Title

Genetic Analysis of NifM Interaction with the Fe Protein of Nitrogenase

Date of Award

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Nara Gavini

Abstract

NifM is a peptidyl prolyl cis-trans isomerase and is required for the maturation and activation of the Fe protein of nitrogenase. However, the exact role of NifM in the maturation of Fe protein is unclear. This research project was directed to study the specificity of NifM for the Fe protein. It was first examined whether the Pin1 protein could genetically complement the function of NifM in A.vinelandii BG98, a nifM mutant strain containing a kanamycin resistance cassette inserted in the nifM gene. Since the Pin1 protein could not complement the function of NifM in A. vinelandii BG98, we constructed a chimera of NifM-Pin1 fusion expressing the amino terminus of NifM and the Pin1 protein. This chimeric protein complemented the function of NifM in A.vinelandii BG98 that led to the hypothesis that the amino terminus may be required for its interaction with the NifH. To analyze the role of the amino terminus of NifM the BacterioMatch Two Hybrid System was utilized and the results revealed that the amino terminus of NifM is responsible for its interaction with NifH. Earlier it was determined that mutants of NifM at positions 99 and 100 were deficient in nitrogen fixation and therefore we tested the interaction of this protein with NifH. This mutant NifM could not interact with NifH showing the importance of these amino acids in the interaction between NifM and NifH. Moreover the specific region of NifH involved in the interaction was also determined. A nifM like gene that has sequence similarity to nifM from A. vinelandii was identified from Pseudomonas aeruginosa PAO1, cloned and expressed in A. vinelandii BG98 and this gene was able to complement the function of NifM in A. vinelandii BG98, it also interacted with NifH. The amino acid comparison also revealed that the docking region of NifH is conserved in NarH of Pseudomonas aeruginosa PAO1. Experiments with the Bacteriomatch Two Hybrid system showed that both NifM from A.vinelandii and PA3871 interact with the NarH. Finally, one of mutant nifH gene generated by DNA shuffling was analyzed for genetic complementation in A. vinelandii BG98, and as a result a mutant capable of functioning in the absence of NifM was obtained. This mutant had mutations consisting of four amino acid replacements, replacing aspargine at position 107 with serine, proline at position 232 with alanine, glutamic acid at position 266 with valine and at isoleucine position 273 with valine. Analysis of this mutant provided further insight into the folding of the Fe protein. The amino terminus of the NifM is an interaction domain and the carboxyl terminal is the peptidyl prolyl cis trans isomerase domain and both the domains are required for the maturation of the Fe protein. The parvulin from other organisms can complement the function of NifM if they contain both the interaction domain and the catalytic domain. Various mutations in the NifH render it NifM independent, which suggests that NifM is involved in the proper folding of the Fe protein.