Mol Gen Genet (1993) 236:459-462 © Springer-Verlag 1993 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 Ramesh Raina, Umesh K. Bageshwar, and H.K. Das Genetic EngineeringUnit, Centre for Biotechnology, JawaharlalNehru University, New Delhi - 110067 (India) Received January 20, 1992 / AcceptedJuly 17, 1992 Summary. An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vine- landii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent al- ternative) 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 BgIII site. A kanamycin resistance cartridge was cloned into the BglII site. This construct was mobilized into A. vinelandii CA12 (A 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 sug- gests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitro- gen fixation in A. vinelandii. Key words: Azotobacter vinelandii - ORF2 - Site-di- rected mutagenesis - vnfpathway Introduction The process of biological nitrogen fixation in Azoto- bacter vinelandii can be carried out by one of three differ- ent pathways. These are the molybdenum-dependent (n~ pathway, the vanadium-dependent (vnf) pathway and a third that is dependent on neither molybdenum nor vanadium (anJ) (Joerger and Bishop 1988). The ni- trogenase enzyme for the molybdenum pathway is en- coded by three structural genes nifH, nifD and nifK which form a single operon (Brigle et al. 1985). We have previously reported (Raina et al. 1988) the characteriza- tion of vnfH, which is the nifH equivalent for the vanadi- um pathway. We have also reported (Raina et al. 1988) the presence downstream of vnfH of a 195 bp ORF (ORF2) which Correspondingauthor: H.K. Das could code for a protein of 65 amino acids. The arrange- ment of the cysteine residues (Cys-Xz-Cys-X2- Cys-X3-Cys-Pro; Fig. lb) in this protein shows a pattern typical of small, low-potential ferredoxins. The presence of a similar sequence downstream of the vnfH gene has also been reported in A. chroococcum (Robson et al. 1986). On the basis of nucleotide sequence analysis, we had reported (Raina et al. 1988) that vnfH and ORF2 are in the same operon and that transcription of ORF2 is controlled by the vnfH promoter. It has already been shown in A. vinelandii (Jacobson et al. 1986) and in A. chroococcum (Robson et al. 1986) that vnfH and ORF2 are cotranscribed under conditions where the vnf path- way of nitrogen fixation operates and their transcription is repressed in the presence of fixed nitrogen source and molybdenum. Flavodoxin and ferredoxin I have both been implicat- ed as components of the electron transport chain to ni- trogenase in the aerobic bacterium A. vinelandii. Genes coding for a flavodoxin (nifF; Bennett et al. 1988) and for ferredoxin I (fdxA; Morgan et al. 1988) have been cloned and sequenced and mutants have been con- structed. Neither of the genes is essential for the molyb- denum-dependent nitrogen fixation process in A. vine- landii (Martin et al. 1989). Here we report the construction and characterization of a mutant of the vanadium-dependent pathway of ni- trogen fixation in A. vinelandii, which carries a Km resis- tance gene cartridge in the ORF2. Materials and methods Growth and maintenance of strains. Bacterial strains and plasmids used are listed in Table 1. The bacterial strains were grown and maintained as described earlier (Raina et al. 1988). Site-directed mutagenesis. The oligonucleotides used for site-directed mutagenesis were synthesized in the model 391 DNA synthesizer (Applied Biosystems) by the