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The isolation of nitrogen-fixing bacteria from environmental sources is described. Strains, designated DU1, TP1, WA1 and WC1, were isolated from mostly aquatic samples at sites located in Raleigh, NC, while strains, designated SM1 and SM2, were isolated from a salt marsh located near Beaufort NC. Strain WB3 was isolated from a salt marsh found near Wrightsville Beach, NC. Results indicate that the isolates can be classified in the gamma subdivision of the class Proteobacteria with genes that encode the Mo nitrogenase and the V nitrogenase.

Characterization of a tungsten-tolerant mutant (CA6) of the diazotroph Azotobacter vinelandii based on the results of growth experiments showed that CA6 requires nitrogenases 1 and 3 for growth in nitrogen (N)-free medium containing Na2MoO4. The wild-type strain CA repressed the transcription of the anfHDGK gene cluster, which encode the nitrogenase enzymes. This suggests that expression of nitrogenases is dependent on the presence of molybdenum.

Two open reading frames (orf), designated anfO and anfR, found downstream of anfK are crucial to nitrogenase 3-dependent diazotrophic growth in Azotobacter vinelandii. When in-frame deletions of these orfs are performed in certain strains, an Anf- phenotype appears. This demonstrates that the products of anfO and anfR should be present for N2 reduction by nitrogenase 3 to occur.

Mutation studies of the two genes encoding Azotobacter vinelandii's nitrogenase 2 and 3 delta subunits, vnfG and anfG, indicate the necessity of VafG and AnfG for diazotrophic growth of A. vinelandii in a nitrogenase 2- and 3-dependent manner. The cysteine residue of AnfG is not essential for dinitrogenase 3-dependent AnfG function, while nitrogenase 2-dependent cell growth requires the vnfG gene product.

Expression of an anfH-lacZ fusion in, and growth studies of, mutants that have amino acid substitutions at the probable metal-binding site in the N-terminus suggest that Cys-21 and Cys-26 are necessary for the transcription of nitrogenase 3 genes in Azobacter vinelandii while Ser-19 is not required. Direct or indirect stimulation of VnfA leads to the residual expression of anfH-lacZ fusion in AnfA mutants.

Competition experiments involving a Mo-independent nitrogenase 3-deficient mutant and wild-type Azotobacter vinelandii reveals that nitrogenase 3 promotes diazotrophic growth on agar media containing molybdenum but not in liquid media. Identification of nitrogenase 3 subunits and use of anfM-lacz fusions confirms the presence of nitrogenase 3 expression in the wild-type cells proliferating on agar media. The experiments indicate that nitrogenase 3 is capable of fixing nitrogen even under Mo-deficit conditions.

The Azotobacter vinelandii genes for nitrogenase-2 are organized in two transcriptional units, vnfHorfFd, which codes for dinitrogenase reductase-2, and vnfDGK, which codes for dinitrogenase-2. The effects of ammonium (NH4+), molybdenum (Mo) and vanadium (V) on the expression of these genes were determined. The results showed that the transcription of both genes were repressed by Mo. However, NH4+ appears to act at both the transcriptional and posttranscriptional levels. Lastly, V is required for the translation of vnfDGK products.

The studies about the molybdenum-independent nitrogenases diazotrophs isolated from wood chip mulch, soil, 'paraffin dirt', and sediments from mangrove swamps are described. Most of the isolates are found to be members of gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria.