Facultative H 2-dependent anoxygenic photosynthesis in the unicellular cyanobacterium Gloeocapsa alpicola CALU 743

When cells of the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 are deprived of nitrate, the phycobilisomes are actively degraded by a proteolytic process termed chlorosis, which accompanied by decrease of rates of oxygen evolution and carbon dioxide fixation, increase of amount of stored glycogen and increase of hydrogenase activity. Suspensions of such cells exhibited a capacity for light-dependent inorganic carbon photoassimilation under anaerobic conditions in the presence of hydrogen and DCMU. The rate of 14C incorporation was commensurable with that for nitrate-sufficient cells at oxygenic photosynthesis and reached 35–38μmol 14C h−1 mg−1 Chl a. Incubation of G. alpicola grown aerobically in the presence of limiting concentrations of nitrate under anaerobic conditions (Ar, CO2, DCMU) in the light with addition of nitrate and H2, resulted in the increase of cellular protein, evidencing that G. alpicola cells with high level of hydrogenase activity are able to perform H2-dependent anoxygenic photosynthesis at levels supporting the growth of this cyanobacterium.


INTRODUCTION
Cyanobacteria (blue-green algae) are plant-type phototrophs which carry out water-splitting oxygenic photosynthesis based on light-mediated electron flow through photosystem II (PSII) and photosystem I (PSI) thereby generating ATP and reducing power for photoassimilation of inorganic carbon.In addition to oxygenic photosynthesis a facultative CO 2 photoassimilation using sulfide as electron donor has been demonstrated in many different cyanobacteria [1][2][3].This type of photosynthesis is called anoxygenic photosynthesis, since it involves PSI only and no oxygen is evolved.Detailed studies of this process in Oscillatoria limnetica show that induction of sulfide-dependent CO 2 photoassimilation requires de novo protein synthesis, specifically an inducible sulfide-quinone oxidoreductase [4][5][6][7].Molecular hydrogen, being as sulfide highly reducing electron donor, can be utilized to photoreduction of CO 2 by not only cyanobacteria but by eucaryotic algae as well [8,9].This reaction requires hydrogenase participation and is driven by PSI, although possible involvement of PSII has been indicated [10].
In spite of many species of cyanobacteria have the ability to photooxidize sulfide and H 2 for CO 2 reduction, actual growth exclusively at the expense of sulfide oxidation has been demonstrated only for two species of Oscillatoria [4,7].There is no information about H 2dependent anaerobic phototrophic growth.
The unicellular non-nitrogen-fixing cyanobacterium Gloeocapsa alpicola possesses the reversible-type hydrogenase, which activity increases significantly during light-or nitrate-limited growth [11].The cells grown † E-mail: sereb@issp.serpukhov.suunder nitrate-limitation are able to produce H 2 under dark anaerobic conditions due to fermentation of stored glycogen and to take it up under illumination [11,12].The light-dependent H 2 uptake was observed in the presence of exogenous hydrogen also and was not sensitive to the inhibitor of PSII, DCMU [12].
The present communication reports experiments showing the use of H 2 as electron donor for CO 2 photoassimilation in G. alpicola and the possibility of H 2 -dependent growth of this cyanobacterium in anaerobic conditions.

MATERIALS AND METHODS
The unicellular non-nitrogen-fixing cyanobacterium G. alpicola CALU 743 (=Synechocystis 6308) was obtained from the Alga Collection of St. Petersburg University and was grown in BG11 • [13] supplemented with 1.5 µM NiCl 2 and 2 or 15 mM KNO 3 as nitrogen source.Cultivation was performed at 30 • C in 0.55-l cylindrical glass flasks two-thirds full of medium and exposed to the light of luminescent lamps with an intensity of 165 µE m −2 s −1 .Growing cultures were bubbled with gas mixture (300 ml/min): Air + 2% CO 2 or Ar + 2% CO 2 + 30% H 2 .Cells from late-log-phase cultures were corrected for Chl a content and used in comparative experiments.
For determination of photosynthetic CO 2 assimilation the vessels (15 ml) with cell suspensions (5 ml, 10 µg Chl a ml −1 ) were flushed in the dark with Ar for 15 min and then injected, if noted, with DCMU (10 µM), KNO 3 (10 mM), flushed with H 2 and incubated on the light (120 µE m −2 s −1 ) for 5 min before injection with Na 2 14 CO 3 to a final concentration of 20 mM (0.8 µCi/ml).Each 30 min aliquots (0.5 ml) of cell suspensions were withdrawn for estimation of 14 C incorporation.
Hydrogenase activity was assayed in cell-free extracts by determining the rate of reduced methyl viologen (MV)-dependent H 2 evolution by gas chromatography [11].NAD(P)H-dependent H 2 evolution was assayed by the same method, using NADH or NADPH instead of MV.
Cell-free extracts were prepared by ultrasonic treatment of cell suspensions in 50 mM Tris-HCl buffer, pH 8.0, at 70 W for 10 min with intermittent cooling on ice.The resulting homogenate was centrifuged (4000 × g, 40 min) and supernatant was used in hydrogenase reactions.

RESULTS AND DISCUSSION
The cyanobacterium G. alpicola strain CALU 743 is a unicellular, non-nitrogen-fixing, obligate photoautotroph requiring only inorganic nutrients and light for growth.Like some other cyanobacteria, in cells of G. alpicola deprived of essential nutrient, such as nitrogen (nitrate), the light-harvesting antennae (the phycobilisomes) are actively degraded by a proteolytic process termed chlorosis [14][15][16][17].The absorbance spectra (Figure 1(A) and (B)) demonstrate a significant reduction in phycocyanin absorbance (peak at 620 nm) in cells of G. alpicola, grown under nitrate-limitation in comparison with nitrogen-sufficient cells.Some physiological properties of such cultures are summarized in Table 1.Nitrate-starvation resulted in inactivation of photosystem II (PSII), which the decrease of photosynthetic oxygen evolution rate (it did not compensate the respiration) and photosynthetic CO 2 fixation rate evidence about.Inactivation of PSII was reversible and was recovered when nitrate was replenished (not shown) [18].The low redox potential intracellular medium in nitrate-starved G. alpicola developed due to inactive PSII and unaltered respiration induced the increase of hydrogenase activity (Table 1) [12].Suspensions of such cells exhibited a capacity for in vivo light-dependent hydrogen consumption in the presence of CO 2 .The process was insensitive to DCMU, an inhibitor of PSII, although DBMIB, inhibitor of plastoquinone oxidation, prevented this reaction (Table 1).
The direct measurements of carbon photoassimilation by nitrogen-starved cells under anoxic conditions clearly demonstrate the dependence of the process on the presence of H 2 (Figure 2).The rate of H 2 -dependent 14 C incorporation (H 2 , DCMU) was similar to control (oxygenic photoreduction of CO 2 ).The addition of NO 3 − resulted in the decrease of H 2 -dependent carbon photoassimilation.It might be due to the competition between CO 2 fixation and nitrate reduction for assimilatory power, when electrons from H 2 are spent for both physiological reactions.The H 2 -dependent photoreduction of CO 2 was not observed in the dark, evidencing that hydrogen is taken up by light-dependent reaction of PSI.
The ability to use H 2 for CO 2 photoassimilation is not in itself sufficient for anaerobic growth.For example, the well known photoreduction with H 2 in eucaryotic alga [8,9] has never been proved to support their growth.On the other hand anaerobic photoautotrophic growth with highly reducing electron donor, such as H 2 S, has been demonstrated and studied in details in cyanobacterium Oscillatotia limnetica [4][5][6][7].In order to check the growth of G. alpicola under H 2 -dependent anoxygenic photosynthesis the cyanobacterium was grown aerobically in the presence of limiting nitrate concentration to the onset of the stationary phase, when hydrogenase activity reached high levels.Then, the cultures were diluted by O 2 -free culture medium and incubated anaerobically (Ar, CO 2 ) in the dark for 15 min, when DCMU (final concentration of 20 µM), KNO 3 (10 mM) and H 2 (30%) were added and the incubation was continued under illumination.As a result, the increase of cellular protein was observed, while the chlorophyll a concentration increased insignificantly (Figure 3).The doubling time on the protein basis during anoxygenic growth was similar to the oxygenic one, which was 12 hours.Interestingly, it was possible to observe that the initially yellow culture became green during incubation, evidencing that the phycobiliprotein synthesis seems to take place.It was also detected on absorbance spectrum (Figure 1, (C)).Most likely, the intensive phycobiliprotein synthesis was caused by aspiration "to repair" the potential for constitutive oxygenic photosynthesis which was destroyed by nitrogen deprivation.The primary step of use of H 2 as electron donor for anoxygenic photosynthesis is catalyzed by the hydrogenase.G. alpicola possesses a hydrogenase of the reversible type, which has a high affinity to molecular hydrogen, K m H2 =38 µmol.The genes encoding for this enzyme have been characterized for several Alcaligenes eutrophus [23] and NADP-dependent hydrogenase of Desulfovibrio fructosovorans [24].Sequence comparisons indicated that cyanobacterial enzyme is composed of the H 2 -cleaving dimer Hox YH and the diaphorase moiety HoxFU transferring electrons to NAD(P).These molecular data are confirmed by physiological reactions of NAD(P)-dependent hydrogenase activity in G. alpicola (Table 2).Cell-free extracts were able to evolve molecular hydrogen in the presence of NADH or NADPH.Therefore it may be suggested that the hydrogenase interacts with the photosynthetic electron-transport chain on the plastoquinone level.Accordingly, in the case of inactive PSII and in the presence of molecular hydrogen, a plastoquinone pool can be provided for electrons from H 2 via hydrogenase and NAD(P)H-dehydrogenase (complex I). Figure 4 demonstrates the hypothetical model for electron transport at H 2 -dependent anoxygenic photosynthesis in G. alpicola cells.The reaction mixture contained 1.2 mg protein per ml.100% H 2 evolution was equivalent to 315 nmol min −1 mg −1 protein.

CONCLUSIONS
The results obtained evidence that G. alpicola is capable to perform facultative anoxygenic photosynthesis using molecular hydrogen as an electron donor, at a level sufficient to support at least anaerobic photoautotrophic protein synthesis in this cyanobacterium.It is unlikely that conditions favorable for H 2 -dependent anoxygenic photosynthesis in cyanobacteria may exist in natural ecosystems.However this study may be important for the understanding of the place of cyanobacteria in evolution of the phototrophs.

Figure 2 .ml − 1 Figure 3 .Figure 4 .
Figure 2. Kinetics of CO 2 photoassimilation in cell suspensios of nitrogen starved G. alpicola under different conditions.For comparison an aerobic CO 2 photoassimilation in nitrogen-sufficient cells (control) is present.DCMU and KNO 3 were added in final concentration 10 µM and 5 mM, respectively.

Table 1 .
Comparative characterization of G. alpicola cultures grown under nitrogen-sufficient and nitrogenlimited conditions.

Table 2 .
Hydrogen evolution by cell-free extracts of G. alpicola.