Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii.

Abstract

The nifF gene encoding flavodoxin from Azotobacter vinelandii OP was cloned and its DNA sequence determined. It is located adjacent to, or possibly within, the major nif cluster and it is preceded by nif-specific regulatory elements. Southern hybridization analysis revealed that there is only a single copy of the nifF gene on the A. vinelandii OP genome. Mutant strains were constructed which have an insertion mutation or an insertion and a deletion mutation within the nifF gene coding sequence. These mutant strains are capable of diazotrophic growth, indicating that flavodoxin is not the unique physiological electron donor to nitrogenase. The results of nifF-lacZYA gene fusion experiments and Northern hybridization analyses indicated that the nifF gene is both transcribed and translated under nitrogen fixing and non-nitrogen fixing conditions. However, under nitrogen fixing conditions a substantial increase in both nifF synthesis and in accumulation of an approximately 800-base pair nifF-encoding mRNA species was observed. Furthermore, strains mutated within the nifF gene have only 70% of the wild type in vivo nitrogenase activity as determined by whole cell acetylene reduction assays. These data demonstrate that the nifF-encoded flavodoxin of A. vinelandii OP, although not essential for nitrogen fixation, is required for maximum in vivo nitrogenase activity.