Acclimation to the growth temperature and thermosensitivity of photosystem II in a mesophilic cyanobacterium, Synechocystis sp. PCC6803

Differences in the temperature dependence and thermosensitivities of PSII activities in Synechocystis sp. PCC6803 grown at 25 and 35 degrees C were studied. Hill reactions in cells, thylakoid membranes and purified PSII core complexes were measured at high temperatures or at their growth temperatures after high-temperature treatments. In the presence of 2,5-dichloro-p-benzoquinone as an electron acceptor, which can accept electrons directly from Q(A), the temperature dependence of the oxygen-evolving activity was almost the same in thylakoid membranes and in the purified PSII complexes from cells grown at 25 or 35 degrees C. When duroquinone, which accepts electrons only through Q(B) plastoquinone, was used as an electron acceptor, the temperature dependence was the same for purified PSII core complexes but was different between thylakoids isolated from the cells grown at 25 and 35 degrees C. No remarkable difference was observed in protein compositions between thylakoids and between purified PSII complexes from cells grown at 25 or 35 degrees C. However, the fluidity of thylakoids, measured by electron flow to P700, was affected by the growth temperature. These results suggest that one of the major factors which cause the changes in the thermosensitivity of PSII is the change in the fluidity of thylakoid membranes. As for the acclimation of PSII in thylakoids to high temperatures, one of the main causes is the decrease in the high-temperature-induced formation of non-Q(B) PSII due to the decreased fluidity in the cells grown at 35 degrees C.     

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis

Phosphatidylglycerol (PG) is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus in thylakoid membranes. However, its function in photosynthesis remains poorly understood. In this study we have identified a pgsA gene of Synechocystis sp. PCC6803 that encodes a PG phosphate synthase involved in the biosynthesis of PG. A disruption of the pgsA gene allowed us to manipulate the content of PG in thylakoid membranes and to investigate the function of PG in photosynthesis. The obtained pgsA mutant could grow only in the medium containing PG, and the photosynthetic activity of the pgsA mutant dramatically decreased with a concomitant decrease of PG content in thylakoid membranes when the cells grown in the presence of PG were transferred to the medium without PG. This decrease of photosynthetic activity was attributed to the decrease of photosystem (PS)II activity, but not to the decrease in PSI activity. These findings demonstrate that PG is essential for growth of Synechocystis sp. PCC6803 and provide the first direct evidence that PG plays an important role in PSII.     

Irreversible photoinhibition of photosystem II is caused by exposure of Synechocystis cells to strong light for a prolonged period

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 degrees C for 2 h with light at an intensity of 2,500 micromol photons m(-2) s(-1), the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.     

Membrane lipid unsaturation modulates processing of the photosystem II reaction-center protein D1 at low temperatures.

The role of membrane lipid unsaturation in the restoration of photosystem II (PSII) function and in the synthesis of the D1 protein at different temperatures after photoinhibition was studied in wild-type cells and a mutant of Synechocystis sp. PCC 6803 with genetically inactivated desaturase genes. We show that posttranslational carboxyl-terminal processing of the precursor form of the D1 protein is an extremely sensitive reaction in the PSII repair cycle and is readily affected by low temperatures. Furthermore, the threshold temperature at which perturbations in D1-protein processing start to emerge is specifically dependent on the extent of thylakoid membrane lipid unsaturation, as indicated by comparison of wild-type cells with the mutant defective in desaturation of 18:1 fatty acids of thylakoid membranes. When the temperature was decreased from 33 degrees C (growth temperature) to 18 degrees C, the inability of the fatty acid mutant to recover from photoinhibition was accompanied by a failure to process the newly synthesized D1 protein, which accumulated in considerable amounts as an unprocessed precursor D1 protein. Precursor D1 integrated into PSII monomer and dimer complexes even at low temperatures, but no activation of oxygen evolution occurred in these complexes in mutant cells defective in fatty acid unsaturation.     

Assembly and disassembly of the photosystem II manganese cluster reversibly alters the coupling of the reaction center with the light-harvesting phycobilisome

The light-driven oxidative assembly of Mn (2+) ions into the H 2O oxidation complex (WOC) of the photosystem II (PSII) reaction center is termed photoactivation. The fluorescence yield characteristics of Synechocystis sp. PCC6803 cells undergoing photoactivation showed that basal fluorescence, F 0, exhibited a characteristic decline when red, but not blue, measuring light was employed. This result was traced to a progressive increase in the coupling of the phycobilisome (PBS) to the PSII reaction center as determined by observing the changes in room temperature and 77 K fluorescence emission spectra that accompany photoactivation. The results support the hypothesis that strong energetic coupling of the PBS to the PSII reaction center depends upon the formation of an active WOC, which presumably diminishes the likelihood of photodamage to reaction centers that have either lost an intact Mn cluster or are in the process of assembling an active WOC.     

The BtpA protein stabilizes the reaction center proteins of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 at low temperature

Specific inhibition of photosystem I (PSI) was observed under low-temperature conditions in the cyanobacterium Synechocystis sp. strain PCC 6803. Growth at 20 degrees C caused inhibition of PSI activity and increased degradation of the PSI reaction center proteins PsaA and PsaB, while no significant changes were found in the level and activity of photosystem II (PSII). BtpA, a recently identified extrinsic thylakoid membrane protein, was found to be a necessary regulatory factor for stabilization of the PsaA and PsaB proteins under such low-temperature conditions. At normal growth temperature (30 degrees C), the BtpA protein was present in the cell, and its genetic deletion caused an increase in the degradation of the PSI reaction center proteins. However, growth of Synechocystis cells at 20 degrees C or shifting of cultures grown at 30 degrees C to 20 degrees C led to a rapid accumulation of the BtpA protein, presumably to stabilize the PSI complex, by lowering the rates of degradation of the PsaA and PsaB proteins. A btpA deletion mutant strain could not grow photoautotrophically at low temperature, and exhibited rapid degradation of the PSI complex after transfer of the cells from normal to low temperature.     

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

Increase in the rate of photosynthetic linear electron transport in cyanobacteruim <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 lacking phycobilisomes

Compensating changes in the pigment apparatus of photosynthesis that resulted from a complete loss of phycobilisomes (PBS) were investigated in the cells of a PAL mutant of cyanobacterium Synechocystis sp. PCC 6803. The ratio PBS/chlorophyll calculated on the basis of the intensity of bands in the action spectra of photosynthetic activity of two photosystems in the wild strain was 1: 70 for PSII and 1: 300 for PSI. Taking into consideration the number of chlorophyll molecules per reaction center in each photosystem, these ratios could be interpreted as association of PBS with dimers of PSII and trimers of PSI as well as greater dependence of PSII as compared with PSI on light absorption by PBS. The ratio PSI/PSII determined by photochemical cross-section of the reactions of two photosystems was 3.5: 1.0 for wild strain of Synechocystis sp. PCC 6803 and 0.7: 1.0 for the PAL mutant. A fivefold increase in the relative content of PSII in pigment apparatus corresponds to a 5-fold increase in the intensity of bands at 685 and 695 nm as related to the band of PSI at 726 nm recorded in low-temperature fluorescence spectrum of the PAL mutant. Inhibition of PSII with diuron resulted in a pronounced stimulation of chlorophyll fluorescence in the PAL mutant as compared to the wild strain of Synechocystis sp. PCC 6803; these data suggested an activation of electron transfer between PSII and PSI in the mutant cells. Thus, the lack of PBS in the mutant strain of Synechocystis sp. PCC 6803 was compensated for by the higher relative content of PSII in the pigment apparatus of photosynthesis and by a rise in the rate of linear electron transport.     

Membrane dynamics as seen by fourier transform infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803. The effects of lipid unsaturation and the protein-to-lipid ratio

The roles of lipid unsaturation and lipid-protein interactions in maintaining the physiologically required membrane dynamics were investigated in a cyanobacterium strain, Synechocystis PCC 6803. The specific effects of lipid unsaturation on the membrane structure were addressed by the use of desaturase-deficient (desA(-)/desD(-)) mutant cells (which contain only oleic acid as unsaturated fatty acid species) of Synechocystis PCC 6803. The dynamic properties of the membranes were determined from the temperature dependence of the symmetric CH(2) stretching vibration frequency, which is indicative of the lipid fatty acyl chain disorder. It was found that a similar membrane dynamics is maintained at any growth temperature, in both the wild-type and the mutant cell membranes, with the exception of mutant cells grown at the lower physiological temperature limit. It seems that in the physiological temperature range the desaturase system of the cells can modulate the level of lipid desaturation sufficiently to maintain similar membrane dynamics. Below the range of normal growth temperatures, however, the extent of lipid disorder was always higher in the thylakoid than in the cytoplasmic membranes prepared from the same cells. This difference was attributed to the considerable difference in protein-to-lipid ratio in the two kinds of membranes, as determined from the ratio of the intensities of the protein amide I band and the lipid ester C&z.dbnd6;O vibration. The contributions to the membrane dynamics of an ab ovo present 'structural' lipid disorder due to the protein-lipid interactions and of a thermally induced 'dynamic' lipid disorder could be distinguished.     

Dissection of photodamage at low temperature and repair in darkness suggests the existence of an intermediate form of photodamaged photosystem II

Irradiation of the photosynthetic machinery with strong light induces damage to the photosystem II complex (PSII), and this phenomenon is referred to as photodamage. In an attempt to characterize the mechanism of photodamage to PSII, we examined the events associated with photodamage by monitoring the phenomenon in Synechocystis sp. PCC 6803 at a low temperature. After the activity of PSII had been reduced to 10% of the original activity by exposure of Synechocystis cells to strong light at 10 degrees C, recovery was allowed to proceed at 34 degrees C in darkness. Under these conditions, approximately 50% of the activity of PSII was restored within 60 min. The recovery in darkness did not require protein synthesis, as demonstrated by Western blotting analysis and a radiolabeling experiment with [(35)S]methionine. We also observed a similar recovery of PSII in darkness in isolated thylakoid membranes. Our findings, together with those of other studies, suggest the presence of an intermediate form of photodamaged PSII that is generated prior to the formation of photodamaged PSII.     

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 °C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 °C to 38 °C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.     

Interrelation between cyanophycin synthesis, L-arginine catabolism and photosynthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

Ultrastructural and immunocytochemical investigations gave evidence that cyanophycin (multi-L-arginyl-poly-L-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on L-arginine or L-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of L-arginine or L-asparagine with nitrate only caused minor cyanophycin accumulation. Growth of Synechocystis PCC 6803 on L-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on L-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. L-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on L-arginine as sole N-source. In both cells types the L-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on L-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of L-arginine to the total nitrogen pool, and the intracellular L-arginine concentration is greatly influenced by the activity of the L-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, L-arginine catabolism, and in addition photosynthesis in Synechocystis PCC 6803.     

PratA, a periplasmic tetratricopeptide repeat protein involved in biogenesis of photosystem II in Synechocystis sp. PCC 6803

The light reactions of oxygenic photosynthesis are mediated by multisubunit pigment-protein complexes situated within the specialized thylakoid membrane system. The biogenesis of these complexes is regulated by transacting factors that affect the expression of the respective subunit genes and/or the assembly of their products. Here we report on the analysis of the PratA gene from the cyanobacterium Synechocystis sp. PCC 6803 that encodes a periplasmic tetratricopeptide repeat protein of formerly unknown function. Targeted inactivation of PratA resulted in drastically reduced photosystem II (PSII) content. Protein pulse labeling experiments of PSII subunits indicated that the C-terminal processing of the precursor of the reaction center protein D1 is compromised in the pratA mutant. Moreover, a direct interaction of PratA and precursor D1 was demonstrated by applying yeast two-hybrid analyses. This suggests that PratA represents a factor facilitating D1 maturation via the endoprotease CtpA. The periplasmic localization of PratA supports a model that predicts the initial steps of PSII biogenesis to occur at the plasma membrane of cyanobacterial cells.     

The PsbY protein is not essential for oxygenic photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

A tetra-manganese cluster in the photosystem II (PSII) pigment-protein complex plays a critical role in the photosynthetic oxygen evolution process. PsbY, a small membrane-spanning polypeptide, has recently been suggested to provide a ligand for manganese in PSII (A.E. Gau, H.H. Thole, A. Sokolenko, L. Altschmied, R.G. Herrmann, E.K. Pistorius [1998] Mol Gen Genet 260: 56-68). We have constructed a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with an inactivated psbY gene (sml0007). Southern-blot and polymerase chain reaction analysis showed that the mutant had completely segregated. However, the DeltapsbY mutant cells grew normally under photoautotrophic conditions. Moreover, growth of the wild-type and mutant cells were similar under high-light photoinhibition conditions, as well as in media without any added manganese, calcium, or chloride, three required inorganic cofactors for the oxygen-evolving complex of PSII. Analysis of steady-state and flash-induced oxygen evolution, fluorescence induction, and decay kinetics, and thermoluminescence profiles demonstrated that the DeltapsbY mutant cells have normal photosynthetic activities. We conclude that the PsbY protein in Synechocystis 6803 is not essential for oxygenic photosynthesis and does not provide an important binding site for manganese in the oxygen-evolving complex of PSII.     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.