Genetic Analysis of the nifM

生物固氮的遗传是非常复杂的，需要大约20个nif基因的协调表达。氮酶由两种不同的蛋白质组成，一种是由nifH基因编码的同型二聚体fe蛋白，另一种是由nifDK基因编码的异源四聚体mofe蛋白。除了这些结构基因（nifHDK）外，还鉴定了一些nif附属基因，其产物对氮酶的成熟和组装至关重要。其中之一是nifM基因，其产物是克雷伯氏肺炎菌和文氏偶氮杆菌生产功能性铁蛋白所必需的。对化学诱变、定点诱变和非对称nifM:PpiC蛋白产生的nifM突变体的初步遗传分析表明，nifM蛋白具有肽基脯氨酸顺/反式异构酶（PPIase）的功能。此外，对纯化的NifM蛋白的研究也表明该蛋白具有PPIase活性。基于这些观察结果以及ppiase与NifM c端区域的高度同源性，我们假设NifM的肽基脯氨酸顺/反式异构酶活性是该蛋白对铁蛋白成熟（活性和稳定性）发挥作用所必需的。本研究的目的是对nifM基因进行分子和遗传分析，以了解nifM在固氮中的功能。为了实现这一目标，将追求以下具体目标：1)表征以前通过化学诱变分离的肺炎克雷伯菌nifM点突变体，以了解nifM功能的分子基础；2)鉴定A. vinelandii NifM蛋白中对PPIase活性至关重要的区域；3)鉴定NifM蛋白中与fe蛋白相互作用所必需的区域；4)分离和鉴定不依赖nifm的Nif A. vinelandii突变体。这些实验旨在了解nifM基因的遗传组织如何促进其产物在铁蛋白成熟中的作用。突变分析的初步数据表明，NifM的n端区域和c端区域的突变都会影响NifM参与铁蛋白成熟的能力。这些研究将有助于评估NifM的c端区域和NifM的PPIase活性在NifM参与铁蛋白成熟的能力中的作用。此外，这些研究将有助于描述NifM的n端区域在决定NifM能力中的作用。此外，这里列出的研究项目是很好的教学工具，使用了现代分子微生物学的许多技术，并且易于掌握目标。项目负责人已经开始了一系列关于生物技术的讲座课程，在此资助期间的主要目标之一是开设分子生物学实验室课程，以开展主动学习，实验学习和指导活动，指导鲍灵格林州立大学的本科生。

Genetics of biological nitrogen fixation is very complex and requires a coordinated expression of about 20 nif genes. The enzyme nitrogenase is composed of two separate proteins designated the Fe-protein, a homodimer coded by the nifH gene and the MoFe-protein, a heterotetramer coded by the nifDK genes. Apart from these structural genes (nifHDK), a number of nif accessory genes whose products are essential for the maturation and assembly of nitrogenase are identified. One of them is the nifM gene whose product is necessary for the production of the functional Fe-protein in Klebsiellapneumoniae and in Azotobacter vinelandii. Initial genetic analyses of nifM mutants generated by chemical mutagenesis, site-directed mutagenesis, and of achimeric NifM:PpiC protein, indicate that the NifM protein functions as a peptidyl-prolyl cis/trans isomerase (PPIase). Moreover, studies on purified NifM protein also showed that this protein has PPIase activity. Based on these observations and also on the high homology shared by the PPIases and C-terminalregion of the NifM, it is hypothesized that the peptidyl-prolyl cis/trans isomerase activity of NifM is essential for this protein to exert its effect on the maturation (activity and stability) of the Fe-protein. The aim of this research is to carry out molecular and genetic analysis on the nifM gene in order to understandthe functions of the nifM in nitrogen fixation. To achieve this goal, the following specific objectives will be pursued: 1) to characterize the nifM point mutants of K. pneumoniae that were previously isolated by chemical mutagenesis in order to understand the molecular basis of NifM function; 2) to identify region(s) of the A. vinelandii NifM protein that are essential for PPIase activity; 3) to identify theregions of NifM protein that are essential for interaction with the Fe-protein; and 4) to isolate and characterize nifM-independent Nif A. vinelandii mutants. These experiments are directed toward understanding how the Genetic organization of the nifM gene contributes to the role of its product in the maturation of Fe-protein. Preliminary data from mutational analysis indicate that mutations in both the N-terminal region of NifM and the C-terminal region of NifM affect the ability of NifM to participate in the maturation of the Fe-protein. These studies will help to assess the role of the C-terminal region of NifM and the PPIase activity of the NifM in the NifM ability to participate in the maturation of the Fe-protein. Moreover, these studies will help to delineate the role of the N-terminal region of the NifM in determining the NifM ability. Besides, the research projects outlined here make excellent teaching tools, utilizing many techniques in modern molecular microbiology and having easily grasped objectives. The PIs have started a series of lecture courses that deal with biotechnology and one of major goals during this grant period is to start a molecular biology laboratory course to conduct active learning, experimental learning and mentoring activities with undergraduate students at Bowling Green State University.