Nif- phenotype of Azotobacter vinelandii UW97. Characterization and mutational analysis

We have identified the molecular basis for the nitrogenase negative phenotype exhibited by Azotobacter vinelandii UW97. This strain was initially isolated following nitrosoguanidine mutagenesis. Recently, it was shown that this strain lacks the Fe protein activity, which results in the synthesis of a FeMo cofactor-deficient apodinitrogenase. Activation of this apodinitrogenase requires the addition of both MgATP and wild-type Fe protein to the crude extracts made by A. vinelandii UW97 (Allen, R.M., Homer, M.J., Chatterjee R., Ludden, P.W., Roberts, G.P., and Shah, V.K. (1993) J. Biol. Chem. 268 23670-23674). Earlier, we proposed the sequence of events in the MoFe protein assembly based on the biochemical and spectroscopic analysis of the purified apodinitrogenase from A. vinelandii DJ54 (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1994) Biochemistry 33, 11842-11849). Taken together, these results imply that the assembly process of apodinitrogenase is arrested at the same step in both of these strains. Since A. vinelandii DJ54 is a delta nifH strain, this strain is not useful in identifying the features of the Fe protein involved in the MoFe protein assembly. Here, we report a systematic analysis of an A. vinelandii UW97 mutant and show that, unlike A. vinelandii DJ54, the nifH gene of A. vinelandii UW97 has no deletion in either coding sequence or the surrounding sequences. The specific mutation responsible for the Nif- phenotype of A. vinelandii UW97 is the substitution of a non-conserved serine at position 44 of the Fe protein by a phenylalanine as shown by DNA sequencing. Furthermore, oligonucleotide site-directed mutagenesis was employed to confirm that the Nif- phenotype in A. vinelandii UW97 is exclusively due to the substitution of the Fe protein residue serine 44 by phenylalanine. By contrast, replacing Ser-44 with alanine did not affect the Nif phenotype of A. vinelandii. Therefore, it seems that the Nif- phenotype of A. vinelandii UW97 is caused by a general structural disturbance of the Fe protein due to the presence of the bulky phenylalanine at position 44.