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: 3131921
• 负责人: ROBERT P GUNSALUS
• 金额: $ 21.71万
• 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 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3131921
• 关键词: DNA footprinting; Escherichia coli; anaerobic bacteria; bacterial genetics; electron transport; enzyme complex; enzyme induction /repression; gene expression; genetic manipulation; genetic promoter element; genetic transcription; laboratory rabbit; microorganism growth; mutant; oxidoreductase; respiratory enzyme

The aim of this research project is to understand the general mechanisms responsible for controlling anaerobic gene expression in the enteric bacterium, Escherichia coli. We are using the fumarate reductase (frdABCD) genes of this organism as a model to examine how the presence of alternative cellular electron acceptors are able to affect gene expression. The frdABCD genes encode the fumarate reductase enzyme complex that functions in the final step of anaerobic respiration to convert fumarate, as the terminal electron acceptor, to succinate. It is membrane bound and contains both flavin and non-heme iron centers. Expression of theses genes occurs optimally under anaerobic growth conditions in the presence of fumarate and in the absence of nitrate. As yet, little is known about the mechanisms responsible for this phenomenon. We wish to elucidate the molecular and biochemical basis of the anaerobic induction and the nitrate repression processes. We will study the role of the fnr, narR, and narL gene products in these processes and determine the mechanisms by which they act. An analysis of cis-acting sites for oxygen/redox state control of frdABCD gene expression will be performed to identify their locations(s) in the frd regulatory region. Chemical and enzymatic methods will be used to establish the domains for the FNR binding. The FNR protein will also be characterized to understand its role in sensing the anaerobic state. Mutagenesis of the fnr gene will be done to identify essential regions in FNR for activating transcription during anaerobiosis. An analysis of cis-acting sites for nitrate control of frdABCD gene expression will also be performed to understand where the narR and narL gene productions act to control also be performed to understand where the narR and narL gene products act to control frdABCD expression. The narR and NarL genes will be characterized with respect to sequence, transcription, and function via mutational analysis to obtain mutations that are altered for nitrate sensing and signaling. The gene products will be purified and characterized to understand the interactions necessary for the repression process. We believe that fumarate reductase is an excellent model system to examine the induction and regulation of genes whose products are required under anaerobic growth conditions. Little is known about how these genes are induced or repressed in response to oxygen and other electron acceptors. This oxygen phenomenon is involved in regulation of the electron transport process, photosynthesis and nitrogen fixation in a variety of bacterial species.

这项研究项目的目的是了解其一般机制。 负责控制肠道中厌氧基因的表达 细菌，大肠埃希菌。我们用的是富马酸还原酶 (FrdABCD)基因作为模型来考察这种有机体的存在 可替代的细胞电子受体能够影响基因 表情。FrdABCD基因编码富马酸还原酶 在无氧呼吸的最后一步起作用的复合体 将作为末端电子受体的富马酸转化为琥珀酸。它是 膜结合并含有黄素和非血红素铁中心。 这些基因在厌氧生长条件下的最佳表达 在富马酸盐存在和不存在硝酸盐的情况下。AS 然而，人们对导致这一现象的机制知之甚少。 现象。我们希望阐明其分子和生化基础。 厌氧诱导和硝酸盐抑制过程。我们会 研究FNR、narR和narL基因产物在这些疾病中的作用 过程，并确定它们的行动机制。 FrdABCD氧/氧化还原态调控的顺式作用位分析 将进行基因表达以确定它们在体内的位置(S) FRD管理区域。将使用化学和酶方法来 建立FNR绑定的域。FNR蛋白也将被 以理解其在感知厌氧状态中的作用为特征的。 将对FNR基因进行突变，以确定 在厌氧过程中激活转录的FNR。 FrdABCD基因硝酸盐调控的顺式作用位点分析 还将执行表达式以了解narr和narL的位置 基因生产作为控制也被执行，以理解在哪里 NarR和narL基因产物调控frdABCD的表达。《柳叶刀》 而NarL基因将根据序列进行表征， 转录，并通过突变分析获得突变的功能 为了硝酸盐感应和信号而改变的。基因产品 将被提纯和表征以理解相互作用 对于镇压过程来说是必要的。 我们认为富马酸还原酶是一个很好的模型系统 检查需要其产物的基因的诱导和调控 在厌氧生长条件下。关于这些基因是如何 对氧和其他电子的反应被诱导或抑制 接受者。这种氧现象参与了对 黄曲霉的电子传递过程、光合作用和固氮作用 细菌种类繁多。