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A spontaneous pleiotropic Nif- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171: 1017-1027]. In addition to being unable to grow on N2 as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH4+, (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH4+ up-shift, and (f) an inability to modify (32P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH4+. Activity of the Fe protein component of nitrogenase required both Mn2+ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.

In the photosynthetic bacterium Rhodopseudomonas capsulata, NH4+ switch-off of nitrogenase activity can be mimicked by its analog, methylamine. Like NH4+, methylamine appeared to require processing by glutamine synthetase (GS) before it was effective; gamma-glutamylmethylamide was shown to be the product of this reaction. Evidence that this glutamine analog functioned directly to initiate nitrogenase inactivation was suggested first by the fact that it was a poor substrate for glutamate synthase (i.e., it was not further metabolized by this pathway) and secondly, azaserine which blocks the transfer of the glutamine amide group had no effect on CH3NH3+ (or NH4+) switch-off. These observations are taken as preliminary evidence to suggest that when NH4+ inhibits nitrogenase activity, inactivation is initiated by glutamine itself, and not a molecule derived from it. Finally, evidence was presented that R. capsulata would use CH3NH3+ as a nitrogen substrate, but lag periods and generation times increased with subsequent passages.

In vivo switch-off of nitrogenase activity by NH4+ is a reversible process in Rhodobacter sphaeroides and Methylosinus trichosporium OB3b. The same pattern of switch-off in Rhodospirillum rubrum is explained by ADP-ribosylation of one of the Fe protein subunits, however, no evidence of covalent modification could be found in the subunits from either R. sphaeroides or M. trichosporium. Fe protein subunits from these organisms showed no variant behaviour on SDS-PAGE, nor were they 32P-labeled following switch-off. These observations suggest either that the attachment of the modifying group to the Fe protein in these organisms is quite labile and does not survive in vitro manipulation, or that the mechanism of switch-off is different than that seen in Rhodospirillum.