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The principal initial product of metabolism of ['3N]N2 and '3NH4' by five diverse cyanobacteria is glutamine. Methionine sulfoximine inhibits formation of [l:JN]glutamine except in the case of Gloeothece sp., an organism with a thick sheath through which the inhibitor may not penetrate. Thus, glutamine synthetase appears to catalyze the initial step in the assimilation of N2-derived or exogenous NH4' by these organisms. ['3N]Glutamate is, in all cases, the second major product of assimilation of "IN-labeled N2 and NH4+. In all of the N2-fixing cyanobacteria studied, the fraction of "N in glutamine declines and that in glutamate increases with increasing times of assimilation of ['3N]N2 and l NH4', and (Gloeothece again excepted) methionine sulfoximine reduces incorporation of`N into glutamate as well as into glutamine. Glutamate synthase therefore appears to catalyze the formation of glutamate in a wide range of N2-flxing cyanobacteria. However, the major fraction of [I:JN]glutamate formed by Anacystis nidulans incubated with "NH4' may be formed by glutamic acid dehydrogenase. The formation of ['3N]alanine from ":3NH4+ appears to be catalyzed principally either by alanine dehydrogenase (as in Cylindrospermum licheniforme) or by a transaminase (as in Anabaena variabilis). Experiments using the radioisotope '3N haveshown that in the heterocyst-forming cyanobacterium Anabaena cylindrica the major enzymatic pathway for the asimilation of NH4', whether derived from N2 or supplied exogenously, consists of glutamine synthetase (L-glutamate:ammonia ligase [ADP forming], EC 6.3.1.2) and glutamate synthase (L-glutamate:ferredoxin oxidoreductase [transaminating], EC 1.4.7.1) (11,19). Glutamic acid dehydrogenase and alanine dehydrogenase also function in the assimilation ofexogenously supplied NH41 in this cyanobacterium, but at a much lower rate even in the presence of relatively high levels of NH4+ (11). In this respect, A. cylindrica differs from some heterotrophic, dinitrogen-fixing bacteria in which N2-derived NH4+ is assimilated principally by the glutamine synthetase/ glutamate synthase pathway, whereas, during growth in the presence of high exogenous concentrations of NHEV, the NHE is assimilated via glutamic acid dehydrogenase (6).When A. cylindrica is grown aerobically, t Preent address:

When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate 113NIN2, the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; 13N in alanine becomes detectable at I minute of fixation and increases rapidly between I and 2 minutes. After 15 minutes of fixation, the major 13N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas 113Niglutamine comprises only a small fraction of organic 13N, and very little 13N is detected in asparagine. The fixation of 113NIN2 into organic products was inhibited more than 99% by C2H2 (10%, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules.Ammonium is the initial product of dinitrogen fixation by symbiotic (1) and free-living (19, 21) prokaryotes and by nitrogenase in vitro (16). Activities of the ammonium-assimilating enzymes glutamic acid dehydrogenase and glutamine synthetase, as well as glutamate synthase, have been detected in bacteroid fractions isolated from root nodules of soybean (2, 3) and lupin (2, 9).Nevertheless, free-living N2-fixing Rhizobiumjaponicum and bacteroids of R. japonicum isolated from soybean release 90 to 94% of the products of fixation of '5N2 into the medium as ammonium (1, 13). Dilworth and Brown (2) suggested that ammonium produced by bacteroids is assimilated in nodules through either the glutamine synthetase-glutamate synthase or glutamic acid dehydrogenase pathways, and favored the latter. In serradella, the major amino acids formed from '5N2 were glutamate and glutamine, but the initial organic product, and thereby the primary assimilatory route, was not determined (8 Pathology, Michigan State University, was maintained on YM agar (1.5%) slants and transferred every 3 months. Liquid cultures, started from slants in 20 ml of YM medium, were transferred to 500 ml of YM medium in 1-liter flasks, and were aerated by agitation on a reciprocating shaker. Cells from 2-day-old cultures were harvested by centrifugation at 6,000g for 10 min, washed and resuspended in ("N-free") legume nutrient medium free of combined nitrogen (6), and used as inoculum.Glycine max (L.) Merr. cv. Hark seeds were surface-disinfected for 20 sec with I % NaOCl, washed in running tap water, and planted in a Vermiculite-Perlite bed. The bed was inoculated with the Rhizobium suspension. Aerated N-free medium was circulated through the bed of germinating seeds for I week. The beds were illuminated with two 40-w Gro-lux fluorescent lamps (Westinghouse