Studies on the structure and function of various nif and nif- associated gene products encoded within the Azotobacter vinelandii nif gene cluster

The present study investigates the structural and functional roles of the metalloclusters present within the MoFe protein of nitrogenase from Azotobacter vinelandii. A gene replacement strategy was developed for oligonucleotide-directed mutagenesis of these proteins and the resulting biological and biochemical effects of these changes were examined. Identification of structurally important regions in the MoFe protein subunits and assignment of specific amino acid residues as potential metal cluster ligands were based upon several criteria: i. metallocluster extrusion requirements; spectroscopic properties of the MoFe protein; interspecies and intersubunit comparisons; iv. comparison of the MoFe protein subunit sequences to iron-molybdenum cofactor biosynthetic gene products. This mutagenesis strategy has permitted the construction of thirty-three mutant strains having specific amino acid substitutions within the MoFe protein subunits. Based on the diazotrophic growth characteristics and substrate reduction capabilities of these mutant strains, a model is presented in which potential metallocluster binding sites within the MoFe protein subunits are defined. In addition to analysis of the MoFe protein subunits, this site-directed mutagenesis and gene replacement strategy can be used to place specific mutations into any gene product encoded within the A. vinelandii nif gene cluster.

Finally, nucleotide sequence analysis of the regions flanking the nifEN genes revealed the presence of three nif genes (nifT, nifY, and nifX) and four open reading frames (ORF1, ORF2, ORF3, and ORF4). Two of these genes, nifX and ORF3, were shown to be under nif control and synthesis of their products elevated in response to a demand for fixed nitrogen. Mutant strains with deletions in ORF3 appeared to accumulate an excess amount of MoFe protein when compared to wild type. The ORF3 gene product has been overproduced in E. coli. This provides an important step toward characterizing the protein and elucidating the molecular basis for its control of nifDK gene expression.