Anaerobic Expression of Fumarate Reductase in E. coli

描述(申请人提供)：本研究项目的主要目的是进一步阐明在大肠杆菌中依赖硝酸盐和钼酸盐的基因表达所需的调控机制。这种肠道细菌是一种模式致病和共生肠道微生物：它可以检测环境中的硝酸盐和其他各种呼吸底物，并通过合成不同的呼吸复合体来响应环境信号的变化。这一策略确保了能量的最佳生产，以支持细胞维护反应、生物合成和细胞生长。在本文提出的研究中，我们将使用该生物体中几个模型硝酸盐/亚硝酸盐控制的基因(例如富马酸还原酶(FrdABCD)，NARG和NAPF硝酸盐还原酶(narGHJI和NAPA)亚硝酸盐还原酶(NRF和Nirb)，以及DMSO/TMAO还原酶(DmsABC)基因来研究替代环境信号是如何被检测到的，以及所产生的信息是如何被用来调节基因表达的。由于这些过程对许多兼性动植物病原体来说是共同的，拟议的分子和生化研究应该提供关于不太了解的微生物如何管理与硝酸盐有关的厌氧过渡的见解。我们将研究依赖硝酸盐、亚硝酸盐和钼酸盐通过NAR和MODE调节电路控制基因表达的基础。我们将扩展我们的研究，进一步研究NarX、NarQ、Narp和NarL蛋白如何检测和转导信号来调节基因控制。我们将研究模式蛋白如何为钼酸盐依赖的控制含钼酸盐的酶提供微调厌氧呼吸途径基因的表达。NAR和模式基因家族将通过微阵列分析进行鉴定。最后，将使用连续培养的方法来检验硝酸盐控制的生理学。

DESCRIPTION (provided by applicant): The major aims of this research project are to further elucidate the regulatory mechanisms required for nitrate- and molybdate-dependent gene expression in Escherichia coli. This enteric bacterium is a model pathogenic and commensal enteric microorganism: it can detect nitrate and a variety of other respiratory substrates in the environment and respond accordingly by synthesizing different respiratory complexes in response to changing environmental signals. This strategy ensures optimal production of energy to support of cell maintenance reactions, biosynthesis, and cell growth. In the studies proposed herein, we will use several model nitrate/nitrite-controlled genes in this organism (e.g., fumarate reductase (frdABCD), NarG and NapF nitrate reductase (narGHJI, and napA) nitrite reductase (nrf and nirB), and DMSO/TMAO reductase (dmsABC) genes to examine how the alternative environmental signals are detected and how the resulting information is then utilized to modulate gene expression. Since these processes are common to many facultative animal and plant pathogens, the proposed molecular and biochemical studies should provide insight concerning how less well understood microbes manage the anaerobic transitions with respect to nitrate. We will examine the basis for nitrate, nitrite, and molybdate-dependent control of gene expression by the Nar and ModE regulatory circuits. We will extend our studies to further examine how the NarX, NarQ, NarP, and NarL proteins detect and transduce signals to modulate gene control. We will examine how the ModE protein provides for the molybdate-dependent control molybdate-containing enzymes to fine-tune expression of the anaerobic respiratory pathway genes. The Nar and Mode gene families will be identified by microarray analysis. Finally, the physiology of nitrate control will be examined by use of continuous culture methods.