The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation

An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique BglII site. A kanamycin resistance cartridge was cloned into the BglII site. This construct was mobilized into A. vinelandii CA12 (Δ nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.