vnfG and anfG encode the ␦ subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf ؊ Anf ؊ strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the ␦ subunits are not required for acetylene reduction by nitrogenases 2 and 3.
Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfl-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.Azotobacter vinelandii is an aerobic nitrogen-fixing soil bacterium. This diazotroph is able to reduce N2 to NH4+ by using any of three distinct nitrogenases. These enzymes are encoded by three separate sets of structural genes. niflDK encodes the subunits of the molybdenum (Mo)-containing nitrogenase (nitrogenase 1), vnfHDGK encodes the subunits of the vanadium (V)-containing nitrogenase (nitrogenase 2), and anJfH-
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