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

研究肠道肺炎克雷伯菌固氮（nif）基因表达的调控机制。重点将放在nifA基因（指定nifA）的蛋白质产物的体外研究上，这是激活nif操纵子转录所必需的。目标是确定NIFA激活转录的机制。根据NIFA在体外转录-翻译耦合系统中刺激nifHDK启动子（编码氮酶和氮酶还原酶的结构操纵子的启动子）表达的能力，将对粗制剂中NIFA的活性进行测定。该实验将用于纯化NIFA蛋白。NIFA的活性将在转录系统和DNA结合蛋白中被表征。遗传证据表明，NIFA是以一种活性形式合成的，即一种刺激nif操纵子表达的形式，但它可以被基因nifL的产物（nifL）在固定氮或氧的存在下灭活。一旦纯化，将尝试在体外证明NIFL对NIFA的失活作用，并确定这种失活的基础。氨是大多数微生物的首选氮源。当在没有氨或其他首选来源的情况下生长时，像肺炎克雷伯菌这样的细菌会发展出获取利用大气氮（固氮）的能力。催化这个过程的酶是由一组被称为nif系统的基因编码的。本项目将研究nif基因被激活的机制。重点将放在表征nif簇（nifA）内基因的蛋白质产物，其表达对其他nif基因的激活至关重要。这项研究将阐明允许细菌将大气中的氮转化为生物有用形式的关键调控事件。

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.