JOtlHNAI, OF BA TPtluoio(x;Y, Apr. 1978, p. 125-130 Vol. 134, No. 1 0021-9193/78/0134-0125$02.00/0 Copyright © 1978 American Society for Microbiology Printed in U.S.A. Pathways of Assimilation of [13N]N2 and 13NH4+ by Cyanobacteria with and without Heterocysts JOHN C. MEEKS,t C. PETER WOLK,I* WOLFGANG LOCKAU,tt NORBERT SCHILLING,' PAUL W. SHAFFER,' AND WAN-SHEN CHIEN2 MSU-ERDA Plant Research Laboratory' and Cyclotron Laboratory and Department ofPhysics,2 Michigan State University, East Lansing, Michigan 48824 Received for publication 9 January 1978 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 synthe- tase 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 Anacys- tis nidulans incubated with "NH4' may be formed by glutamic acid dehydrogen- ase. 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 have much, and perhaps all, of the dinitrogen reduc- shown that in the heterocyst-forming cyanobac- tion occurs in the heterocysts, where it is coupled terium Anabaena cylindrica the major enzy- to the formation of glutamine by the action of matic pathway for the asimilation of NH4', glutamine synthetase (17). Glutamate synthase- whether derived from N2 or supplied exoge- mediated fonnation of glutamate appears to oc- nously, consists of glutamine synthetase (L-glu- cur only in vegetative celLs of N2-grown fila- tamate:ammonia ligase [ADP forming], EC ments, whereas glutmine synthetase functions 6.3.1.2) and glutamate synthase (L-gluta- in both cell types (17). mate:ferredoxin oxidoreductase [transaminat- The pattern of positioning of heterocysts in ing], EC 1.4.7.1) (11, 19). Glutamic acid dehydro- cyanobacteria varies among different genera. genase and alanine dehydrogenase also function For example, spaced sequences of intercalary in the assimilation ofexogenously supplied NH41 heterocysts are present in Anabaena, whereas in this cyanobacterium, but at a much lower rate only terminal heterocysts are normally found in even in the presence of relatively high levels of Cylindrospernum. In addition, nitrogenase ac- NH4+ (11). In this respect, A. cylindrica differs tivity in the cyanobacteria is not confined to from some heterotrophic, dinitrogen-fixing bac- heterocyst-forming species. Thus, Pkectonema teria in which N2-derived NH4+ is assimilated boryanum reduces acetylene and grows with N2 principally by the glutamine synthetase/ as the sole nitrogen source under microaerobic glutamate synthase pathway, whereas, during conditions (16), and Gloeocapsa sp. can reduce growth in the presence of high exogenous con- acetylene and grow with N2 as the sole nitrogen centrations of NHEV, the NHE is assimilated via source aerobically (20; and cf. 7). Yet other glutamic acid dehydrogenase (6). cyanobacteria, e.g., Anacystis nidulans (syn. Sy- When A. cylindrica is grown aerobically, nechococcus 6301 [15]), appear unable to reduce acetylene or N2. Anabaena, Cylindrospermun, t Preent address: Department of Bacteriology, University and Plectonema are filamentous, Gloeocapsa is of Calfornia, Davis, CA 96616. tt Present address: Fachbereich Biologie und Vorklinische colonial or unicellular, and Anacystis is unicel- Medizin, Institut fir Botanik, Univer it Regensburg, D-40 lular. We have sought to determine whether the Regensburg, Federal Republic of Germany. diversity of morphological and physiological 125 126 MEEKS ET AL. J. BACTERIOL. types of cyanobacteria is paralleled by a diver- RESULTS of NH4+. sity in the pathway(s) of assimilation Assimilation of Incubation for up The results are compared with results reported ['"'N]N2. to 120 s with ["N]N, resulted in incorporation earlier (11, 19) for A. cylindrica Lemmermann of '"'N into three organic constituents by each of (ATCC 29414). the four N2-fixing cyanobacteria examined. The MATERIALS AND METHODS compounds were tentatively identified as gluta- mine, glutamate, and either citrulline or, after of Cultures cyanobacteria. Cylindrospermum and 120 s of fixation C. licheniforme Kutzing (ATCC 29412) and Anabaena 60 by licheniforme, variabilis Kutzing (ATCC 29413) are strains that have alanine, on the basis of their comigration with been studied in our laboratory for a period of years. stable amino acids during electrophoresis at pH Gloeothece sp. 6909 (ATCC 27152; Gloeocapsa sp. 9.2 (19). After 15 s of fixation of [':N]NN, gluta- [15]) was obtained from the American Type Culture mine was in all cases the most highly radioactive Collection. A. nidulans (UTCC 625) and P. boryanum compound, accounting for 71 to 88% of the total Gomont (UTCC 594) were obtained from the Univer- organic 'sN recovered (Fig. 1). The fraction of sity of Texas culture collection. '3N in glutamine decreased and that in gluta- C. licheniforme and A. variabilis were grown aer- mate increased during longer incubation periods obically, with N2 as the nitrogen source, as semicontin- until, after 60 or 120 s of fixation, glutamate was uous cultures in photosynthetic fermentors in an eight- fold dilution of the medium of Allen and Arnon (2). P. more highly radioactive than glutamine. Citrul- boryanum and A. nidulans were grown in the same line became detectably radioactive after 60 s of manner except that the 5-liter culture of P. boryanum was sparged with N2-CO2 (99:1, vol/vol) at a rate of a b 900 to 1,000 ml/min, and the medium for the culture of A. nidulans was supplemented with 1 mM NaNO3 0.8 C and 1 mM KNO3. Gloeothece sp. was also grown in the same manner but in the medium of Allen (3), modified by substitution of NaCl for NaNO3 at the 0.6 same molar concentration. A short time before expo- sure to '3N, the cyanobacteria were concentrated to 27 jg of chlorophyll per ml by centrifugation at 200 x g 0.4 (1,000 x g for A. nidulans) for 5 min and, except for P. boryanum, were incubated in an atmosphere of80% Ar, 0.1 or 1.0% C02, and the balance 02, until use. P. z 0.28 boryanum was concentrated and incubated under Ar- CO2 (99:1, vol/vol). In some experiments, L-methio- nine-DL-sulfoximine (MSX; Sigma Chemical Co., St. 0 Louis, Mo.) or aminooxy acetate (Sigma) was added C d to the cyanobacterial suspensions at the time of resus- 0- o- pension to a concentration of 2.0 mM (1.0 mM final concentration upon dilution with '"'NH4+). Labeling with 'N. "N was generated by irradia- tion of 18.6 mg of "C with protons and ['I:N]N, was formed by subjecting the "C target to Dumas com- bustion (18, 19). ':'NH,+ was generated by acid diges- tion of the "C target and vacuum distillation (17). ["3N]N2 was fixed in the light under an atmosphere of Ar-N2-CO2 (97:2:1, vol/vol/vol) by 0.25 ml of cyano- bacterial suspension in 1.0-ml Reactivials (Pierce Chemical Co., Rockford, Ill.) fitted with stopcocks (19). Assimilation of ''NH4+ took place in the light under air in 15-ml conical centrifuge tubes (11). Re- actions were initiated by the addition of the cyanobac- Fixation Time (s) terial suspension and were terminated by mixing the Fi(;. 1. Distribution of '"'N in organic products ex- suspension with 4 volumes of methanol (19). The tracted with 80% methanol after fixation of [" N]N2 radioactive organic products in the methanolic ex- for 15, 60, and 120 s by (a) A. variabilis, (b) C. tracts were separated by high-voltage (3 kV) electro- licheniforme, (c) P. boryanum, and (d) Gloeothece sp. phoresis with 70 mM borate buffer (pH 9.2) on thin The radioactivity in the constituents of extracts sub- layers of cellulose (19). In certain experiments, electro- jected to electrophoresis at pH 9.2 was quantitated phoresis was followed by chromatography in an or- by integration of peaks in radioscans, with correc- thogonal direction in phenol-water (3:1, vol/vol) equil- tions applied for decay. Values presented are means, ibrated with 3% aqueous NH4OH (11, 19). Radioactive from two experiments, of the fraction of organic ' 'N constituents were localized and quantitated by scan- comigrating with stable glutamine (A), glutamate ning of thin-layer plates after one- and two-dimen- (0), and citrulline (x) (or, in the case of C. licheni- sional separations (11, 19). forme, alanine). VOL. 134, 1978 ASSIMILATION OF [';N]N2 AND '$NH4+ BY CYANOBACTERIA 127 incubation of A. variabilis and Gloeothece sp. observation that a major fraction of the "3N in and after 120 s of incubation of P. boryanum. that product could, in the case of each organism, The mean rates of fixation of [l:IN]N2 into be distilled (11, 19) as amide nitrogen. After 1 s total extractable material by C. licheniforme of assimilation of 13NH4', 77 to 97% of the or- and A. variabilis, 36.6 ± 7.1 and 34.5 ± 6.0 dpm ganic 13N extracted was found in glutamine (Fig.