GENETIC CONTROL OF NITRATE RESPIRATION IN E. COLI

适应厌氧是一种复杂的生理反应， 被兼性细菌如大肠杆菌感染。 的一个重要方面 这种适应是呼吸交替终端电子的能力 受体代替氧气。 硝酸根是重要的交替电子 受体，并合成呼吸酶，硝酸还原酶， 在缺氧的情况下由硝酸盐引起。 此外，功能 硝酸盐呼吸抑制其他兼性呼吸的形成 和发酵途径。 该项目的长期目标是 确定和表征控制这些活动的监管要素和网络， 硝酸还原酶和其他兼性呼吸酶的形成。 这项建议描述了一种分子遗传学方法， 控制硝酸盐呼吸。 它专注于nar（ch 1C）基因座， 含有硝酸盐的结构基因和调控基因 还原酶。 nar基因座将被详细检查，结合缺失 作图、互补分析、分子克隆和检测 基因-蛋白质关系 一个nar点，插入和 缺失突变将通过多种方法和筛选分离 程序. 这些突变将用于构建详细的 nar地区的精细结构遗传图。 选择的突变将是 进行基因互补测试， 映射到互补组中。 nar区域将被克隆， 通过限制性酶切图谱和Southern印迹分析进行分析。 这些研究 将允许遗传精细结构的相关性，互补性 和物理地图，并将用于精确定位基因的nar 地区 硝酸还原酶的结构基因将通过以下方法鉴定： 测试用于生产硝酸还原酶的各种突变体和亚克隆 多肽。 最后，操纵子融合将用于分析 调节硝酸还原酶和其它兼性呼吸基因， 并且还测试NAR调节基因中的特定突变的影响。

Adaptation to anaerobiosis is a complex physiological response demonstrated by facultative bacteria such as Escherichia coli. One important aspect of this adaptation is the ability to respire alternate terminal electron acceptors in place of oxygen. Nitrate is an important alternate electron acceptor, and synthesis of the respiratory enzyme, nitrate reductase, is induced by nitrate in the absence of oxygen. In addition, functioning nitrate respiration inhibits the formation of other facultative respiratory and fermentation pathways. The long-range goal of this project is to identify and characterize regulatory elements and networks that control the formation of nitrate reductase and other facultative respiratory enzymes. This proposal describes a molecular genetic approach toward understanding the control of nitrate respiration. It focuses on the nar (ch1C) locus, which contains both structural genes and regulatory genes for nitrate reductase. The nar locus will be examined in detail, combining deletion mapping, complementation analysis, molecular cloning and examination of gene-protein relationships. A collection of nar point, insertion and deletion mutations will be isolated by a variety of methods and screening procedures. These mutations will be used to construct a detailed fine-structure genetic map of the nar region. Selected mutations will be tested for genetic complementation, allowing division of the fine-structure map into complementation groups. The nar region will be cloned and analyzed by restriction mapping and Southern blot analysis. These studies will allow the correlation of the genetic fine-structure, complementation and physical maps, and will serve to precisely locate the genes of the nar region. The structural genes for nitrate reductase will be identified by testing various mutants and subclones for production of nitrate reductase polypeptides. Finally, operon fusions will be used to analyze the regulation of nitrate reductase and other facultative respiratory genes, and also to test the effects of specific mutations in nar regulatory genes.