Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977

THE POSITION OF IN THE WORLD OF .

by

ROGER Y STANIER

D~partementde Biochimieet G6n6tiqueMicrobienne 28, rue du Dr Roux, 75015 Paris, France

EMIL CHR. nANSENlecture delivered at the Carlsberg Laboratory Centennial and the Inauguration of the Carlsberg Research Center on September 28th, 1976

Key words: prokaryotic photosynthetic apparatus; obligate photoautotrophy; oxidative and reductive pentose phosphate cycles; nitrogen fixation; oxygenic ; facultative anoxygenic photosynthesis; cyanobacterial ecology; cyanobacterial developmental patterns

Recent advances in our knowledge of the biological properties of the cyanobacteria (former ,blue-green algae,) are reviewed. The subjects discussed include: cyanobacterial cell structure and development; comparative as- pects of photosyntetic structure and function in prokaryotes; carbon metabolism and its relation to obligate photoautotrophy; and special properties of cyanobacteria that are of ecological significance (temperature rela- tionships, nitrogen fixation and facultative anoxygenicphotosynthesis).

1. INTRODUCTION seated functional differences between the two The largest evolutionary discontinuity among kinds of cells could be appreciated only after contemporary organisms lies at the cellular the molecular biological revolution. level; it permits us to distinguish two biological In addition to the organisms long recognized superkingdoms, eukaryotes and prokaryotes as , the superkingdom of prokaryotes (46, 43). The recognition of this fact is quite re- includes a large and widely distributed group of cent, since the differences between the phototrophic organisms, the cyanophytes or eukaryotic and the prokaryotic cell could not blue green algae. They can be defined as be rigorously formulated prior to 1960. A major microorganisms that harbor, within a typically technical advance, the application of electron prokaryotic cell, a photosynthetic apparatus microscopy to the study of biological fine struc- similar in structure and function to that located ture, was necessary to reveal the nature and ex- in the chloroplast of phototrophic eukaryotes. tent of the structural differences between pro- Their traditional classification as algae was, karyotic and eukaryotic cells; and the deep- accordingly, based on the resemblances be- R. Y. STANIER: Cyanobacteria

Figure 1. Thin section of a unicellular cyanobacterium, Synechocystis ATCC 27178, in the course of division. 60,000 x. The cell membrane (CM) is surrounded by a wall composed of three layers: the peptidoglycan (Pg) layer, the outer membrane (Om) layer, both universal in cyanobacteria; and an additional structured (St) layer. The regularly spaced thylakoids (th) bear rows of phycobilisomes (pbs), attached to both surfaces. Cs, carboxysome; ri, 70S ribosome.

80 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANIER: Cyanobacteria tween the cyanophytan cell and one constituent with the dimensions and fine structure of a unit of the algal cell, its chloroplast. These orga- membrane, containing proteins and lipopoly- nisms are in reality bacteria, and I shall here- saccharides (16). These two wall layers, com- after refer to them collectively as cyanobacte- mon to all Gram negative bacteria, are accom- ria. panied in many cyanobacteria by additional The cyanobaeteria differ from the two other wall layers, an example of which is evident in groups of phototrophic prokaryotes, which figure 1. have always been classified among the bacteria The only form of cellular locomotion in cya- -namely, purple and green bacteria - in many nobacteria is gliding movement, widespread but respects: the mechanism of photosynthesis; the not universal. A layer of fibrils, intercalated be- fine structure and chemical composition of the tween the two layers of the Gram negative cell photosynthetic apparatus; nutritional require- wall, has recently been demonstrated in some ments and growth physiology. gliding cyanobacteria (24), and may possibly be Fifteen years ago, when we began to work on the organelle responsible for this type of move- cyanobacteria, these organisms were still con- ment. strued as algae, and their study was largely The cell membrane, usually of simple con- conducted, as it always had been, by phycolog- tour, is closely appressed to the peptidoglycan ists, with a heavy emphasis on descriptive wall layer. The most conspicuous internal struc- and ecology. It was commonly be- tures of the cell are the regularly spaced photo- lieved that the isolation of pure cultures is ex- synthetic thylakoids, which occupy much of the ceptionally difficult, even though M. B. ALLEN cytoplasmic region, and bear attached to their (1) had made a promising start on this technical outer surfaces disc-shaped structures about 40 problem about 1950: indeed, most of the pure nm in diameter, known as phycobilisomes. The strains then in existence had been isolated by phycobilisome is a light-harvesting organelle, her. Accordingly, much of our initial work was and contains the cellular complement of phyco- directed to the isolation and purification of biliproteins, the major components of the cy- additional members of this group (47), an essen- anobacterial light-harvesting pigment antenna tial prerequisite for comparative experimental (21). The nucleoplasm has a typically prokary- studies. The news that these organisms are real- otic fine structure. The cytoplasm contains a ly bacteria has spread, thus encouraging other comparatively sparse population of 70S ribo- microbiologists to take up work on cyanobac- somes, a reflection of the low growth rates cha- teria. More significant information about their racteristic of most cyanobacteria. biological properties has emerged in the past Two classes of inclusions characteristic of cy- decade than in the preceding century. Some of anobacteria are carboxysomes and cyanophy- these recent advances will be reviewed here. cin granules. The former - polyhedral bodies bounded by a thin, non-unit membrane - occur in many other prokaryotes which use CO2 as 2. CELL STRUCTURE AND their major carbon source. They have been re- DEVELOPMENT cently isolated from a chemoautotroph, Thio- 2.1. Organization of the cyanobacterial cell. bacillus neapolitanus (38), and shown to be fil- Like the other photosynthetic prokaryotes, led with closely packed molecules of carboxy- cyanobacteria belong to the major sub-class of dismutase, the enzyme responsible for CO2 fixa- Gram negative bacteria, an assemblage now no tion in autotrophs. The carboxysome might be longer defined by the staining reaction of the considered the prokaryotic counterpart of the cell, but rather by the fine structure and mole- pyrenoid. cular composition of its wall. The cyanobacte- Cyanophycin granules (27) have been detect- rial cell wall contains at least two structurally ed only in cyanobacteria. The recent isolation distinct layers (fig. 1). The relatively thin and of these granules (39) has led to the interesting electron-dense innermost layer is composed of discovery that they are composed of a branched peptidoglycan (18). It is surrounded by a layer polypeptide which contains only two amino

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 81 R. Y. STANIER"Cyanobacteria

Figure 2. Rod-shaped unicellular cyanobacteria of the Synechococcus. Phase contrast, 2000 x. A, ATCC 27194; B, ATCC 27145; C, ATCC 29534. acids: a polyaspartic acid core, bearing lateral cell division into a vegetative cell which can arginyl residues (40). This material accumulates attain a diameter of 20-30 ~tm, this development in the cell as cultures approach the stationary being accompanied by the formation and pro- phase, and probably serves as a nitrogen re- gressive thickening of a fibrous outer wall layer. serve. A rapid series of cell divisions unaccompanied by growth then takes place within the fibrous 2.2. Patterns of development wall layer, which breaks open to release as The simplest cyanobacteria are unicellular many as 1000 reproductive cells. In Dermo- rods or cocci, which reproduce by binary fission carpa, this process of multiple fission is the only (figs. 2 and 3). A few unicellular cyanobacteria mode of cell division. In other pleurocapsalean (fig. 4) reproduce by budding (51), a variant of cyanobacteria, exemplified by Pleurocapsa (fig. binary fission which also occurs in many other 6), enlargement of the reproductive cell is fol- bacterial groups (44). lowed by a series of binary fissions, to produce The pleurocapsalean cyanobacteria have a an aggregate of vegetative cells, held together remarkable developmental cycle, which is illu- by the contiguous fibrous outer wall layers; strated in figure 5 for one of the simplest repre- some (or all) of the vegetative cells then under- sentatives, Democarpa (52). A small reproduc- go multiple fission and release small reproduc- tive cell, 2-3 ttm in diameter, enlarges without tive cells.

82 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANIER:Cyanobacteria

Figure 3. Coccoid unicellular cyanobacteria of the genera Gloeocapsa (A, B), the cells of which are enclosed by sheaths, and Synechocystis (C, D), the cells of which are not ensheathed. Phase contrast, 2000 x. A, ATCC 29159; B, ATCC 29113; C, ATCC 27171 ; D, ATCC 27819.

Many cyanobacteria have a filamentous structure. The filament of cells, or trichome, elongates by intercalary divisions and repro- duces by breakage. In oscillatorian cyanobac-

Figure 4. A unicellular budding cyanobacterium, Chamaesiphon ATCC 29397. Phase contrast, 2000 x. Three cells with polar buds, and two recently released small daughter cells.

Carlsberg Res. Commun, Vol, 42, p. 7%98, 1977 83 R. Y. STANIER:Cyanobacteria

Figure 5. Dermocarpa ATCC 29368. 500 x. Time lapse photomicrographs of the growth of three vegetative cells over a period of 288 hr. One cell has undergone multiple fission, and released small daughter cells.

teria (fig. 7) the trichome is an unbranched chome being converted into non-reproductive chain, composed only of vegetative cells. Other cells with a special physiological function, filamentous cyanobacteria can undergo cellular known as heterocysts. Certain heterocystous differentiation, some vegetative cells in the tri- cyanobacteria also form thick-walled resting

84 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANmg: Cyanobacteria

Figure 6. Pleurocapsa ATCC 29388. 1000 x. An aggre- gate of vegetative cells, some of which have under- gone multiple fission. The arrow points to a small daughter cell.

cells known as akinetes, which likewise diffe- rentiate from vegetative cells of the filament (fig. 8). Cell divisions in all oscillatorian and many heterocystous cyanobacteria occur in only one plane, at right angles to the long axis of the tri- chome. However, some members of the latter group have a more complex pattern of vegeta- tive growth. As the trichome develops, it be- comes branched and in part multiseriate, a con- sequence of changes in the plane of division of some of the constituent cells (fig. 9).

3. STRUCTURE AND FUNCTION OF THE PROKARYOTIC PHOTOSYNTHETIC APPARATUS As already mentioned, there is a dichotomy among phototrophic prokaryotes with respect to the mechanism of photosynthesis. In cyano- bacteria, as in phototrophic eukaryotes, photo- synthesis is oxygenic: molecular oxygen is a major metabolic product. In purple and green bacteria, on the other hand, photosynthesis is anoxygenic. This gross metabolic difference is the expression of a profound difference in the underlying photochemical mechanisms.

3.1. A comparative analysis of photosynthetic function The photosynthetic apparatus can be dissect- ed experimentally into three major compo- nents, which are closely integrated both func- tionally and structurally (fig. 10). These are: an antenna of light-harvesting pigments; a limited number of photochemical reaction centers, which always contain in a special state; and an electron transport chain. The re-

Figure 7. Three filamentous cyanobacteria of the genus Oscillatoria. 500 x. A, ATCC 29209; B, ATCC 29081; C, ATCC 29169.

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 85 R. Y. STAN[ER: Cyanobacteria action centers and the carriers of the electron transport system are always incorporated into a unit membrane. In most phototrophs, this is also the site of the light-harvesting pigments. However, part or all of these pigments may be located in peripheral structures, attached to the unit membrane, as exemplified by the phycobi- lisomes which contain the major cyanobacterial light-harvesting pigments. Light energy absorb- ed by the antenna pigments is channelled by radiationless transfer to a reaction center, where a charge separation is effected by ejec- tion of an electron from the reaction center . This initiates a cyclic electron flow in the associated photosynthetic electron trans- port chain, coupled with the synthesis of ATP. The light-mediated synthesis of ATP is the com- Figure 8. Cylindrospermum ATCC 29535, a cyano- mon denominator of all modes of photosynthe- bacterium which produces both heterocysts (HC) and sis. In the anoxygenic photosynthesis perform- akinetes (AK). 500 x. In this genus, the heterocysts ed by purple and green bacteria, it is the sole are terminal, and the akinetes sub-terminal. chemical consequence of light absorption (fig. 11). In oxygenic photosynthesis, however, the

Figure 9. Fischerella ATCC 29538, a heterocystous cyanohacterium which forms branched and in part multi- seriate trichomes. 500 x. HC, heterocyst.

86 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R, Y. STANIER:Cyanobactr photolysis of water, The two reaction centers are functionally interconnected through the electron transport chain. If both are simul- taneously excited, a non-cyclic electron flow occurs, linking the oxidation of water at reac- tion center I[ with the reduction of pyridine nucleotide at reaction center I, and accom- panied by ATP synthesis. In oxygenic photosyn- thesis, energy transfer from the light-harvesting pigment antenna to the two reaction centers is to some extent compartmentalized. For ex- ample, in cyanobacteria, the energy of light quanta harvested by chlorophyll a is largely

Figure 10. A schematic diagram to show the functions of the three components of a photosynthetic appa- ratus.

NON-CYCLIC ELECTRON FLOW IN CYANOBACTERIA AND RHODOPHYTAN CHLOROPLASTS,

Figure 12. A diagram of the cyanobacterial photosyn- thetic apparatus, showing non-cyclic electron flow. RC, reaction center.

Figure 11. A schematic diagram of the function of the photosynthetic apparatus of purple and green bacteria. RC BCHL, reaction center bacteriochloro- phyll.

chemical consequences of light absorption are considerably more complex, as a result of the fact that the photosynthetic apparatus contains two different types of reaction centers, known as type ! and type II reaction centers (fig, 12). The type [ reaction center, like the single reac- tion center associated with the performance of Figure 13. Operation of the cyanobacterial photosyn- anoxygenic photosynthesis, can mediate ATP thetic apparatus when reaction center It function is synthesis by cyclic electron flow. The specific selectively inhibited, for example by treatment with function of the type II center is to mediate the DCMU.

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 87 R. Y. STANIER" Cyanobacteria channelled to reaction center I, whereas the 3.2. The biosynthetic consequences of oxygenic energy of light quanta harvested by phycobili- and anoxygenic photosynthesis. proteins is transmitted to both reaction centers What are the consequences for biosynthesis (25). of the chemical differences between oxygenic Photosynthetic energy conversion can still and anoxygenic photosynthesis? Most, if not all, proceed in cyanobacteria if reaction center II phototrophs can synthesize organic cell mate- activity is destroyed, for example, by treatment rial from CO2, a process that requires an input with an appropriate concentration of the herbi- both of ATP and of reducing power, the latter cide DCMU (42). Under these special circum- in the form of reduced pyridine nucleotide. stances, photosynthesis becomes anoxygenic, Both are provided by the reactions of oxygenic photophosphorylation occurring though cyclic photosynthesis (25). However, anoxygenic pho- electron flow associated with reaction center I tosynthesis provides only ATP (42). Hence the (fig. 13). synthesis of cell material from CO2 by purple

Table I

The chemical differencesbetween oxygenic and anoxygenic photosynthesis

Source of Source of Source of carbon ATP reducing power

COs Photochemical H2S, H2 Purple and (enzymatic) green bacteria Organic compounds Photochemical Not required

Cyanobacteria CO2 Photochemical H~O and chloroplasts (photochemical)

Table II

Comparative chemistry of the photosynthetic apparatus

Light-harvesting Principal Membrane glycolipids pigments electron carriers (diglycerides)

Purple None bacteria ; ,Bacterial, ferredoxins; group-specific group-specific Green aliphatic or monocyclic quinones and bacteria carotenoids cytochromes Monogalactosyl-

Cyanobacteria and Chlorophyll a; rhodophytan chloroplast phycobiliproteins; Plant, ferredoxin; 13-carotene and derivatives plastoquinone; Monogalactosyl-; plastocyanin; digalactosyl- cytochrome f and sulfoquinovosyl- Chlorophytan Chlorophylls a and b chloroplast 13-carotene and derivatives

88 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANIER:Cyanobacteria and green bacteria is dependent on the genera- tion of reduced pyridine nucleotide by non- photochemical means; specifically by the en- zymatic oxidation of H2S, other reduced in- organic compounds, or molecular hydro- gen. A suitable chemical electron donor is thus required by these phototrophs for the synthesis of cell material from COs. Some purple and green bacteria can use organic compounds in place of COs as a source of carbon in the light: under these circumstances, biosynthesis is de- pendent only on an input of ATP, and the re- quirement for an inorganic electron disappears (45). These differences are summarized in Table I.

3.3. Comparative anatomy of the prokaryotic pho- tosynthetic apparatus. The mechanistic dichotomy between oxy- genic and anoxygenic photosynthesis is paral- leled by differences in both the chemical com- position and the fine structure of the photosyn- Figure 14. Thin section of the purple bacterium, thetic apparatus. Major chemical differences Rhodopseudomonas palustris, showing the regular exist with respect to the antenna pigments, the infoldings of the cell membrane (IM) which bears the components of the electron transport system, photosynthetic apparatus. 120,000 x. Courtesy of Dr. and the lipids of the photosynthetic apparatus Germaine Cohen-B azire. (Table II). The anatomical differences are equally striking (9). In purple bacteria (fig. 14) all elements of the photosynthetic apparatus are integrated into the cell membrane, which has a very complex topology (10). It is deeply infold- ed into the cytoplasm, and consequently has a very large area, this being the only physical means to accomodate a high level of light-har- vesting pigments. In green bacteria (fig. 15) the reaction centers and the electron transport system are located in the cell membrane, which has a simple topology (20): the antenna of light-harvesting pigments is adjacent to the membrane and is located in a series of cortical ovoid structures known as chlorobium vesicles (11). Among cyanobacteria, the photosynthetic apparatus can assume two configurations (figs.

Figure 15. Thin section of the green bacterium, Chlo- robium limicola. CW, cell wall, bearing pili (P). CM, cell membrane. CV, chlorobium vesicle. 135,000 x. Courtesy of Dr. Germaine Cohen-Bazire.

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 89 R. Y. STANIER:Cyanobacteria ratus has become internalized. An extensive system of flattened membranous sacs or thy- lakoids, topologically separated from the cell membrane, contains the reaction centers and the electron transport system. The light-har- vesting antenna of phycobiliproteins is borne in a regular array of granules known as phycobi- lisomes, attached to the outer membrane sur- face of the thylakoids. In such cyanobacteria, the cell membrane probably does not contain any component of the photosynthetic machi- nery. In the red algal chloroplast (22), the phy- cobitiproteins also serve as major light-har- vesting pigments, and the fine structure of the photosynthetic apparatus is homologous with that of most cyanobacteria. The various modes of organization of the photosynthetic apparatus are compared diagrammatically in fig. 18.

3.4. Physiological correlates of anoxygenic and oxygenic photosynthesis. In purple and green bacteria, anoxygenic photosynthesis is associated with an anaerobic life style. The majority of these organisms are strict anaerobes. Some purpte bacteria and one small group of green bacteria can tolerate mole- cular oxygen, and use it as a respiratory elec- tron acceptor. However, even these organisms are unable to grow photosynthetically in the presence of air, as a result of the fact that bacte- Figure 16. Thin section of a cyanobaeterium without riochiorophyll synthesis is completely repressed thylakoids, Gloeobaeter violaceus ATCC 29082. CW, by low partial pressures of oxygen (12). cell wall. CM, cell membrane. CL, cortical layer of phycobiliproteins. P, polyphosphatr granule. C, The physiological properties of purple and cyanophycin granule. Cs, carboxysomr 49,000 x. green bacteria effectively confine them to illu- From (37). minated, oxygen-free aquatic habitats (32). These reduced environments contain H2S, Ha and organic end-products formed by fermenta- tive anaerobes, the nutrients required for pho- 16 and 17). In a unicellular cyanobacterium tosynthetic growth of purple and green bacteria. which we have recently described, Gloebacter Competition for solar energy with oxygenic violaceus (37), its structure is analogous to that phototrophs which inhabit the superficial aero- in green bacteria. A cell membrane of simple bic water layer is not a problem, since the prin- topology is the site of the reaction centers and cipal absorption bands in vivo of the bacterio- the electron transport system; the light-har- chlorophylls are situated in the far red or infra- vesting antenna of phycobiliproteins is located red region, well beyond the spectral range used in a continuous subcortical layer, in contact for oxygenic photosynthesis. with the inner surface of the celt membrane. For cyanobacteria, as for eukaryotic photo- The second configuration, characteristic of all trophs, the ability to grow photosynthetically in other cyanobacteria so far studied, resembles the presence of molecular oxygen is a physio- that of the chloroplast: the photosynthetic appa- logical necessity. The cyanobacteria according-

90 Carlsherg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANmR:Cyanobacteria ly belong to the physiologico-nutritional cate- gory of aerobic photoautotrophs. Their adapta- tion to an aerobic environment has several se- condary expressions. Unlike many purple and green bacteria, eyanobacteria cannot generate ATP by fermentative means; their dark meta- bolism is purely respiratory. They also share with the great majority of algae the ability to use nitrate as a nitrogen source; no purple or green bacteria can perform an assimilatory re- duction of nitrate.

4. CYANOBACTERIAL CARBON METABOLISM AND THE PHENOMENON OF OBLIGATE PHOTOAUTOTROPHY Under photoautotrophic growth conditions, cyanobacteria assimilate CO2 through the re- ductive pentose phosphate cycle (30) and fixed carbon in excess of biosynthetic needs is stored as glycogen (fig. 19). In the dark, the endoge- nous glycogen reserve is mobilised and respired through the oxidative pentose phosphate cycle (6, 31), as shown in fig. 20. The two dehydro- genases of this metabolic pathway, glucose-6- phosphate and 6-phosphogluconate dehydro- genases, are both NADP-specific in cyanobac- teria (31). Dark ATP synthesis is thus associated with the reoxidation of NADPH via an 05- link- Figure 17. Thin section of a filamentous cyanobacte- ed respiratory transport chain (3), the compo- rium of the Lyngbya-Plectonema-Phormidiurngroup, nents of which have not so far been characte- ATCC 29344, with a cortical parallel array of rized. There is, accordingly, a very close inter- thylakoids bearing phycobilisomes. 50,000 x. relationship between the primary pathways of dark and light carbon metabolism in cyanobac- teria (fig. 21). A distinctive feature of the intermediary car- (33). Many of them are obligate photoauto- bon metabolism of cyanobacteria is that the tri- trophs; and even those which can grow carboxylic acid cycle does not operate as a in the dark do so at the expense of a small range respiratory pathway (41). This reflects the ab- of organic substrates, all carbohydrates which sence of the enzyme a-ketoglutarate dehydro- can be metabolized through the oxidative pen- genase; levels of malic and succinic dehydro- tose phosphate pathway (36). Dark growth of genases are also very low (41). The role of the facultative heterotrophs is always much slower remaining enzymes normally associated with than photoautotrophic growth. the tricarboxylic acid cycle is purely biosyn- The utilizability of sugars as exogenous sub- thetic in cyanobacteria. strates is dependent on the possession by the The nature of cyanobacterial carbon metab- cell of the appropriate permeases; and the ab- olism is reflected in the very limited heterotro- sence of carbohydrate transport systems appears phic capacity characteristic of these organisms to account in large part, though perhaps not

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 91 R. Y. STANIER:Cyanobacteria

LOCATION OF; PHOTOSYNTHETIC REACTION PIGMENT ANTENNA CENTER (S)AND SCHEMATIC OlAGRAMI PROKA RYOTES E.T. SYSTEM ,qv--- C.W. Purple bacteria Cell membrane Cell membrane .•--• c.m. ~C.W. Chlorobium vesicle Cell membrane Green bocteria g h?.'v e s. Cyonobocteria:

Gloeobacter Subcortical layer Cell membrane subc. layer

,6_.-.-~ C- W, All others Phycobilisome Thylokoid membrane -4------c.m. ~----- t h,yl, posome CHLOROPLASTS chl. ore. pbsome Rhodophytan Phycobili some Thylakoid membrane .~, ~'~'~--- thyl. chl. am. Chlorophytan Thylokoid membrane Thylokoid membrane ~,~ ~ thyl,

COMPARATIVE ANATOMY OF THE PHOTOSYNTHETIC APPARATUS

Figure 18. Comparative anatomy of the photosynthetic apparatus in prokaryotes and chloroplasts. CW, cell wall; CM, cell membrane; chl. ves., chlorobium vesicle; subc. layer, phycobiliprotein-cortaining cortical layer; thyl, thylakoid; pbsome, phycobilisome; chl. am., chloroplast outer membrane.

(light INTRACELLULAR entirely, for the prevalence of obligate photo- reactions) GLYCOGENPOOL autotrophy among cyanobacteria (31). The f ADP-.~ active transport system which mediates glucose ADP- glucose uptake by facultatively heterotrophic cyano- ~ATP~p-p bacteria has recently been characterized (2); it G~ucose- I - P permits the intracellular concentration, as free sugars, of glucose and non-metabolizable glu- Fructose - 6 - P----=,-- Glucose- 6 - P cose analogs. t

(light 2 Those- P 5. PHYSIOLOGICAL AND METABOLIC reactions) FACTORS THAT GOVERN THE NATURAL DISTRIBUTION OF CYANOBACTERIA I2 ADP'~.,~ The cyanobacteria are very highly specialized 2 ATP -j for a photoautotrophic mode of life, based on 2 NADP~k RibulOSe- 5 - P the performance of oxygenic photosynthesis. This brings them into direct competition in na- NAD;H~ycerate ~r ATP~ tural environments with eukaryotic algae, from which they do not differ significantly, as do CO2 ~'~ Ribulose di-P "4"-"~'-S "~ADPrea(i!htn) purple and green bacteria, in the spectral pro- perties of their light-harvesting pigments. Their Figure 19. The primary paths of autotrophic carbon competitive success is incontestable; no major assimilation by cyanobacteria in the light. group of algae has an ecological range as exten-

92 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANIER:Cyanobacteria INTRACELLULAR sive and varied as that of cyanobacteria. To GLYCOGENPOOL what do cyanobacteria owe their ability to find a place in the sun? Glucose-I-Pl It is not attributable to intrinsically high pho- tosynthetic growth rates, since the mean gene- Fructose-6-P Glucose-6-P.i EXOGENOUSGLUCOSE ration times in culture of most cyanobacteria ADP range from 12-48 hr. and are thus considerably / longer than those of many unicellular algae. r NADP~ Although this ecological question cannot be 6_ p _ glu!o'~n aN~ D P H -'~ ERLENCTpRONT"~ 2H20 fully answered, the factors which lead to the predominance of cyanobacteria in some envir- NADP~, CHAIN/ ~ 02 onments are now understood. They reflect, CO2.~"1~ NADPH ~ without exception, the possession by cyano- bacteria of physiological or metabolic proper- L Ributose-5 - P ATP ties that are widely distributed among other prokaryotes, but wholly absent from eukary- Figure 20. The pathways of cyanobacterial respira- otes. tory metabolism in the dark.

INTRACE LLULAR GLYCOGEN POOL

A DP-lucose

Glucose- I -P~.

1L EXOGENOUS Fructose-6-P ~ ..- Glucose-6-P GLUCOSE 1l "~ ,.,o

/ P.~TE \ ~-P-i,uco.ot. _ | 1L o, 2 P-Glycerote Ri , - - CO 2

Figure 21. The interrelations between the primary photosynthetic and respiratory pathways of carbon metabolism in cyanobacteria. Heavy white arrows indicate step-reactions specific to light metabolism; heavy black arrows, those specific to dark metabolism.

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 93 R. Y. STANmR:Cyanobacteda 5.1. Temperature relationships the cells become converted to heterocysts. The Certain cyanobacteria are thermophiles, with formation of mature heterocysts takes about 24 temperature maxima as high as 75 ~ C (4,5). The hours, and is paralleled by the development of upper temperature limit for growth of any pho- nitrogenase activity (29). During differentia- totrophic eukaryote lies at 55-60 ~ C (14). As a tion, the poles of the heterocyst become drawn result, cyanobacteria constitute the dominant, out; a small and structurally specialized contact indeed almost the exclusive, photosynthetic area with the adjacent vegetative cells is form- population of hot springs. Thermotolerance ed. A complex outer envelope, greatly thicken- appears to be the key factor which makes ed and multilayered in the region of the cell-to- cyanobacteria the major agents of photosynthe- cell junction, is laid down around the develop- sis in deserts, where some of these organisms ing heterocyst (26). The mature heterocyst have found a remarkable ecological niche. cannot divide or de-differentiate (53). It has They develop as a subsurface layer in desert lost photosystem II activity, but still retains rocks, in microfissures where water is trapped photosystem I activity, and hence can perform by condensation at night. The cyanobacteria cyclic photophosphorylation (15). from this habitat are thermoduric, but not ther- The structural and functional changes that mophilic; their diurnal period of growth is con- accompany the development of the heterocyst sequently confined to the early morning, devel- confer on it the ability both to maintain nitro- opment being arrested by the rise of tempera- genase in an active state, and to meet by photo- ture during the day (19). chemical means the large ATP requirement for the reduction of N 2 to ammonia. However, the 5.2. Nitrogen fixation heterocyst, having lost the ability to assimilate Many cyanobacteria can fix atmospheric ni- CO2, can no longer synthesize the organic com- trogen. This property is absent from all photo- pounds required both as electron donors for the trophic eukaryotes, with the exception of the reduction of Nz, and for the synthesis of organic various plant groups which have entered into nitrogenous compounds from ammonia. For symbiotic relations with N2-fixing prokaryotes. these, the heterocyst is dependent on the intra- Nitrogen-fixing cyanobacteria thus dominate cellular transfer of organic metabolites formed the microbial phototrophic population in envir- through CO2 fixation in the neighboring vege- onments where the supply of combined nitro- tative cells (54). In turn, the vegetative cells gen is limiting (49). obtain from the heterocyst their combined ni- At first sight, oxygenic photosynthesis and trogen supply. The form in which nitrogen is nitrogen fixation appear to be incompatible transferred from the heterocyst to the adjacent metabolic processes, since nitrogenase, the en- vegetative cells of the filament has been identi- zyme which catalyzes the reduction of N2 to fied very recently as glutamine (28). According- ammonia, is rapidly and irreversibly denatured ly, in this large sub-group of cyanobacteria, the by low partial pressures of oxygen (13). Yet all phys!ological incompatibility between nitrogen cyanobacteria which produce heterocysts are fixation and oxygenic photosynthesis has been active aerobic nitrogen-fixers (48). For many circumvented by a cellular separation of the decades, the heterocyst remained a biological two activities, achieved through a unique type enigma: it was a structurally specialized cell to of cellular differentiation. which the botanists were unable to assign any As recently as 1969, heterocystous cyanobac- clear-cut function (27). Through work in several teria were the only cyanobacteria known to fix laboratories, this puzzle has been largely solved nitrogen. Aerobic nitrogen fixation, permitting during the past ten years. photoautotrophic growth in a medium free of Heterocyst formation is repressed, as a rule combined nitrogen, has since been demon- completely, in cultures grown with an excess of strated in a few unicellular cyanobacteria, all nitrate or ammonia. If such filaments of vege- belonging to the genus Gloeothece (55,34). tative cells are transferred to a medium lacking These organisms must possess a mechanism a combined nitrogen source, from 5 to 10% of which permits the maintenance of an active

94 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 R. Y. STANIER:Cyanobacteria nitrogenase in vegetative cells which are en- major modes of cyanobacterial energy-yielding gaged in the performance of oxygenic photo- metabolism heretofore recognized - oxygenic synthesis, but its physiological basis is not photosynthesis and oxygen-linked respiration - known. preclude the expression of the property by In 1970, STEWARTand LEX (50) showed that these cyanobacteria. As described below, this the oscillatorian cyanobacterium Plectonema paradox has now been resolved. can synthesize nitrogenase, even though it is incapable of growing aerobically at the expense 5.3. Facultative anoxygenic photosynthesis of N2. By placing filaments in a nitrogen-free A mode of cyanobacterial photosynthesis medium in the light under an anaerobic atmos- which does not lead to oxygen production has phere, renewed at intervals, they were able to been recently discovered by COHEN (7), as the induce detectable nitrogenase activity. This ac- outcome of a detailed ecological analysis of a tivity rapidly disappeared when the filaments shallow hypersaline lagoon, the Solar Lake, were exposed to air; the nitrogenase of Plec- situated on the Gulf of Eilat. In the winter, stra- toma is thus oxygen-sensitive in vivo. tification produces an anaerobic layer with a RIPPKA (35) has recently extended the search steep H2S gradient, inhabited by three popula- for non-heterocystous cyanobacteria able to tions of photosynthetic prokaryotes. In the synthesize nitrogenase, by the development of a upper parts of the anaerobic layer, purple and technique which permits the synthesis of the form two largely distinct enzyme in the complete absence of molecular horizontal layers. Oscillatorian cyanobacteria oxygen. The cultures, grown with a combined are present at all levels, but form a dense mat at nitrogen source, are pre-incubated aerobically, the bottom, where the H2S concentration is in the light in the presence of CO2, but in the maximal. This is a complete reversal of the absence of combined nitrogen. Under these usual situation in a stratified lake, where oxy- conditions, since general biosynthesis is pre- genic phototrophs normally occur in the aero- vented by nitrogen starvation, the cells accumu- bic surface layer. Cohen accordingly suspected late a massive glycogen reserve. The cultures that the cyanobacteria in this particular eco- are then placed under anaerobic conditions system might be engaged in anoxygenic photo- (argon-acetylene atmosphere) in the light, in synthesis, a hypothesis confirmed by isolating the presence of 10-5 M DCMU, which abolishes the predominant species, Oscillatorian limne- photosystem II activity and thus prevents en- tica, and studying its physiological properties dogenous oxygen production. Under these (8). Like all other cyanobacteria, this organism special conditions, strains which possess the can develop as an oxygenic photoautotroph. necessary genetic information can both synthe- Exposure of filaments to a concentration of size nitrogenase, and express its activity 4mM- H2S totally inhibits photosystem II activ- (measured by acetylene reduction). The cellular ity, and immediately arrests CO2 assimilation. glycogen reserve provides a source of carbon However, after a short lag, the assimilation of and of reducing power; and ATP can be synthe- CO2 is resumed, now accompanied by a stoi- sized by cyclic photophosphorylation. Such chiometric oxidation of H2S to elemental sulfur, experiments have shown that approximately which is deposited outside the filament. Since half the oscillatorian and pleurocapsalean this occurs even in the presence of DCMU, it strains in our collection are potential nitrogen- cannot be attributed to a resumption of photo- fixers, although none of them can maintain ni- system II activity. Sulfide evidently has two trogenase activity when exposed to air. effects on the metabolism of this organism, one The widespread occurrence of the genetic immediate and one delayed. The immediate information for nitrogen fixation among non- effect is to act as a selective inhibitor of photo- heterocystous cyanobacteria revealed by this system II, converting the photosynthetic ma- survey is difficult to explain, unless the property chinery into a phenocopy of the purple or green can achieve effective phenotypic expression bacterial one. The delayed effect is the induc- under natural conditions. However, the two tion of a sulfide-oxidizing enzyme system,

Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977 95 R. Y. STANIER: Cyanobacteria which makes possible the resumption of photo- which had very largely escaped previous detec- synthetic CO2 assimilation. The same group has tion, when ecological studies were based on a since found (23) that several other non-hete- purely descriptive analysis of field populations. rocystous cyanobacteria respond similarly to sulfide, although the sulfide concentrations which permit anoxygenic photosynthesis are considerably lower than those tolerated by Oscillatoria limnetica. REFERENCES This important discovery clarifies certain hi- 1. ALLEN,M. B.: The cultivation of Myxophyceae. therto completely obscure aspects of cyanobac- Arch. Mikrobiol. 17, 34-53 (1952) terial ecology. Many illuminated natural micro- 2. BEAUCLERK, A. A. D. & A. J. SMITH: Carbo- environments either fluctuate periodically be- hydrate uptake by cyanobacteria. Abstr. 2nd tween the aerobic and anaerobic states, or are International Symposium on Photosynthetic situated on a sharp boundary line between the Prokaryotes Aug. 1976. University of Dundee, Scotland (in press) oxygenated and the anaerobic, sulfide-rich re- 3. BIGGINS,J.: Respiration in blue-green algae. J. gions of the biosphere; and oscillatorian cyano- Bact. 99,570-575 (1969) bacteria are frequently abundant in these habi- 4. CASTENHOLZ, R. W. : The thermophilic cyano- tats (for example, muddy areas in the intertidal phytes of Iceland and the upper temperature zone). The ability to alternate readily between limit. J. Phycol. 5,360-368 (1969a) the performance of oxygenic and anoxygenic, 5. CASTENHOLZ, R. W.: Thermophilic blue-green sulfide-dependent photosynthesis, a metabo- algae and the thermal environment. Bact. Rev. lic capacity confined to cyanobacteria, confers 33,476-504 (1969b) a unique advantage in such micro-environ- 6. CHEtmG, W. Y. & M. GIBBS: Dark and photo- ments. The aerobic facet is closed to purple and metabolism of sugars by a blue green algae Toly- pothrix tenuis. Plant Physiol. 41, 731-737 (1966) green bacteria, while the anaerobic sulfide-rich 7. COHEN, Y.: Dynamics of prokaryotic photosyn- facet is closed to eukaryotic alagae. Moreover, thetic communities of the Solar Lake. Thesis, when growing in the anaerobic facet, cyanobac- Hebrew University, Jerusalem (1975) teria which do not produce heterocysts can ex- 8. COHEN, Y., E. PADAN & M. SmLO: Facultative press the genetic capacity to fix nitrogen. In this anoxygenic photosynthesis in the cyanobacterium type of micro-environment, oscillatorian cyano- Oscillatoria limnetica. J. Bact. 123, 855-861 (1975) bacteria may well play an important and hi- 9. COHEN-BAZIRE,G: The photosynthetic apparatus therto unsuspected role as agents of nitrogen of prokaryotic organisms. In ,Biological Ultra- fixation. structure: The Origin of Cell Organelles,. Harris, P. J. ed., 30th Annual Biology Colloquium, Oregon state University Press, U. S. A., pp. 65-90 (1971) 6. CONCLUDING REMARKS 10. COHEN-BAZIRE, G. & R. KUNISAWA:The fine structure of Rhodospirillum rubrum. J. Cell Biol. As E. C. HANSEN showed almost a century 16,401-419 (1963) ago, many useful insights into problems of l l. COHEN-BAZIRE,G., N. PFENNIG ~: R. KUNISAWA: microbial ecology can be derived from experi- The fine structure of green bacteria. J. Cell Biol. mental studies on pure cultures, performed 22, 207-225 (1964) under controlled conditions. The development 12. COHEN-BAZIRE,W. R. SISTROM & R. Y. STANIER: of research on cyanobacteria, following the Kinetic studies of pigment synthesis by non-sulfur very tardy application to these organisms of purple bacteria. J. Cell Comp. Physiol. 49, 25-68 pure methodology, provides a most instructive (1957) 13. illustration of Hansen's working principle. In DALTON, H. & L. E. MORTENSON:Dinitrogen (Nz) fixation (with a biochemical emphasis). Bact. the past ten years, we have at last begun to Rev. 36,231-260 (1972) understand the physiological and metabolic fac- 14. DOEMEL, W. N. & T. D. BROCK: The upper tem- tors which account for the exceptionally wide perature limit of Cyanidium caldarium. Arch. natural distribution of the cyanobacteria, and Mikrobiol. 72, 326-332 (1970)

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98 Carlsberg Res. Commun. Vol. 42, p. 77-98, 1977