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The Mo-nitrogenase is responsible for most biological nitrogen fixation activity in the biosphere and due to its great agronomical importance it has been the subject of thorough genetic and biochemical studies. With the goal of providing a framework to engineer N2 fixation in non-diazotrophs, CBGP researchers have investigated the time course of expression of nitrogenase components in cells of Azotobacter vinelandii initiating diazotrophic growth by using quantitative real time PCR and quantitative immunoblotting. Expression timing, levels, and NifA-dependence greatly varied among the nif operons. Moreover, the exact concentrations of Nif proteins, and their changes over time, were determined for the first time showing that FeMo-cofactor biosynthetic proteins accumulated at levels 50-100 fold lower than structural proteins. At least two negative feedback regulatory mechanisms appeared to control nif expression.
This work has been selected by F1000 faculty member Prof. Ray Dixon as very important to the field. In his commentary, Professor Dixon states that: “In this paper, the authors have carried out detailed kinetic analysis of nif mRNA and protein levels during nif derepression in Azotobacter vinelandii. They demonstrate that synthesis of the enzyme is exquisitely sensitive to negative feedback regulation and that protein stoichiometry is delicately balanced. These findings have important implications for engineering nitrogen fixation in non-diazotrophic hosts such as crop plants”.