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: 6169941
• 负责人: ROBERT P GUNSALUS
• 金额: $ 32.09万
• 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 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6169941
• 关键词: Escherichia coli; X ray crystallography; anaerobiosis; bacterial genetics; bacterial proteins; biological signal transduction; dicarboxylate; gene expression; gene mutation; genetic promoter element; molybdenum; nitrate reductases; nuclear magnetic resonance spectroscopy; oxidoreductase; phosphorylation; 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.

这项研究的目的是阐明机制 负责调节无氧呼吸途径基因， 包括富马酸还原酶（frdABCD）、硝酸还原酶（narGHJI） 和DMSO/TMAO还原酶（damsABC）基因，其编码膜缔合 参与电子传递反应的酶复合物， 厌氧条件 这些过程产生化学渗透势 用于随后通过质子易位ATP酶产生ATP，和 用于质子驱动的溶质摄取和用于细胞运动。 的诱导 呼吸基因表达对厌氧的反应主要是由于 fnr基因产物Fnr。 我们最近成功地纯化了Fnr， 一种生物活性形式，仅在厌氧条件下结合DNA 条件 我们计划改进Fnr纯化方案， 以生物化学方式表征Fnr，以了解它如何检测厌氧微生物， 状态 我们还将描述DNA结合所需的属性， Fnr激活基因表达。 无氧呼吸的控制 响应硝酸盐的基因由narX/narQ/narL基因提供 包括双组分调节系统的产品。 NarX和NarQ 需要检测硝酸盐，它们可以独立地将 通过磷酸化向NarL发出信号，NarL与DNA负结合， 积极调节基因表达。 为了更好地理解这些 在机械层面的过程，突变，将被引入到 narX和narQ以及所得蛋白质将使用体内 和体外方法。 磷酸化在NarL激活中的作用 和NarL-DNA结合也将被检查。的转录 narX、narQ和narL基因将被表征，以确定 产物的水平在细胞中变化。 最后，基础 frdABCD和narGHJI基因的额外需氧/厌氧控制 将审查对法国国家铁路公司来说独立的情况。 我们认为 frdABCD、dmsABC和narGHJI基因。coli提供了一个很好的模型， 一种检测细菌基因厌氧控制过程的系统 表情 在其他肠道中对这些过程知之甚少。 是人类和动物的病原体。 这些过程也 常见于大量的土壤微生物，可以适应， 当环境中的氧气变得有限时进行无氧呼吸。