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 a chimeric 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-terminal region 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 understand the 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 the regions 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.
