Genetic Studies of Glutamine Synthetase in Bacteria

描述(申请人提供)：我们对微生物的氮代谢及其调控一直很感兴趣，并专注于肠道细菌中的转录激活因子NTRC(氮调节蛋白C)。在NTRC控制下表达最高的两个操纵子，glnK amtB和rut操纵子的产物，是这项提案的重点。AmtB(铵转运体B)蛋白和AMT蛋白通常似乎是NH3的生物气体通道，而不是像以前认为的那样是NH4+的活性转运体。AmtB蛋白是肠道细菌快速生长所必需的，当外界NH3浓度有限时(gt；或等于50 nM)，AmtB蛋白在功能上与高亲和力NH3同化酶谷氨酰胺合成酶(GS)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在其细胞质膜上的非介导性扩散。我们将使用遗传和生化方法来验证这样的假设，即AmtB和GS必须处于物理接触才能发挥作用，尽管它们的结合必须是不稳定的。我们将首先分离中断AmtB和GS之间接触的突变-分别称为amtB*和glnA*-然后分离通过以补偿方式改变配对蛋白来恢复联系的抑制突变。我们将启动对光合作用红色蛋白杆菌的两个AMT蛋白的研究，以确定一个是否与GS偶联，另一个与生物合成的谷氨酸脱氢酶偶联，谷氨酸脱氢酶是另一种主要的NH3同化酶。在更广泛的背景下，AMT蛋白(在一些生物体中称为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。它们是已知的唯一特定的“铵”转运体(用于指定NH3+NH4+)，这是许多微生物首选的氮源。AMT/MEP蛋白与其他蛋白质的结合关系到气体通道如何改善气体通过膜的非介导性扩散，以及不稳定的超分子结构在细胞生理中的作用。我们的第二个长期目标是继续研究B1012操纵子。这个由7个基因组成的操纵子编码了一条先前未被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。邻近的基因b1013编码一个负的RUT转录辅助调节因子，现在被称为RutR。我们将使用各种遗传和生化方法来鉴定RUT途径的中间产物和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。新的生化途径很少见，它们的特征在基因组的注释和对细胞自我复制重要的代谢和调节联锁的研究中是有用的。

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.