CHARACTERISTICS OF ORF1 AND ORF2 IN THE ANFHDGK GENOMIC REGION ENCODING NITROGENASE-3 OF AZOTOBACTER-VINELANDII

In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2 ) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions, We have des ignated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the sub units for nitrogenase 3 under Mo-deficient diazotrophic conditions. An fO and AnfR are required for nitrogenase 3-dependent diazotrophic grow th and N-15(2) incorporation but not for acetylene reduction. AnfO con tains a putative heme-binding domain that exhibits similarity to presu med heme-binding domains of P-450 cytochromes. Amino acid substitution s of Cys-158 show that this residue is required for fully functional A nfO as measured by diazotrophic growth under Mo- and V-deficient condi tions. The nucleotide sequence of the region located immediately downs tream of anfR has been determined. A putative rho-independent transcri ption termination site has been identified 250 bp from the 3' end of a nfR. A third open reading frame (orf3), located downstream of anfR, do es not appear to be required for diazotrophic growth under Mo- and V-d eficient conditions.