Nitrogen fixation is a tightly regulated trait. Switching from N2-fixation repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high-energy demand it imposes. By using quantitative real time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, levels, and NifA-dependence greatly varied among the nif operons. Moreover, the exact concent... More
Nitrogen fixation is a tightly regulated trait. Switching from N2-fixation repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high-energy demand it imposes. By using quantitative real time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, 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. Nif protein concentrations were exquisitely balanced; with FeMo-cofactor biosynthetic proteins accumulating at levels 50-100 fold lower than structural proteins. Mutants lacking nitrogenase structural genes, or impaired in FeMo-cofactor biosynthesis, showed over enhanced responses to derepression proportionally to the degree of nitrogenase activity impairment that were consistent with the existence of at least two negative feedback regulatory mechanisms. The first of such mechanisms would respond to the levels of fixed nitrogen whereas the second mechanism appears to respond to the levels of matured NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in non-diazotrophs.
