N2 and 13NH4+ by Cyanobacteria with and Without Heterocysts JOHN C

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 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 Anacys- tis 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 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 glutamine, 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 exposure 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-methionine-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 cyanobacterial 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. per 106 dpm added per min, respectively, are of 3). The second major radioactive product of the same order of magnitude as the rates ob- assimilation of 13NH4' by all of the cyanobac- served with A. cylindrica (19). The rate of in- teria studied was glutamate. Aspartate was de- corporation by P. boryanum was usually four- tectably radioactive in all of the organisms as of fold lower (9.2 ± 1.3 dpm per 106 dpm added per 120 or 900 s of assimilation. After 900 s of assim- min), although a substantially higher rate was ilation, glutamine, glutamate, and aspartate still observed in one experiment, and that by Gloeo- accounted for 70% (in A. variabilis) to 88% (in thece sp. was ca. 15-fold lower (2.3 ± 0.3 dpm P. boryanum) ofthe total, 80% methanol-soluble per 106 dpm added per min). radioactivity in metabolites. However, of that After 15 s of fixation of ['3N]N2, the ratio of total of 100%, ['3N]glutamine accounted for 72% '3N in glutamate to 13N in glutamine was 0.14 in P. boryanum (and 76% in A. nidulans), but (C. licheniforme) to 0.38 (Gloeothece sp.). This for only 14% in A. variabilis. ratio rapidly increased until after 120 s offixation Alanine was detectably radioactive after as it was 1.29,4.15,4.99; and 5.68 in C. licheniforme, little as 1 s of assimilation by A. variabilis, C. A. variabilis, P. boryanum, and Gloeothece sp., licheniforme, and A. nidulans and after 15 s of respectively (Fig. 2). assimilation by Gloeothece sp., but was detected Assimilation of 13NH4. Products of assimi- only as a shoulder on peaks of citrulline at 120 lation observed in methanolic extracts after 900 and 900 s in the case of P. boryanum. Con- s of incubation with 13NH4' included glutamine, versely, radioactivity in citrulline was detectable glutamate, aspartate, citrulline, alanine, and ar- only as a shoulder on the alanine peak following ginine. These radioactive constituents were 900 s of assimilation by A. variabilis, C. lichen- identified by comigration with stable amino iforme, and A. nidulans and was also clearly acids during one- and two-dimensional separa- detectable only after 900 s of assimilation by tions (11, 19). The identification of one of those Gloeothece. No (A. nidulans) or low radioactiv- constituents as glutamine was supported by the ity (<7% of the total in A. variabilis, <3% of the total in the other organisms) was detected in 6 10 l00 1Q00 arginine after 900 s of assimilation. An unidentified substance migrating between alanine and arginine at pH 9.2, present after all periods of 5- exposure to 13NH4' and approximately as radioactive as glutamate after 1 s of labeling, was observed in extracts of P. boryanum. Less-con- spicuous, unidentified 13N-labeled substances 04.) were also observed in extracts of the other organisms studied. Inclusion of 1 mM MSX in the cyanobacterial suspensions resulted in 85 to 95% inhibition of total 13NH4+ assimilation by C. licheniforme, A. variabilis, and P. boryanum after 900 s of incubation; formation of glutamine was more strongly inhibited than was formation of gluta- Assimilation Time (s) mate (Table 1). Formation of [13N]glutamine by FIG. 2. Time course of the ratio of the sum of A. nidulans was inhibited 81% in the presence [3NJglutamate plus f'3NJaspartate to /"Nlgluta- of MSX, but formation of ['3N]glutamate was mine during fixation of 3N]N2 (open symbols) and increased about sevenfold, so that net assimila- assimilation of '3NH4' (closed symbols) by N2-grown tion was decreased only about 35%. Assimilation A. variabilis (0, 0), C. licheniforme (A, A), P. bor- of 13NH4+ by Gloeothece sp. (at 900 s, "3N-labeled yanum (0, U), Gloeothece sp. 6909 (V, V), and NO3-- metabolites extracted accounted for 3.4% of the grown A. nidulans (x). 3NJAspartate accountedfor 13N added) was not inhibited by up to 10 mM a significant fraction of the '3N in the glutamate- MSX, and the distribution of 13N among metab- plus-aspartate band only after 120 and 9(K s ofassimilation of 13NH4'. The radioactivity in the amino olites was essentially unaffected. MSX greatly acids was determined as in Fig. 1 and 3, and the reduced radioactivity in alanine in A. variabilis, ratios were then calculated. A logarithmic scale is but did not decrease formation of [13N]alanine used on the abscissa topermit clear representation of by C. licheniforne. Similarly, 1 mM aminooxy a large range of tines. acetate inhibited formation of ['3N]alanine by 128 MEEKS ET AL. J. BACTERIOL.

0.2LL~

Assimilation Tme (s) FIG. 3. Distribution of ":N in organic products extracted with 80% methanol after assimilation of ';NH4+ for 1, 15, 120, and 900 s by (a) A. variabilis, (b) C. licheniforme, (c) P. boryanum, (d) Gloeothece sp., and (e) A. nidulans. Samples were processed, and the radioactivity in constituents was quantitated, as in Fig. 1. Values presented are means from two experiments of the fraction of organic ";N comigrating with stable glutamine (A), glutamate plus aspartate (0), alanine plus citrulline (x), and arginine plus other compounds (Ol). After 9(X) s of assimilation, alanine was more highly labeled than citrulline in A. variabilis and C. licheniforme, whereas citrulline was more highly labeled than alanine in P. boryanum. In all of the cyanobacteria, [' 'NJaspartate accounted for a significant but minor fraction of the "N in the glutamate-plus-aspartate peak after 120) and 900s.

A. variabilu about 40%, despite an increase in uct of assimilation of ["N]N2 and/or "3NH4' by formation of ['3N]glutamate, but did not reduce all six N2- or N03 -grown cyanobacteria that we the formation ofV'3N]alanine by C. licheniforme. have examined is glutamine (Fig. 1 and 3; 11, The ratio of '3N in glutamate plus aspartate 19). After 15 s of fixation of ["N]N2 or 1 s of to I3N in glutamine is in all cases lower after 120 assimilation of '3NH4', glutamine accounts for s of incubation with 13NH4' than with ["N]N2 at least 71% of the total 13N incorporated into (Fig. 2). The ratio remains below, or close to, organic metabolites extractable with 80% meth- unity up to 900 s of assimilation of "NH4' in all anol. Addition of 1 mM MSX (which inhibits of the cyanobacteria except A. variabilis (Table the glutamine synthetase of A. cylindrica [11, 1, Fig. 3). 17, 19]) reduces incorporation of 13N into glutamine by 94 to 98% after 900 s of incubation with DISCUSSION 13NH4+ in A. variabilis, C. licheniforme, and P. Glutamine synthetase/glutamate syn- boryanum and by 81% in A. nidulans (cf. 99.85% thase pathway. The first major organic prod- in NH4+-grown A. cylindrica; 11). The fact that Vou, 134, 1978 ASSIMILATION OF [";'N]N2 AND '"NH4+ BY CYANOBACTERIA 129 TABLE 1. Principal radioactive constituents observed after 900 8 ofassimilation of 13NH4' by N2- or N03-- grown cyanobacteria in the absence andpresence of) mM MSXa '3N found in compound as % of the '3N added Cyanobacterium Treatment Total Asp + Glu Gln Cit + Ala Arg + others A. variabilis -MSX 2.82 1.64b 0.34 0.56c 0.09 +MSX 0.35 (88)d 0.25' (85) 0.01 (97) O.09f (84) 0 (100) C. licheniforme -MSX 3.29 1.27" 1.70 0.24c 0.08 +MSX 0.47 (86) 0.11F (91) 0.09 (95) 0.26f (0) 0 (100) P. boryanum -MSX 5.35 0.86 3.97 0.52' 0.01 +MSX 0.53 (90) 0.14e (84) 0.32 (92) 0.07f (86) 0 (100) 'A. nidulans -MSX 1.82 0.10 1.67 0.05 0 +MSX 1.22 (31) 0.84 (0) 0.32 (81) 0.06 (0) 0 a Values are means of two experiments and were determined by integration and time correction of peaks of radioactivity from '3N after electrophoresis at pH 9.2, in comparison with '3NH+ radioactivity added, determined by scintillation counting. Asp, Aspartate; Glu, glutamate; Gln, glutamine, Cit, citrulline; Ala, alanine; Arg, arginine. b Asp + Glu was calculated as one peak, but in all cases the peak was primarily Glu. c Peak was primarily Ala with a trace of Cit. d Numbers in parentheses refer to percent inhibition. e No Asp observed. f No Cit observed. ' Peak was primarily Cit with a trace of Ala. MSX failed to inhibit glutamine formation in may be transferred rapidly from glutamine to Gloeothece sp. may possibly be due to an inabil- glutamate. Second, as was also observed earlier ity of MSX to permeate the thick sheath sur- for A. cylindrica (11, 19), MSX greatly reduces rounding the celis of the organism. Thus, it incorporation of UN into glutamate in the three appears that the most important initial step in filamentous cyanobacteria examined. This inhi- the assimilation of N2-derived or exogenously bition would not be expected if the major frac- supplied NH4' by a wide range of cyanobacteria tion of glutamate were formed by the action of is catalyzed by glutamine synthetase. These ob- glutamic acid dehydrogenase. servations extend previous results obtained with Other pathways. However, a fraction- two strains ofA. cylindrica and based on enzym- varying with the species-of the assimilation of ological data (5, 11, 17) and on tracer experi- NH4' is also attributable to the action of glu- ments with 14C (9) and 13N (11, 19). tamic acid dehydrogenase and alanine dehydro- The initial kinetics of '3N incorporation into genase. Activities of both of these enzymes have glutamate observed in the experiments pre- been detected in numerous cyanobacteria (8, sented here are similar to those previously re- 12-14) and appear to be responsible to a minor ported for A. cylindrica (11, 19). In the case of extent for the asimilation of exogenously sup- A. cylindrica, with nitrogen supplied as N2 or plied NH4' by NH4+-grown A. cylindrica (11). NH4', it was established by means of pulse- The fact that in A. nidulans glutamate becomes chase experiments and studies with inhibitors much more radioactive in the presence of MSX that glutamate synthase rather than glutamic than in its absence indicates that, in this orga- acid dehydrogenase mediates most of the for- nism, glutamate may be formed principally by mation of glutamate (11, 19). Two observations glutamic acid dehydrogenase and catabolized reported in the present paper imply that gluta- principally by glutamine synthetase. mate is also formed principally by glutamate Alanine appears to be fonned principally by synthase in A. variabilis, C. licheniforme, and alanne dehydrogenase in C. licheniformne, be- P. boryanum, and perhaps in Gloeothece sp. cause the very extensive inhibition of fonnation First, although the actual amount of ['3N]glu- of glutamine and glutamate by MSX is not ac- tamine formed increases with increasing times companied by an inhibition of formation of ala- of assimilation, the fraction of the total '3N nine, and because formation of alanine is also observed in glutamate (plus aspartate at 120 s of not reduced by the presence of aminooxy ace- assinailation of '3NH4' and thereafter) increases, tate, an inhibitor of amino transferase reactions while the corresponding fraction in glutmine in A. cylindrica (17). On the other hand, A. decreases. This observation suggests that 13N variabilis appears to form alanine principally by 130 MEEKS ET AL. J. BACTERIOL. an amino transferase reaction, probably from 2. Allen, M. B., and D. I. Arnon. 1955. Studies on nitrogen- glutamate, because the reactions incorporating fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol. "3N into glutamate (plus aspartate) and into 30:366-372. alanine were inhibited strongly and to a similar 3. Allen, M. M. 1968. Simple conditions for growth of uni- extent by MSX, and because aminooxy acetate cellular blue-green algae on plates. J. Phycol. 4:1-4. inhibited alanine formation. These results illus- 4. Carpenter, E. J., and C. C. Price. 1976. Marine Oscil- latoria (Trichodesmium): explanation for aerobic nitro- trate a diversity in the nitrogen metabolism of gen fixation without heterocysts. Science the cyanobacteria. 191:1278-1280. Relative rates ofincorporation of '3N into 5. Dharmawardene, M. W. N., A. Haystead, and W. D. glutamate (plus aspartate) and into gluta- P. Stewart. 1973. Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica. Arch. Mikrobiol. mine. In A. cylindrica, 13NH4' derived from 90:281-295. ['3N]N2 in heterocysts is assimilated by gluta- 6. Dilworth, A. J., and C. M. Brown. 1976. Chemostat mine synthetase, and the resulting glutamine studies on control of ammonia incorporation in Rhizo- moves into vegetative cells, where glutamate is bia, p. 426-488. In W. E. Newton and C. T. Nyman (ed.), Proceedings of the First International Symposium synthesized by the action of glutamate synthase on Nitrogen Fixation. Washington State University (17). Only the glutamate that moves back to the Press, Pullman. cells where N2 fixation is taking place can react 7. Gallon, J. R., W. G. W. Kurz, and T. A. Larue. 1975. to form additional ['3N]glutamine. When exog- The physiology of nitrogen fixation by a Gloeocapsa sp., p. 159-173. In W. D. P. Stewart (ed.), Nitrogen enous 13NH4+ is the nitrogen source, newly syn- fixation by free-living micro-organisms. Cambridge Uni- thesized glutamate is rapidly utilized by all veg- versity Press. etative cells to produce more glutamine, result- 8. Haystead, A., M. W. N. Dharmawardene, and W. D. ing in rapid labeling of both the a-amino and the P. Stewart. 1973. Ammonia assimilation in a nitrogen- fixing blue-green alga. Plant Sci. Lett. 1:439-445. amide groups of glutamine (11). It follows that 9. Lawrie, A. C., G. A. Codd, and W. D. P. Stewart 1976. in a heterocyst-forming cyanobacterium with The incorporation of nitrogen into products of recent glutamate synthase present only in vegetative photosynthesis in Anabaena cylindrica Lemm. Arch. cells and glutamine synthetase in both vegeta- Microbiol. 107:15-24. 10. Lorch, S. K., and C. P. Wolk. 1974. Application of gas- tive cells and heterocysts, the ratio of 13N in liquid chromatography to study of the envelope lipids glutamate (plus aspartate) to 13N in glutamine of heterocysts. J. Phycol. 10:352-355. should increase more rapidly when nitrogen is 11. Meeks, J. C., C. P. Woll, J. Thomas, W. Lockau, P. provided as [13N]N2 than when it is provided as W. Shaffer, S. M. Austin, W.-S. Chien, and A. Galonsky. 1977. The pathways of assimilation of 13NH4+. '3NH4' by the cyanobacterium, Anabaena cylindrica. In fact, that ratio is greater after 120 s of J. Biol. Chem. 252:7894-7900. fixation of [13N]N2 than after an equal period of 12. Neilson, A. H., and M. Doudoroff. 1973. Ammonia assimilation of 13NH4' by Plectonema and assimilation in blue-green algae. Arch. Mikrobiol. 89:15-22. Gloeothece as well as by the heterocyst-forming 13. Pearce, J., C. K. Leach, and N. G. Carr. 1969. The species studied (Fig. 2; 11, 19). It has been sug- incomplete tricarboxylic acid cycle in the blue-green gested that nitrogenase of the filamentous, non- alga Anabaena variabilis. J. Gen. Microbiol. heterocystous cyanobacterium Trichodesmium 55:371-378. 14. Rowell, P., and W. D. P. Stewart. 1976. Alanine dehy- sp. may be compartmentalized in cells that func- drogenase of the N2-fixing blue-green alga, Anabaena tion in a manner similar to the heterocysts of cylindrica. Arch. Microbiol. 107:115-124. other N2-fixing cyanobacteria (4). In addition, 15. Stanier, R. Y., R. Kunisawa, M. Mandel, and G. there are hints that lipids characteristic of het- Cohen-Baire. 1971. Purification and properties of unicellular blue-green algae (order Chroococcaks). Bac- erocysts may be present in Gloeothece sp. (1, teriol. Rev. 35:171-205. 10). Further work is required to clarify whether 16. Stewart, W. D. P., and M. Lex. 1970. Nitrogenase activ- or not nitrogenase is compartmentalized in such ity in the blue-green alga Plectonema boryanum strain species. 594. Arch. Mikrobiol. 73:250-260. 17. Thomas, J., J. C. Meeks, C. P. Wolk, P. W. Shaffer, S. M. Austin, and W.-S. Chien. 1977. Formation of ACKNOWVLEDGMENTNS glutamine from ['3N]ammonia, ['3N]dinitrogen, and We thank Marga Stya and Brian Uechi for assistance with I14CJglutamate by heterocysts isolated from Anabaena the two-dimensional scanner. cylindrica. J. Bacteriol. 129:1545-1555. N.S. received partial support from the Deutsche For- 18. Wolk, C. P., S. M. Austin, J. Bortins, and A. Galon- schungsgemeinschaft. This work was supported by the U.S. sky. 1974. Autoradiographic localixation of '3N after Energy Research and Development Administration under fixation of '3N-labeled nitrogen gas by a heterocyst- contract EY-76-C-02-1338 and by the U.S. National Science forming blue-green alga. J. Cell Biol. 61:440-453. Foundation. 19. Wolk, C. P., J. Thomas, P. W. Shaffer, S. M. Austin, and A. Galonsky. 1976. Pathway of nitrogen metabolism after fixation of '3N-labeled nitrogen gas by the LITERATURE CITED cyanobacterium, Anabaena cylindrica. J. Biol. Chem. 1. Abreu-Grobois, F. A., T. C. Billyard, and T. J. Wal- 251:5027-5034. ton. 1977. Biosynthesis of heterocyst glycolipids of An- 20. Wyatt, J. T., and J. K. G. Silvey. 1969. Nitrogen fixation abaena cylindrica. Phytochemistry 16:351-354. by Gloeocapsa. Science 165:908-909.