JOURNAL OF BACTERIOLOGY, Sept. 1975, p. 855-861 Vol. 123, No. 3 Copyright i 1975 American Society for Microbiology Printed in U.SA.

Facultative Anoxygenic in the Cyanobacterium limnetica

YEHUDA COHEN,* ETANA PADAN, AND MOSHE SHILO Department ofMicrobiological Chemistry, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 9 May 1975

An isolate from H2S-rich layers of the Solar Lake, the cyanobacterium Oscillatoria limnetica, exhibits both oxygenic and anoxygenic photosynthesis. It can use Na2S as an electron donor for CO2 photoassimilation (photosystem I supplies the energy) in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 700-nm light. A stoichiometric ratio of approximately 2 is observed between the Na2S consumed and the photoassimilated CO2. The anoxygenic phototrophic capability of this cyanobacterium explains its growth in nature in high sulfide concentrations and indicates a selective advantage.

That ("blue-green algae") The role of H2S in cyanobacterial metabolism occur in anaerobic habitats has long been is of particular interest with respect to the known (4-6, 13, 14, 22, 23, 27, 28). Furthermore, evolution of photosynthesis. Cyanobacteria are growth under reducing conditions by species of among the most ancient organisms on earth, Oscillatoria (22, 23, 29), Simploca, Mastigo- appearing in middle Precambrian rock and cladus (29), Anacystis nidulans (17), Gleocapsa perhaps even earlier (26). Thus, cyanobacteria (34), and Anabaena flos-aquae (32) has been were the earliest phototrophic organisms having recorded. oxygenic photosynthesis like eukaryotic algae In most of the anaerobic habitats containing and higher plants. Nevertheless, they have a cyanobacteria, H2S was demonstrated to be the prokaryotic cellular structure like other bacte- main reducing component. Hence, the question ria. Therefore, it is generally agreed (5) that the arises whether the cyanobacteria are capable of cyanobacteria may represent the evolutionary anoxygenic photosynthesis using electron do- link between anoxygenic photosynthesis (typi- nors such as H2S. Hinze (13) and Nakamura cal of ) and oxygenic photosynthesis (22) reported the presence of sulfur granules (typical of higher plants). However, so far there within the cells of H2S-grown Oscillatoria fila- has been no conclusive report on anoxygenic ments, similar to that observed in some photo- photosynthesis among cyanobacteria. trophic sulfur bacteria. Stewart and Pearson We recently isolated pure cultures of several (32) could not confirm this but found that H2S cy4nobacteria from an anaerobic H2S-rich layer disappears from the medium during growth, of the hypolimnion of the Solar Lake located although no growth occurred when photosystem near the Gulf of Elat (20). The highest rates of II was totally inhibited by 3-(3,4-dichlorophe- primary production ever reported for a nonpol- nyl)-1,1-dimethylurea (DCMU). These authors luted water body were measured in this layer of suggested that it is the photolysis of water by the Solar Lake and have been ascribed mainly photosystem II that is essential for the growth of to the activity of the cyanobacteria. Anabaena flos-aquae in the presence of H2S. In the present work we investigated the role of Nevertheless, when photolysis of water is re- H2S in the matabolism of one of these isolates, duced but not inhibited completely, H2S may identified as Oscillatoria limnetica. act simultaneously with water as a source of elec- trons. Furthermore, they suggested that H 2S MATERIALS AND METHODS is oxidized mainly by the photosynthetically Cyanobacteria strain and culture conditions. 0. evolved oxygen, thereby reducing the oxygen limnetica was isolated in pure culture from samples tension that seems to be deleterious to several taken from the H,S-rich lower hypolimnion of Solar cyanobacteria. Preliminary notes indicate the Lake. The culture medium used was similar to that possibility of H2S serving as an electron donor designed for Chromatium by K. J. Eimhjellen (Tech- for CO2 photoassimilation in cyanobacteria (9). nical University of Norway, Trondheim, Norway) and 855 856 COHEN, PADAN, AND SHILO J. BACTERIOL. consists of the following major elements (grams per Sulfide measurements. Determinations of sulfide liter): KH2PO2, 0.33; NH4CL, 0.33; MgCl2-6H20, ions was carried out by using an Orion Research 0.33; KCI, 0.33; NaHCO,, 1.50; Na2S 9H2O, 0.75; (Cambridge, Mass.) setup including a sulfide-silver- CaCl2-2H2O, 0.33; vitamin B12, 10-I M; and the trace specific electrode (model 94-16), a double-junction elements SL-4 described by Pfennig and Lippert (25). reference electrode (model 90-02-00), an ion analyzer The sulfide concentration of this medium was varied (model 801), and a Goerz recorder (model Mini in the different experiments as noted. Solar Lake GOAR, Vienna, Austria). Standard curves were ob- water of 96.7 o/oo chlorinity and 174.1 o/oo salinity tained by volumetric titration of AgNO,. Total con- was used at the suspension water. The composition of centration of the sulfide ion was calculated according this water corresponds to standard seawater concen- to the calibration curves provided by Orion, taking trated 5.27 times (7). The final pH was adjusted to 6.8 into account the ionic strength, the pH values, and with HC1. the temperature of the medium. Liquid cultures were grown in completely filled, The experimental system for simultaneous deter- glass-stoppered bottles and incubated under continu- mination of sulfide consumption from the medium ous illumination provided by white fluorescent lamps and CO2 photoassimilation consisted of the growth (4,300 K, 20 W, incident intensity of 5 x 102 to 7 x 103 medium containing 200 mM tris(hydroxymethyl)- ergs/cm2 per s) at 35 C. Cell density was followed by aminomethane buffer (pH 7), 1.2 mM Na2S, and determination of cell proteins after ethanol washing of 22 mM NaHCO,, at a cell density of 330 Mg of cell the cells to remove sulfur by the method of Lowry et protein per ml. The sulfide electrode was introduced al. (21). When the inoculum (5%) consisted of a 4- to to the reaction mixture (30 ml) in an air-free sealed 6-day-old culture, the logarithmic phase of growth cell, and the sulfide ion concentration was recorded lasted 7 days, after a lag period of 48 h. continuously. After a 1-h preincubation at 35 C with CO2 photoassimilation measurements. When stirring and illumination (500-W reflector flood tung- CO2 photoassimilation was determined continuously, sten lamp, 10' ergs/cm2 per s) and DCMU (10-i M), the experimental system consisted of: the growth NaH14CO. (0.14 MCi/Mmol) was added. Aliquots (1 medium at various sulfide concentrations supple- ml) were removed anaerobically with a syringe at the mented with NaHCO, (22 mM) and NaHl4CO, indicated intervals for determination of CO2 photoas- (Amersham, England) at a final specific activity of similation. 0.14 gCi/pmol, and logarithmic-phase algal cells DCMU was obtained from DuPont; the Na2S used at a cell density of 8 to 20 gg of protein per ml. was analytical grade (BDH, England). The cell suspensions in completely filled 25-ml flasks were incubated with shaking at 35 C and illumi- RESULTS nated by tungsten lamps (60 W, incident intensity of 2.104 ergs/cm2 per s). Aliquots (1 ml) of the cell The 0. limnetica isolate photoassimilates suspensions were filtered on glass filter paper (What- CO2 in the presence of Na2S in a pattern that is man GF/C) and washed with 40 ml of trichloroacetic dependent on the sulfide concentration. The acid (10%, 4 C). The radioactivity within the cells rate in the sulfide-free medium (control) is was counted in a gas flow counter (Nuclear-Chicago, about 3,000 nmol of CO,/mg of protein per h, model 181B). whereas in the presence of 0.5 mM Na2S this When the rate of CO, photoassimilation was deter- mined at different wavelengths of actinic light, sus- rate is about 1,000 nmol/mg of protein. Increas- pensions of algal cells (8 Ag of cell protein per ml) were ing the sulfide concentration to 4 mM Na2S prepared in the same reaction medium but without increased the rate of the photoassimilation to the radioactive label in stoppered vials (5 ml), and almost that of the control although after a lag preincubated for 1 h under the same experimental period of 0.5 to 1 h. conditions. NaH '4O,2 was then introduced to give a DCMU, the specific inhibitor of photosystem final specific activity of 1.14 MCi/Mmol, and the II in plants (1) as well as in cyanobacteria (24, experiment was conducted for 5 min at the specific 33), at a 10-7 M concentration completely wavelength (35 C; stirring), after which the cellular radioactivity was determined as described above. blocks CO2 photoassimilation in the system Illumination was provided by a 100-W tungsten-halo- lacking Na2S (Fig. 2). However, an increasing gen photographic lamp (Atlas P1/15,). The light was Na2S concentration allows for progressively in- filtered through Baird Atomic interference filters and creased photoassimilation even in the presence blocked to infinity, peaking at 580 nm (15-nm half- of higher concentrations of DCMU. Whereas band width) and 700 nm (25-nm half-band width). 10-6 M DCMU completely blocks photoassimi- The short-wavelength light was prefiltered by 5 cm of lation in the sulfide-free system, this concentra- a saturated solution of CuS04, and the long- tion has no effect on the reaction when 2.1 mM wavelength light was prefiltered by a sharp cut-off Na2S is present. Corning H. R. 2-60 filter. The intensity of the actinic light, varied by using a powerstat, was measured by a To rule out the possibility that high Na2S Yellow Springs Instrument radiometer, model 65. concentrations cause irreversible inactivation of The extent of light absorption by the algae was the capacity of DCMU to inhibit the photoas- measured by the method of Shibata et al. (30) in a similation, we tested the activity of DCMU in Cary 14 spectrophotometer in a cuvette containing systems in which the sulfide concentration was the reaction mixture used in the assays. reduced after a period of incubation (Fig. 3). In VOL. 123, 1975 FACULTATIVE ANOXYGENIC PHOTOSYNTHESIS 857

2500k

3.3 xld 6 DCMU C 2.1mM No2S cm ° 2000

C E 8000 / OmM Na2S

0. 0 Lo 1500 01~~~~~~~~~~ to E )F N~~ ~ ~ ~ ~ ~ ~~M% O 6000- 21mMN0S c 0~ .?1 1000 - 0~~~~~~~~~- E c /

0 0 u

/I1- . I . I I . . I 1 2 3 4 5 6 78 9

T i m e (h rs) 1 2 3 4 T ime (hrs) FIG. 3. Reversibility of DCMU inhibited CO2 photoassimilation of 0. limnetica in the presence of FIG. 1. Photoassimilation of CO2 by 0. limnetica in cells (20 Mg of protein per ml) were the presence of Na2S. For continuous determination Na,S. Oscillatoria of CO2 photoassimilation with NaH"4C03, Oscilla- prepared in the experimental system containing 2.1 mM Na,S described in the legend to Fig. 1, but light toria cells at a cell density of 20 ,ug of cell protein per ml served in the experimental system. The incubation was provided by a battery of cold white (4,300 K, 20-W) fluorescent lamps (5.108 ergs/cm2 per s), which was at 35 C with shaking and light was provided by rate battery of 60- W tungsten lamps (2.104 ergs/cm2 per s). accounts for the lower of photoassimilation when to 1. was Initial concentrations of Na2S indicated on the graph compared Fig. DCMU (10- 6 M) added, the was 4 (arrow) half of were determined at the beginning of the experiment system incubated for h, and then was in Na2S by using an Orion model 94-16 sulfide ion electrode. the reaction mixture diluted threefold medium, giving a DCMU concentration of3.3.10- 6 M, Symbols: 0, sulfide-free control; x, 0.5 mM Na2S; , whereas the other half was diluted into sulfide-free 1.4 mM Na2S; A, 2.1 mM Na2S; A, 4.0 mM Na2S. medium, giving final concentrations of 0.5 mM Na2S (concentration corrected according to measurements) 100 ol or and 3.3.10-6 M DCMU.

80 _ the initial reaction mixture containing 10-8 M Z 0 DCMU and 2.1 mM Na2S, the photoassimila- showed 10% inhibition, which 0 tion reaction only 0 accords with Fig. 2. After 4 h, half the reaction mixture was diluted threefold in the medium ._o . containing Na2S; this reduced the final DCMU 0 concentration to 3.3 x 10-6 M. The CO 2 0. v10 \ continued in this reaction 0 photoassimilation mixture at the same rate. At the same time, the other half of the reaction mixture was diluted in Na2S-free medium, giving a final concentration DCM U (M) of 0.5 mM Na2S and 3.3 x 10-' M DCMU. It is FIG. 2. Effect of Na S concentration on the DCMU evident from Fig. 3 that CO2 photoassimilation inhibition of the CO2 photoassimilation reaction in 0. is inhibited (90%) immediately after the shift to limnetica. Experimental system as in Fig. 1. The rates of CO2 photoassimilation obtained for each of the the lower Na2S concentration. This accords corresponding Na2S concentrations are expressed as a with data in Fig. 2, which show that this DCMU percentage of the corresponding maximal rates with- concentration indeed inhibits photoassimilation out DCMU (see Fig. 1). Symbols as in Fig. 1. at the low sulfide concentration. Similar shifts 858 COHEN, PADAN, AND SHILO J. BACTERIOL. of Na2S concentrations, but in the presence of a lower DCMU concentration (10-6 to 3.3 x 10- .z M), yielded 85% inhibition of the photoassimi- cx lation rate with 0.5 mM Na2S, whereas no inhibition was observed with 2.1 mM Na2S (not shown). In control systems (not shown), similar shifts of Na2S concentrations without DCMU had no effect on the relation between the photoassimilation rates expected from Fig. 1. It can be concluded that DCMU remains com- pletely active after incubation in the presence of Na2S. It thus appears that photosystem II is inhibited by DCMU even at a high Na2S 60 90 120 concentration and that an alternative photoas- T m e (min) similation reaction may be operative, in which FIG. 4. Relation between C02 photoassimilation Na2S serves as the electron donor in place of and sulfide ion consumption by 0. limnetica. 0. water. limnetica (330 1sg of protein per ml was suspended Figure 4 shows the kinetics of CO2 photoas- in the growth medium containing 22 mM NaHCO,, similation and sulfide depletion at initial con- 200 mM tris(hydroxymethy0)aminomethane buffer centrations of 1.2 mM Na2S and DCMU (10-5 (pH 7), and 1.2 mM Na,S. The sulfide-specific M). As the photoassimilation of CO2 proceeds, electrode was introduced to the reaction mixture the sulfide ion concentration is reduced. Both (30 ml) in an air-free sealed cell, and the sulfide reactions proceed at such rates (at 30 to 60 min) ion concentration was recorded continuously (un- broken line). After 1 h of preincubation at 35 C that the ratio between sulfide consumption and with stirring and illumination (500-W reflector flood photoassimilation is 2.0 in this experiment and tungsten lamp, 10' ergs/cm2 per s) with DCMU 1.6 to 2.5 in three other experiments (not (10-5 M), NaH14CO3 (0.14 MCi/Mmol) was added, and shown). CO2 photoassimilation was arrested 1-ml aliquots were removed anaerobically by a syringe when the sulfide ion concentration dropped to for determination of CO2 photoassimilation (0). When less than 300 AM. Photoassimilation recom- the sulfide concentration dropped to 12M&M, Na,S was menced at an even higher rate upon addition of added again (arrow) to bring it up to the initial fresh Na2S. concentration. The demonstration that Na2S supports CO2 photoassimilation in 0. limnetica in the pres- ence of DCMU implicates photosystem I in driving the light reactions required for the CO, photoassimilation. To test the functioning of photosystem I and II in the presence of Na2S (2.1 mM) and to rule out reversible inactivation of DCMU by sulfide, we determined the pho- toassimilation of CO2 at two wavelengths: 580-nm light to excite mostly phycocyanin and, thus, both photosystems; 700-nm light to excite preferentially photosystem I-localized chloro- phyll. This experiment was run under limiting light intensity so that the rate of CO2 photoas- similation would be linearly related to the actinic light intensity. In addition, low cell lo (PJE /cm/min ) densities (with stirring) were maintained to FIG. 5. Photoassimilation of CO, in the presence or avoid a "self-shading" effect. From Fig. 5, it is absence of Na,S (2.1 mM) at 580- or 700-nm incident evident that 0. limnetica photoassimilates CO2 light. 0. limnetica (8 gg of cell protein per ml) was at 700 nm when Na2S is present, whereas the preincubated in 5-ml vials for 1 h under the conditions rate of photoassimilation at this wavelength is of continuous photoassimilation determination, but without radioactive label. NaH"4CO, (1.14 MCi/Mmol) almost negligible in the absence of Na2S. Con- was added, and actinic light, either 580- or 700-nm sidering the low CO2 photoassimilation (the wavelength, was provided by filtered light. After 5 only electron acceptor in the system), we can min, the cell radioactivity was determined (0, 580 assume that 02 evolution is very low under nm; A, 700 nm; 0, A, sulfide-free medium; 0, A, these conditions. Thus, actinic light, which Na,S medium). VOL. 123, 1975 FACULTATIVE ANOXYGENIC PHOTOSYNTHESIS 859 preferentially activates photosystem I, supports the long wavelength (above 680 nm) in exciting photoassimilation of CO2 in the presence of photosystem II and, consequently, inability to Na,S. However, when 580-nm light served as extract electrons from water. However, when the exciting light, photoassimilation proceeds Na,S served as the electron donor to the pho- both in absence and presence of Na,S. toassimilation reaction, the rates at 700 nm To compare the rates of CO, photoassimila- were drastically increased, giving a ratio be- tion at the different wavelengths, we evaluated tween rates with the corresponding electron the absorbed intensity. Absorption spectra of donors (Na,S-H,O) of 11:19. It can be con- the pigments of whole cell of 0. limnetica were cluded, therefore, that 0. limnetica can pho- determined in the reaction mixtures by the toassimilate CO2 with Na,S as the electron method of Shibata et al. (30), which minimizes donor and photosystem I as the energy donor. artifacts due to light scattering. This measure- ment showed that the absorption at 580 nm is 1.55 times higher than at 700 nm. This factor DISCUSSION was then used for comparing the photoassimila- The cyanobacterium 0. limnetica tested here tion rates under the same absorbed quanta. was isolated from the lower part of the hypolim- Thus, rates of photoassimilation at 580 nm nion of the Solar Lake, where it is the major corresponding to incident actinic intensity (IO) component of the massive cyanobacterial popu- of 0.2 gE/cm2 per min were compared to the lation developing in conditions of high H,S rates of incident actinic intensity of 1.55 x 0.2 concentration under two discrete phototrophic MAE/cm2 per min at 700 nm (Table 1). Although bacterial populations (20). The results indicate this calculation does not give the absolute that, although this cyanobacterial strain has absorbed intensity, it allows comparison of the the typical oxygenic photosynthesis, it can also rates at the two wavelengths. photosynthesize anoxygenically by using Na.S When 580-nm light served as the exciting as the electron donor with the operation of light, the ratio of photoassimilation rates in photosystem I alone. Although the detailed presence and absence of Na2S ranged from 0.55 mechanism of the observed photooxidation of to 1.0 (Table 1). When water served as the Na,S is still unknown, it is apparent that there electron donor, the photoassimilation rate in is a similarity to sulfide utilization by photo- 700-nm light was very low in comparison to trophic sulfur bacteria. Our experiments showed rates with 580-nm light. Thus, the ratio of the that when photosystem II is inhibited in the rates at the two wavelengths (700:580) amounts presence of DCMU or is not excited by 700-nm to 0.08 to 0.095. These results agree with the light, so that there is no electron flow from the "red-drop" phenomenon observed in cyanobac- water, the photoassimilation reaction proceeds terial whole cells and thoroughly studied in in the presence of Na,S. The long-wavelehgth- green algae and chloroplasts (12). This red-drop light-driven photoassimilation is as efficient as phenomenon demonstrates the low efficiency of that of the short wavelength (580 nm). TABLE 1. Comparison of the rates of CO2 photoassimilation supported by 580- or 700-nm light and an Na,S or water electron donora Electron donor Wavelength Io (nmol of CO,/mg of protein per h) Proportion of rates (nm) (oE/cm' per min) Na2S:water Water Na,S 580 0.2 200, 184, 123 110, 161, 125 0.55, 0.9, 1.0 700 0.31 17, 15, 13 180, 250, 248 11.0, 16.7, 19.1 Proportion of rates 700:580 0.085, 0.08, 0.11 1.6, 1.6, 2.0 a Experimental systems as in Fig. 5 (each repeated three times). To compare the rates of CO, photoassimilation at the different wavelengths, the absorbed intensity was evaluated. The absorption spectra of the pigments of the algal cells, determined in the reaction mixtures according to Shibata et al. (30), showed that the absorption at 580 nm is 1.55 times higher than at 700 nm. Thus, incident light intensity (IO) of 0.2 ME/cm2 per min at 580 nm is equivalent to I. of 0.31 uE/cm' per min at 700-nm light with respect to the absorbed intensity. In addition, these experiments were run at a very low cell density with stirring and under limiting light intensity so that 4"self-shading" is avoided and the rate of CO2 photoassimilation would be linearily related to the actinic light intensity. 860 COHEN, PADAN, AND SHILO J. BACTERIOL. As expected for an electron donor, the concen- preventing the detrimental accumulation of tration of Na2S in the medium decreases at a oxygen in the medium during oxygenic photo- constant rate as CO2 photoassimilation pro- synthesis. Sulfide may provide electrons for the ceeds linearly with time. There is a stoichiomet- CO2 photoassimilation by the Anabaena only ric relationship of about 2 between the con- when the reducing power from photolysis of sumed Na2S and the photoassimilated CO2. water is lowered, but not completely inhibited. This stoichiometric ratio is often observed with In addition to the anoxygenic photosynthesis, phototrophic bacteria (31). It should be added 0. limnetica is capable of oxygenic photosyn- that extracellular granules resembling the ele- thesis and can readily shift from one to the mentary sulfur granules of the sulfide-oxidizing other. It is known that certain strains of green phototrophic bacteria (Chlorobiaceae and some algae and of cyanobacteria can carry out pho- Chromatiaceae) are observed accumulating on toreduction of CO2 with H2 in addition to their the outside of these algal cells in nature and in oxygenic photosynthesis (2, 3, 9-11, 15). There cultures at high-H2S conditions. In addition, are also indications that H2S may serve as an B.B. Jorgensen (Aarhus University, Denmark) electron donor for photoreductions in several has found that elemental sulfur is the ultimate species of green algae and cyanobacteria (9, product of H2S oxidation in this strain. 17-19, 23, 32). CO2 photoassimilation proceeds at the same Experiments conducted on green algae (15, rate with 3 to 4 mM Na2S as in the control 16) showed that, in the photoreduction with (without sulfide), whereas only 30% of this hydrogen, photosystem II may be at least partly photoassimilation is observed with 0.5 mM active, in addition to photosystem I. Both Na2S (Fig. 1). It is possible that at the lower photosystems were shown to be involved in sulfide concentration photosystem II is already photoreduction with H2S in certain green algae inhibited, whereas this concentration is not yet and cyanobacteria (18, 19, 32). In any case, the sufficient to allow efficient electron flow. The photoreduction with hydrogen in green algae lag (0.5 to 1 h) observed for the high-sulfide occurs only under special conditions (weak light system may indicate that a period of adaptation preceded by a period of dark anaerobic adapta- is needed for the shift to sulfide as an electron tion), and no growth occurs during the photore- donor. The similar initial lag period observed in duction (11). The photoreduction recorded with the presence of DCMU is reduced upon subse- H2S both in green algae and cyanobacteria quent supplementation of Na2S (Fig. 4). The seems also be to limited to special concentration of Na2S that starts limiting the conditions-when electron flow from photosys- rate of the photosystem I-driven photoassimila- tem II is reduced but not completely inhibited tion reaction (in the presence of DCMU) is (18, 32). Hence, the strain of 0. limnetica around 300 gM. Re-addition of Na2S restores studied here seems to be the first organism immediately and completely the photoassimila- reported to grow with either type of photosyn- tion reaction. Concentrations of Na2S (1.2 to 4 thesis. This represents a selective advantage mM) that can sustain anaerobic CO2 photoas- since when the short-wavelength light that similation by 0. limnetica were found in its activates photosystem II is not limiting it can natural ecosystem. The light penetrating to the use water as the electron donor with operation lower hypolimnion of the Solar Lake, where this of both photosystems. This cyanobacterial strain thrives, consists mainly of long- strain is thus not restricted to special ecosys- wavelength light above 700 nm. This long- tems where specific electron donors are availa- wavelength light was shown experimentally to ble, like those of other phototrophic bacteria, be as efficient in extracting electrons from Na2S and is also found under oxygenated conditions as the short-wavelength light (580 nm) was in during holomixis of the Solar Lake in summer extracting electrons from water (Table 1). when phototrophic sulfur bacteria disappear. We have observed growth of 0. limnetica On the other hand, when mainly long- under high concentrations of Na2S (2 mM) and wavelength light is available and when other in the presence of DCMU. We suggest that it is oxygenic phototrophic organisms cannot sur- the capacity to Na2S as an electron donor with vive, the 0. limnetica thrives in the sulfide-rich the operation of photosystem I which accounts environment. for the successful growth of this alga under high In this strain of 0. limnetica, both photosys- sulfide concentrations in nature. Stewart and tems are fully developed, and photosystem I can Pearson (32) suggest another mechanism for the still support life in this cyanobacterium effi- growth of some cyanobacteria in the presence of ciently and independently of photosystem II sulfide. They propose that the main role of H2S when reduced electron donors are available. In in the growth of Anabaena flos-aquae is in this connection it may be noted that photosys- VOL. 123, 1975 FACULTATIVE ANOXYGENIC PHOTOSYNTHESIS 861 tem I of cyanobacteria has been demonstrated mechanisms of green plants. National Academy of Science U.S.A., Washington, D.C. to function in vivo independent of photosystem 13. Hinze, G. 1903. Uber Schwefeltropfen im innern von II. Photosystem I of Plectonema boryanum Oscillarian. Ber. Desch. Bot. Ges. 21:394-398. supports the development of cyanophages at the 14. Kaplan, I. 1955. Quoted by L.G.M. Baas-Becking and same efficiency as both photosystems (24). E.J.F. Wood. 1955. Biological processes in the eustua- rine environment. II. Ecology of the sulphur cycle. Heterocysts, the differentiated cells of cyano- Proc. Acad. Sci. Amst. B. 58:173-181. bacteria thought to be responsible for the aero- 15. Kessler, E. 1957. Stoffwechselphysiologische Untersu- bic nitrogen fixation, have only photosystem I chungen an Hydrogenase enthaltenden Grunalgen. I. (8). Uber die Rolle des Mangans bei Photoreduktion und Because the 0. limnetica isolate displays Photosynthese. Planta 49:435-454. 16. Kessler, E. 1968. Effect of hydrogen adaptation on both oxygenic and anoxygenic types of photo- fluorescence in normal and manganese deficient algae. synthesis and can shift from one to the other, it Planta 81:264-273. is tempting to speculate on its evolutionary 17. Knobloch, K. 1966. Photosynthetische Sulfia. lation position. It is possible that this cyanobacterium griuner Pflanzen. I. Mitteilung. Planta 70:73-b. 18. Knobloch, K. 1966. Photosynthetische Sulfidoxydation represents a secondary specialization, back to- gruiner Pflanzen. II. Wirkung von Stoffwechselinhibi- ward anoxygenic photosynthesis typical of bac- toren. Planta 70:172-186. terium. On the other hand, it is possible that it 19. Knobloch, K. 1969. Sulfide oxidation via photosynthesis is the missing link between the two types of in green algae, p. 1032-1034. In H. Metiner (ed.), Progress in photosynthesis research, vol. 11. Interna- photosynthesis. tional Union of Biological Sciences, Tubingen. ACKNOWLEDGMENTS 20. Krumbein, W. E., and Y. Cohen. 1974. Biogene, klas- tische und evaporitische Sedimentation in einem We are indebted to Norbert Pfennig (Institut fur Mikrobi- mesothermen monomiktischen ufernahen See (Golf ologie, G8ttingen) for his helpful suggestions and to M. Avron von Aqaba). Geol. Rundschau 63:1035-1065. (Weizmann Institute of Science, Rehovot) for his discussions 21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. and critical appraisals of this work. We thank I. Dor (Hebrew Randall. 1951. Protein measurement with the Folin University) for classification of the cyanobacterium and B. phenol reagent. J. Biol. Chem. 193:265-275. Golek for help in the preparation of the manuscript. 22. Nakamura, H. 1937. Uber das Auftreten des Schwefelkii- This study was supported by a grant from the Deutsche gelchens imm Zellinnern von einigen niederen Algen. Forschungsgemeinschaft. Bot. Mag. 51:529-533. 23. Nakamura, H. 1938. Uiber die kohlensaureassimilation LITERATURE CITED bei niederen Algen in Anwesenheit des Schwefelwas- 1. Avron, M. 1967. Mechanism of photoinduced electron serstoffes. Acta Phytochim. 10:271-281. transport in isolated chloroplasts. Curr. Top. Bio- 24. Padan, E., D. Ginzburg, and M. Shilo. 1970. The energ. 2:1-19. reproductive cycle of cyanophage LPP1-G in Plec- 2. Bishop, N. I. 1966. Partial reactions of photosynthesis tonema boryanum and its dependence on photosyn- and photoreduction. Annu. Rev. Plant Physiol. thesis and respiratory system. Virology 40:514-521. 17:185-208. 25. Pfennig, N. and K. D. Lippert. 1966. Uber das Vitamin 3. Bishop, N. I., and H. Gaffron, 1962. Photoreduction at A B,, Bedulrfnis phototropher Schwefelbacterien. Arch. 705 my in adapted algae. Biochem. Biophys. Res. Mikrobiol. 55:245-256. Commun. 8:471-476. 26. Schopf, J. W. 1970. Precambrian microorganisms and 4. Brock, T. D. 1967. Life at high temperatures. Science evolutionary events prior to the origin of vascular 158: 1012-1019. plants. Biol. Rev. 45:319-352. 5. Brock, T. D. 1973. Evolutionary and ecological aspects of 27. Schwabe, G. H. 1960. Uber den thermobioten Kosmopo- the cyanophytes, p. 487-500. In N. G. Carr and B. A. liten Mastigoclaudus laminosus. Cohn. Blaualgen und Whitton (ed.), The biology of blue-green algae. Black- Lebensraum. V. Schweiz. Z. Hydrol. 22:757-792. well Scientific Pub., Oxford. 28. Serruya, C., M. Edelstein, U. Pollingher, and S. Serruya. 6. Castenholtz, R. W. 1973. Ecology of blue-green algae in 1974. Lake Kinneret sediments: nutrient composition hot springs, p. 379-414. In N. G. Carr and B. A. of the free water and mud water examples. Limnol. Whitton (ed.), The biology of blue-green algae. Black- Oceanogr. 19:489-508. well Scientific Pub., Oxford. 29. Setlike, I. 1957. Light-dark transients in oxygen exchange 7. Eckstein, Y. 1970. Physicochemical limnology and geol- of blue-green algae. Biochim. Biophys. Acta ogy of a meromictic pond on the Red Sea shore. 24:436-437. Limnol. Oceanogr. 15:363-372. 30. Shibata, K., A. A. Benson, and M. Calvin. 1954. The 8. Fay, P. 1973. The heterocysts, p. 238-259. In N. G. absorption spectra of suspensions of living micro-orga- Whitton (ed.), The biology of blue-green algae. Black- nisms. Biochim. Biophys. Acta 15:461-470. well Scientific Pub., Oxford. 31. Stanier, R. Y. 1961. Photosynthetic mechanisms in bacte- 9. Frenkel, A., H. Gaffron, and E. H. Battley. 1949. Photo- ria and plants: development of a unitary concept. synthesis and photoreduction by a species of blue- Bacteriol. Rev. 25:1-17. green algae. Biol. Bull. 97:269. 32. Stewart, W. D. P., and H. W. Pearson. 1970. Effects of 10. Gaffron, H. 1940. Carbon dioxide reduction with molecu- aerobic and anaerobic conditions on growth and lar hydrogen in green algae. Am. J. Bot. 27:273-283. metabolism of blue-green algae. Proc. R. Soc. London 11. Gaffron, H. 1944. Photosynthesis, photoreduction and Ser. B. 175:293-311. dark reduction of carbon dioxide in certain algae. Biol. 33. Susor, W. A., and D. W. Krogman. 1964. Hill activity in Rev. 19:1-20. cell free preparations of a blue-green alga. Biochim. 12. Govendjee, R. 1963. Emerson enhancement effect and Biophys. Acta 88:11-19. two light reactions in photosynthesis, p. 318-339. In 34. Wood, E. J. F. 1965. Marine microbial ecology. Chapman B. Kok and A. T. Jagendorf (ed.), Photosynthetic and Hall, Ltd., London.