Artificial activation of nif gene expression in nodule bacteria Ex Planta

Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters. Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state. Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation. Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.

The FixM Flavoprotein Modulates Inhibition by AICAR or 5′AMP of Respiratory and Nitrogen Fixation Gene Expression in Sinorhizobium meliloti

AICAR, a purine-related metabolite, was recently shown to inhibit respiratory and nifA gene expression in Sinorhizobium meliloti. Here, we demonstrate that AICAR has essentially no or little effect in a wild-type S. meliloti strain and inhibits respiratory and nitrogen fixation gene expression only in specific mutant backgrounds. We have analyzed in detail a mutant in which addition of AICAR inhibited fixK, fixN, fixT, and nifA expression. The corresponding gene, fixM, is located just downstream of fixK1 on pSymA megaplasmid and encodes a flavoprotein oxidore-ductase. 5′AMP, a structural analogue of AICAR, mimicked AICAR effect as well as the nucleoside precursors AICAriboside and adenosine. The mode of action of AICAR and 5′AMP in vivo was investigated. We demonstrate that AICAR does not affect FixK transcriptional activity and instead regulates fixK and nifA gene expression. We hypothesize that AICAR and 5′AMP may modulate, possibly indirectly, the activity of the FixLJ two-component regulatory system. The possible physiological roles of AICAR, 5′AMP, and fixM in the context of symbiosis are discussed.