Expression of the nifBfdxNnifOQ region of Azotobacter vinelandii and its role in nitrogenase activity

Abstract

The nifBQ transcriptional unit of Azotobacter vinelandii has been previously shown to be required for activity of the three nitrogenase systems, Mo nitrogenase, V nitrogenase, and Fe nitrogenase, present in this organism. We studied regulation of expression and the role of the nifBQ region by means of translational beta-galactosidase fusions to each of the five open reading frames: nifB, orf2 (fdxN), orf3 (nifO), nifQ, and orf5. Expression of the first three open reading frames was observed under all three diazotrophic conditions; expression of orf5 was never observed. Genes nifB and fdxN were expressed at similar levels. With Mo, expression of nifO and nifQ was approximately 20- and approximately 400-fold lower than that of fdxN, respectively. Without Mo, expression of nifB dropped three- to fourfold and that of nifQ dropped to the detection limit. However, expression of nifO increased threefold. The products of nifB, fdxN, nifO, and nifQ have been visualized in A. vinelandii as beta-galactosidase fusion proteins with the expected molecular masses. The NifB- fusion lacked activity for any of the three nitrogenase systems and showed an iron-molybdenum cofactor-deficient phenotype in the presence of Mo. The FdxN- mutation resulted in reduced nitrogenase activities, especially when V was present. Dinitrogenase activity in extracts was similarly affected, suggesting a role of FdxN in iron-molybdenum cofactor synthesis. The NifO(-)-producing mutation did not affect any of the nitrogenases under standard diazotrophic conditions. The NifQ(-)-producing mutation resulted in an increased (approximately 1,000-fold) Mo requirement for Mo nitrogenase activity, a phenotype already observed with Klebsiella pneumoniae. No effect of the NifQ(-)-producing mutation on V or Fe nitrogenase was found; this is consistent with its very low expression under those conditions. Mutations in orf5 had no effect on nitrogenase activity.