductase (frdABCD), nitrate reductase (narGHJI) and DMSO/TMAO reductase (damsABC) genes which encode membrane associate enzyme complexes that participate in electron transport reaction during anaerobic conditions. These processes generate a chemosmotic potential for subsequent ATP generation via the proton translocating ATPase, and for proton driven solute uptake and for cell motility. The induction of respiratory gene expression in response to anaerobiosis is due primarily to the fnr gene product, Fnr. We recently succeeded in purifying Fnr in a biologically active form which binds DNA only under anaerobic conditions. We plan to improve the Fnr purification protocol, to characterize Fnr biochemically to understand how it detects the anaerobic state. We will also characterize the DNA binding properties required for Fnr activation gene expression. The control of the anaerobic respiratory genes in response to nitrate is provided by the narX/narQ/narL gene products that comprise a two-component regulatory system. NarX and NarQ are required to detect nitrate and they can independently transfer a signal via phosphorylation to NarL which binds DNA to both negatively and positively regulate gene expression. To better understand these processes at the mechanistic levels, mutations, will be introduced into narX and narQ and the resulting proteins will be analyzed using in vivo and in vitro approaches. The role of phosphorylation in NarL activation and in NarL-DNA binding will also be examined. The transcription of the narX, narQ, and narL genes will be characterized to determine how the levels of the products vary in the cell. Lastly, the basis for additional aerobic/anaerobic control of the frdABCD and narGHJI genes that is independent for fnr will be examined. We believe that the frdABCD, dmsABC, and narGHJI genes of E. coli offer an excellent model system to examine the processes for anaerobic control of bacterial gene expression. Little is known about these processes in other enteric bacteria that are human and animal pathogens. These processes are also common to a large number of soil microorganisms that can adapt and respire anaerobically when oxygen becomes limited in the environment.

这项研究计划的目的是阐明这种机制。 负责厌氧呼吸途径基因的调节 包括富马酸还原酶(FrdABCD)、硝酸还原酶(NarGHJI) 和编码膜相关蛋白的DMSO/TMAO还原酶(DamsABC)基因 参与电子传递反应的酶复合体 厌氧条件。这些过程产生一种化学渗透势 对于随后通过质子转移ATPase产生的ATP，以及 用于质子驱动的溶质吸收和细胞运动。的归纳 呼吸基因表达对厌氧菌的反应主要是由于 与FNR基因产物FNR结合。我们最近成功地提纯了FNR 一种生物活性形式，仅在厌氧条件下才与DNA结合 条件。我们计划改进FNR纯化方案，以 对FNR进行生化表征，以了解它是如何检测厌氧的 州政府。我们还将描述所需的DNA结合特性 FNR激活基因的表达。厌氧呼吸的控制 响应硝酸盐的基因是由narX/narQ/narL基因提供的 由两个组成部分组成的监管体系的产品。NarX和NarQ 被要求检测硝酸盐，他们可以独立地转移一个 通过磷酸化到NarL的信号，它将DNA与负性和 正向调节基因表达。为了更好地理解这些 机械层面的过程，突变，将被引入到 NarX和NarQ以及由此产生的蛋白质将在体内进行分析 和体外实验方法。磷酸化在NarL激活中的作用 在NarL中，还将检查DNA结合情况。《纽约时报》的转录 NarX、narQ和narL基因的特征将决定 这些产物的水平在细胞中各不相同。最后， 对frdABCD和narGHJI基因的额外需氧/厌氧控制 这是独立的，因为FNR将被审查。我们相信， 大肠杆菌的frdabcd、dmsabc和narghji基因提供了一个很好的模型 细菌基因厌氧控制过程的检测系统 表情。对其他肠道的这些过程知之甚少。 是人类和动物病原体的细菌。这些过程也是 对大量的土壤微生物来说是共同的，它们可以适应和 当环境中的氧气变得有限时，进行无氧呼吸。