Expression of the nifBfdxNnifOQ region of Azotobacter vinelandii and its role in nitrogenase activity (original) (raw)

1993, Journal of Bacteriology

The niJBQ transcriptional unit ofAzotobacter 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 13-galactosidase fusions to each of the five open reading frames: nifB, orf2 (fdxN), orJ3 (nifO), nifQ, and orf5. Expression of the first three open reading frames was observed under all three diazotrophic conditions; expression of orfs was never observed. Genes nifB andfdxN were expressed at similar levels. With Mo, expression of nifO and nifQ was-20and =400-fold lower than that offdxN, respectively. Without Mo, expression of nifB dropped threeto fourfold and that of nijQ dropped to the detection limit. However, expression of nifO increased threefold. The products of nijB, fdxN, nifO, and nifQ have been visualized in A. vinelandii as 13-galactosidase fusion proteins with the expected molecular masses. The NifBfusion lacked activity for any of the three nitrogenase systems and showed an iron-molybdenum cofactor-deficient phenotype in the presence of Mo. The FdxNmutation 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 (-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 orfS had no effect on nitrogenase activity.