Identification of sequences important for recognition of vnf genes by the VnfA transcriptional activator in Azotobacter vinelandii.

To analyze regulation of the vanadium-dependent nitrogenase of Azotobacter vinelandii, plasmids carrying vnfE-, vnfH-, or vnfD-lacZ fusions were transferred to Escherichia coli. These genes were expressed only if VnfA was present. Deletions of the vnfE upstream region were constructed and comparison of a region necessary for expression with sequences upstream of other vnf genes indicated a substantially conserved motif, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA. This motif was duplicated with 17 or 18 bases lying between each in the vnfH and vnfD promoters. Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression. In footprinting experiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recognition motifs. The active form of VnfA is probably interacting dimers, a tetramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA.