A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4' into medium.Mutant strains capable of producing nitrogenase in the presence of NH4' were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, N03 , or low concentrations of NH4' (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4' produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4' when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4' produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment.In free-living, nitrogen-fixing organisms, nitrogenase synthesis and activity are regulated by the presence of NH4' in the medium (3,10,31,45). That NH4+-mediated regulation of nitrogenase synthesis is not exerted by NH4+ itself but is actually a consequence of metabolic products of NH4+ assimilation has been revealed by several lines of evidence.(i) Mutant strains incapable of NH4' assimilation were derepressed for nitrogenase synthesis in the presence of NH4+ (28,31,43,44). (ii) Addition of amino acids to the growth medium repressed nitrogenase synthesis even in nitrogenase-derepressed mutant strains (30). (iii) Inhibition of glutamine synthetase, a primary enzyme responsible for NH4+ assimilation, by a substrate (glutamate) analog, Lmethionine-D,L-sulfoximine (MSX) as well as by other inhibitors led to derepression of nitrogenase synthesis in the presence of NH4+ in all organisms tested (8,13,14,21,31,39). The above described derepression of nitrogenase synthesis is achieved by alteration of cellular physiology leading to a decrease in the rate of glutamate production, since addition of amino acids to the medium reversed the effect (22,29). This set of conditions also derepressed other NH4+-as well as glutamate-producing enzyme systems (NO3 assimilation; histidine and proline utilization) in the cell (16,29).Mutant strains that are blocked at the level of NH4+ assimilation were first described in Klebsiella pneumoniae (32), and such mutant strains not only derepress nitrogenase synthesis but also excrete the NH4+ produce...