Regulation of Expression of the Nitrogen Fixation (nif) Genes of K. Pneumoniae

将研究调节表达的机制 肠杆菌K. 肺炎。 重点将放在体外研究， nifA基因的蛋白质产物（命名为NIFA）， 需要激活转录的NIF操纵子。 目标 将确定NIFA激活的机制 转录。 测定粗品中NIFA的活性 制剂将根据其刺激 从nifHDK启动子（用于表达的启动子）表达 编码固氮酶和固氮酶还原酶的结构操纵子） 在体外转录-翻译耦合系统中。 这 将使用测定来纯化NIFA蛋白。 NIFA活性 将在转录系统中被表征为DNA- 结合蛋白 基因证据表明NIFA是 以活性形式合成，即刺激 表达的NIF操纵子，但它可以被灭活 基因nifL（NIFL）的产物响应于以下物质的存在： 固定氮或氧。一旦被净化， 证明体外NIFL对NIFA的灭活作用， 确定这种失活的依据。 氨是氮的首选来源， 微生物的 当生长在缺乏氨或其他 优选来源，细菌如K. 肺炎发展 获得利用大气氮的能力（氮 固定）。 催化这一过程的酶由 一组被称为NIF系统的基因。 该项目将 研究nif基因被激活的机制。 重点将放在表征的蛋白质产品， nif簇内的基因（nifA），其表达是 对其他NIF基因的激活至关重要。 本研究将 揭示了关键的调控事件， 将大气中的氮转化为生物有用的形式。

A study will be made of the mechanisms that regulate expression of the nitrogen fixation (nif) genes of the enteric bacterium K. pneumoniae. Emphasis will be place on an in vitro study of the protein product of the nifA gene (designated NIFA), which is required to activate transcription of the nif operons. The goal will be to determine the mechanism by which NIFA activates transcription. An assay for the activity of NIFA in crude preparations will be developed based on its ability to stimulate expression from the nifHDK promoter (the promoter for the structural operon encoding nitrogenase and nitrogenase reductase) in a coupled in vitro transcription-translation system. This assay will be used to purify the NIFA protein. NIFA activity will be characterized in a transcription system and as a DNA- binding protein. Genetic evidence suggests that NIFA is synthesized in an active form-that is, a form which stimulates expression of the nif operons-but that it can be inactivated by the product of gene nifL (NIFL) in response to the presence of fixed nitrogen or oxygen. Once purified, an attempt will be made to demonstrate inactivation of NIFA by NIFL in vitro and to determine the basis for this inactivation. Ammonia is the preferred source of nitrogen for most microorganisms. When grown in the absence of ammonia or other preferred sources, bacteria like K. pneumoniae develop the ability to obtain utilize atmospheric nitrogen (nitrogen fixation). The enzymes that catalyze this process are encoded by a cluster of genes known as the nif system. This project will examine the mechanism by which the nif genes are activated. Emphasis will be placed on characterizing the protein product of a gene within the nif cluster (nifA) whose expression is essential for activation of the other nif genes. This study will shed light on the critical regulatory events that allow bacteria to convert atmospheric nitrogen into biologically useful forms.