Genetic Studies of Glutamine Synthetase in Bacteria

描述（由申请人提供）：我们对微生物氮代谢及其调控有长期的兴趣，并专注于肠道细菌中的转录激活子NtrC（氮调节蛋白C）。NtrC控制下两个最高表达的操纵子的产物，glnK amtB和rut操纵子，是本提案的重点。AmtB（铵转运蛋白B）蛋白和Amt蛋白通常表现为NH3的生物气体通道，而不是像以前认为的那样是NH4+离子的活性转运蛋白。AmtB蛋白是肠道细菌在外部NH3浓度受限（>或等于50 nM）时快速生长所必需的，并与高亲和的NH3同化酶谷氨酰胺合成酶（GS）功能偶联。该项目的一个长期目标是了解大肠杆菌AmtB蛋白如何改善NH3在其细胞质膜上的无介导扩散。我们将使用遗传和生化方法来验证AmtB和GS必须在物理接触中才能发挥作用的假设，即使它们的结合必须是不稳定的。我们将首先分离破坏AmtB和gs之间联系的突变（分别称为AmtB *和glnA*），然后分离通过以补偿方式改变伴侣蛋白来恢复联系的抑制突变。我们将开始对光合变形细菌红螺旋菌的两个Amt蛋白进行研究，以确定一个是否与GS偶联，另一个是否与生物合成谷氨酸脱氢酶偶联，这是另一个主要的NH3同化酶。在更广泛的背景下，Amt蛋白（在某些生物体中称为Mep）广泛存在于微生物，维管植物和无脊椎动物中，是第一个被描述的生物气体通道。它们是唯一已知的“铵”（用于指定NH3 + NH4+）的特定转运体，这是许多微生物首选的氮源。Amt/Mep蛋白与其他蛋白的结合与气体通道如何改善气体通过膜的无介导扩散以及不稳定的超分子结构在细胞生理学中的作用有关。我们的第二个长期目标是继续研究b1012操纵子。这7个基因的操纵子编码先前未描述的嘧啶环降解途径，我们将其命名为rut（嘧啶利用）操纵子。与之相邻的基因b1013编码一种车辙转录的负辅助调节因子，现在称为RutR。我们将使用多种遗传和生化方法来鉴定Rut途径的中间体和RutA-RutF蛋白的酶活性。我们将在体外研究RutR的DNA结合和转录抑制活性。新的生化途径是罕见的，它们的表征对基因组的注释和对细胞自我复制重要的代谢和调控联锁的研究是有用的。

DESCRIPTION (provided by applicant): We have a longstanding interest in microbial nitrogen metabolism and its regulation and have focused on the transcriptional activator NtrC (nitrogen regulatory protein C) in enteric bacteria. Products of two of the most highly expressed operons under NtrC control, the glnK amtB and rut operons, are the focus of this proposal. The AmtB (ammonium transporter B) protein and Amt proteins generally appear to be biological gas channels for NH3 rather than active transporters for the ion NH4+, as was previously believed. The AmtB protein is essential for rapid growth of enteric bacteria when the external concentration of NH3 is limiting (> or equal to 50 nM) and is functionally coupled to the high-affinity NH3 assimilatory enzyme glutamine synthetase (GS). One long-term goal of this project is to understand how the E. coli AmtB protein can improve on unmediated diffusion of NH3 across its cytoplasmic membrane. We will use genetic and biochemical approaches to test the hypothesis that AmtB and GS must be in physical contact for AmtB to function, even though their binding must be labile. We will first isolate mutations that disrupt contact between AmtB and GS-called amtB* and glnA*, respectively-and then isolate suppressor mutations that restore contact by altering the partner protein in a compensatory manner. We will initiate studies of the two Amt proteins of the photosynthetic proteobacterium Rhodospirillum rubrum to determine whether one is coupled to GS and the second to biosynthetic glutamate dehydrogenase, the other major NH3 assimilatory enzyme. In a broader context, Amt proteins (called Mep in some organisms) are found widely in microbes, vascular plants, and invertebrate animals and are the first biological gas channels to be described. They are the only specific transporters known for "ammonium" (used to designate NH3 + NH4+), which is a preferred nitrogen source for many microbes. The association of Amt/Mep proteins with other proteins bears on how gas channels can improve on unmediated diffusion of gases through membranes and on the role of labile supramolecular structures in cell physiology. Our second long-term goal is to continue studies of the b1012 operon. This operon of 7 genes encodes a previously undescribed pathway for degradation of pyrimidine rings, which we have designated the rut (pyrimidine utilization) operon. The adjacent gene, b1013, encodes a negative auxiliary regulator of rut transcription, now called RutR. We will use a variety of genetic and biochemical approaches to identify the intermediates of the Rut pathway and the enzymatic activities of the RutA-RutF proteins. We will study the DNA binding and transcriptional repression activities of RutR in vitro. New biochemical pathways are rare and their characterization is useful in the annotation of genomes and in studies of the metabolic and regulatory interlocks important to cellular self-replication.