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 Photosynthesis in the Cyanobacterium Oscillatoria 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 cyanobacteria ("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 bacteria) 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.