The lack of the cell division protein FtsZ induced generation of giant cells under acidic stress in cyanobacterium Synechocystis sp. PCC6803.

Bacteria exposed to environmental stresses often exhibit superior acclimation abilities to environmental change. Acid treatment causes an increase in the cell length of the cyanobacterium Synechocystis sp. PCC6803 under light conditions. We aimed to elucidate the relationship between acidic stress and cell enlargement. After being synchronized under dark conditions, the cells were cultivated at different pH (pH 8.0 or pH 6.0) levels under light conditions. Synechocystis 6803 cells exhibited only cell growth occurred (cell volume expansion) and slow proliferation under the acidic condition. In the recovery experiment of the enlarged cells, they proliferated normally at pH 8.0, and the cell lengths decreased to the normal cell size under light conditions. Inhibition of cell division might be caused by acidic stress. To understand the effect of acidic stress on cell division, we evaluated the expression of FtsZ via Western blotting. The FtsZ concentration in cells was lower at pH 6.0 than at pH 8.0 and was not sufficient for cell division in the photoautotrophic conditions. ClpXP is well known as a regulator of the Z-ring dynamics in E. coli. The transcriptional level of four clpXP genes was upregulated approximately threefold at pH 6.0 after 24 h compared with that in cells grown at pH 8.0. The lack of FtsZ may be caused by the upregulation of clpXP expression under acidic condition. Therefore, ClpXP may participate in the degradation of FtsZ and be involved in the regulation of cell division via FtsZ under acidic stress in Synechocystis 6803.

Characterization of Sll1558 in environmental stress tolerance of Synechocystis sp. PCC 6803.

So far, the molecular mechanisms underlying the acidic-stress responses of plants are complicated and only fragmentally understood. Here, we investigated the mechanisms responsible for acidic-stress acclimation. Previously, DNA microarray analysis identified the sll1558 gene in Synechocystis sp. PCC 6803 (hereafter called Synechocystis 6803) to be upregulated following short-term acid treatment (1 h at pH 3.0). The sll1558 gene encodes uridine diphosphate-glucose pyrophosphorylase (UDP-glucose pyrophosphorylase), which catalyzes the conversion of glucose-1-phosphate into UDP-glucose. We constructed mutant cells for this gene and analyzed their phenotype. The sll1558 gene did not completely segregate in sll1558 mutant cells; thus, Sll1558 is essential for the survival of Synechocystis 6803. Besides, the partially disrupted sll1558 mutant cells were highly sensitive to acidic stress (pH 6.0) as well as other stress conditions (high salt, high osmolality, high/low temperature, and ultraviolet-B stress); the number of sll1558 transcripts increased under these conditions. UDP-glucose is used for the synthesis of various materials, such as glycolipids. From the membrane lipid composition analysis, digalactosyldiacylglycerol decreased and phosphatidylglycerol increased in the partially disrupted sll1558 mutant cells under acidic stress. These results suggest that sll1558 is important not only for the survival of Synechocystis 6803, but also for tolerance under various stress conditions.

Characterization of ABC transporter genes, sll1180, sll1181, and slr1270, involved in acid stress tolerance of Synechocystis sp. PCC 6803.

Over 50 ATP-binding cassette (ABC) transporter-related genes are detected in the Synechocystis sp. PCC 6803 genome by genome sequence analysis. Deletion mutants of other substrate-unknown ABC transporter genes were screened for their acid stress sensitivities in a low-pH medium to identify ABC transporters involved in acid resistance. We found that a mutant of sll1180 encoding proteins with homology to HlyB in Escherichia coli (E.coli) is more sensitive to acid stress than wild-type (WT) cells and analyzed the abundance of expression of the genes in WT cells under acid stress condition by quantitative real-time reverse transcriptase-polymerase chain reaction. sll1180 expression increased in the WT cells after acid stress treatment. Immunofluorescence revealed that Sll1180 localized in the plasma membrane. These results suggest that Sll1180 has an important role in the growth of Synechocystis sp. PCC 6803 under acid stress conditions. HlyB, HlyD, and TolC complex transport HlyA in E.coli; therefore, we searched for genes corresponding to these in Synechocystis sp. PCC 6803. A BlastP search suggested that HlyA, HlyD, and TolC proteins had homology to Sll1951, Sll1181, and Slr1270. Therefore, we constructed deletion mutant of these genes. sll1181 and slr1270 mutant cells revealed acid stress sensitivity. The bacterial two-hybrid analysis showed that Sll1180 interacted with Sll1181 and Sll1951. Dot blot analysis of Sll1951-His revealed that the sll1180 and sll1181 mutant cells did not transport Sll1951-His from the cytoplasm to the extracellular matrix. These results suggest that Sll1180 and Sll1181 transport Sll1951 and that Sll1951-outside of the cells-might be a key factor in acid stress tolerance.

Sll0751 and Sll1041 are involved in acid stress tolerance in Synechocystis sp. PCC 6803.

The ATP-binding cassette (ABC) transporter is a multi-subunit membrane protein complex involved in lipid transport and acid stress tolerance in the cyanobacterium Synechocystis sp. PCC 6803. This organism has two sets of three ABC transporter subunits: Slr1045 and Slr1344, Sll0751 and Sll1002, and Sll1001 and Sll1041. We previously found that Slr1045 is essential for survival under acid stress condition (Tahara et al. 2012). In the present study, we examined the participation of other ABC transporter subunits in acid stress tolerance using a deletion mutant series of Synechocystis sp. PCC 6803. Although Slr1344 is highly homologous to Slr1045, Δslr1344 cells were not susceptible to acid stress. Δsll0751 and Δsll1041 cells displayed acid stress sensitivity, whereas Δsll1001/sll1002 double mutant cells grew normally. Under high- and low-temperature stress conditions, the growth rate of Δslr1344 and Δsll1001/sll1002 cells did not differ from WT cells, whereas Δsll0751 and Δsll1041 cells showed significant growth retardation, as previously observed in Δslr1045 cells. Moreover, nile red staining showed more lipid accumulation in Δslr1045, Δsll0751, and Δsll1041 cells than in WT cells. These results suggest that Slr1045, Sll0751, and Sll1041 function together as a lipid transport complex in Synechocystis sp. PCC 6803 and are essential for growth under various stresses.

Slr2019, lipid A transporter homolog, is essential for acidic tolerance in Synechocystis sp. PCC6803.

Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse environmental stress. In Synechocystis sp. PCC6803 (Synechocystis), lipopolysaccharide (LPS) may play a role in this protection. To examine the relationship between stress tolerance of Synechocystis and LPS, we focused on Slr2019 because Slr2019 is homologous to MsbA in Escherichia coli, which is related to LPS synthesis. First, to obtain a defective mutant of LPS, we constructed the slr2019 insertion mutant (slr2019) strain. Sodium deoxycholate-polyacrylamide gel electrophoresis indicated that slr2019 strain did not synthesize normal LPS. Second, to clarify the participation of LPS in acid tolerance, wild type (WT) and slr2019 strain were grown under acid stress; slr2019 strain growth was significantly weaker than WT growth. Third, to examine influences on stress tolerance, slr2019 strain was grown under various stresses. Under salinity and temperature stress, slr2019 strain grew significantly slower than WT. To confirm cell morphology, cell shape and envelope of slr2019 strain were observed by transmission electron microscopy; slr2019 cells contained more electron-transparent bodies than WT cells. Finally, to confirm whether electron-transparent bodies are poly-3-hydroxybutyrate (PHB), slr2019 strain was stained with Nile Blue A, a PHB detector, and observed by fluorescence microscopy. The PHB granule content ratio of WT and slr2019 strain grown at BG-11 pH 8.0 was each 7.18 and 8.41 %. At pH 6.0, the PHB granule content ratio of WT and slr2019 strain was 2.99 and 2.60 %. However, the PHB granule content ratio of WT and slr2019 strain grown at BG-11N-reduced was 10.82 and 0.56 %. Because slr2019 strain significantly decreased PHB under BG-11N-reduced compared with WT, LPS synthesis may be related to PHB under particular conditions. These results indicated that Slr2019 is necessary for Synechocystis survival in various stresses.

Genomic analysis of parallel-evolved cyanobacterium Synechocystis sp. PCC 6803 under acid stress.

Experimental evolution is a powerful tool for clarifying phenotypic and genotypic changes responsible for adaptive evolution. In this study, we isolated acid-adapted Synechocystis sp. PCC 6803 (Synechocystis 6803) strains to identify genes involved in acid tolerance. Synechocystis 6803 is rarely found in habitants with pH < 5.75. The parent (P) strain was cultured in BG-11 at pH 6.0. We gradually lowered the pH of the medium from pH 6.0 to pH 5.5 over 3 months. Our adapted cells could grow in acid stress conditions at pH 5.5, whereas the parent cells could not. We performed whole-genome sequencing and compared the acid-adapted and P strains, thereby identifying 11 SNPs in the acid-adapted strains, including in Fo F1-ATPase. To determine whether the SNP genes responded to acid stress, we examined gene expression in the adapted strains using quantitative reverse-transcription polymerase chain reaction. sll0914, sll1496, sll0528, and sll1144 expressions increased under acid stress in the P strain, whereas sll0162, sll0163, slr0623, and slr0529 expressions decreased. There were no differences in the SNP genes expression levels between the P strain and two adapted strains, except for sll0528. These results suggest that SNPs in certain genes are involved in acid stress tolerance in Synechocystis 6803.

Sll0939 is induced by Slr0967 in the cyanobacterium Synechocystis sp. PCC6803 and is essential for growth under various stress conditions.

In this study, the genes expressed in response to low pH stress were identified in the unicellular cyanobacterium Synechocystis sp. PCC 6803 using DNA microarrays. The expression of slr0967 and sll0939 constantly increased throughout 4-h acid stress conditions. Overexpression of these two genes under the control of the trc promoter induced the cells to become tolerant to acid stress. The Δslr0967 and Δsll0939 mutant cells exhibited sensitivity to osmotic and salt stress, whereas the trc mutants of these genes exhibited tolerance to these types of stress. Microarray analysis of the Δslr0967 mutant under acid stress conditions showed that expression of the high light-inducible protein ssr2595 (HliB) and the two-component response regulator slr1214 (rre15) were out of regulation due to gene inactivation, whereas they were upregulated by acid stress in the wild-type cells. Microarray analysis and real-time quantitative reverse transcription-polymerase chain reaction analysis showed that the expression of sll0939 was significantly repressed in the slr0967 deletion mutant. These results suggest that sll0939 is directly involved in the low pH tolerance of Synechocystis sp. PCC 6803 and that slr0967 may be essential for the induction of acid stress-responsive genes.

Slr0967 and Sll0939 induced by the SphR response regulator in Synechocystis sp. PCC 6803 are essential for growth under acid stress conditions.

Two-component signal transduction is the primary signaling mechanism for global regulation of the cellular response to environmental changes. We used DNA microarray analysis to identify genes that were upregulated by acid stress in the cyanobacterium Synechocystis sp. PCC 6803. Several of these genes may be response regulators that are directly involved in this type of stress response. We constructed deletion mutants for the response regulator genes and compared the growth rates of cells transfected with mutant and wild-type genes in a low pH medium. Of these mutants, deletion of sphR affected the growth rate under acid stress (pH 6.0) conditions. We examined genome-wide expression in ΔsphR mutant cells using DNA microarray to determine whether SphR was involved in the regulation of other acid stress responsive genes. Microarray and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses of wild-type cells showed that the expression of phoA, pstS1, and pstS2, which are upregulated under phosphate-limiting conditions, increased (2.48-, 1.88-, and 5.07-fold, respectively) after acid stress treatment for 0.5h. In contrast, pstS2 expression did not increase in the ΔsphR mutant cells after acid stress, whereas the phoA and sphX mRNA levels increased. Furthermore, qRT-PCR and northern blot analysis indicated that downregulation of the acid-responsive genes slr0967 and sll0939 occurred with the deletion of sphR. Indeed, mutants of these genes were more sensitive to acid stress than the wild-type cells. Thus, induction of Slr0967 and Sll0939 by SphR may be essential for growth under acid stress conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Role of Slr1045 in environmental stress tolerance and lipid transport in the cyanobacterium Synechocystis sp. PCC6803.

ATP-binding cassette (ABC) transporter proteins mediate energy-dependent transport of substrates across cell membranes. Numerous ABC transporter-related genes have been found in the Synechocystis sp. PCC6803 genome by genome sequence analysis including H(+), iron, phosphate, polysaccharide, and CO(2) transport-related genes. The substrates of many other ABC transporters are still unknown. To identify ABC transporters involved in acid tolerance, deletion mutants of ABC transporter genes with unknown substrates were screened for acid stress sensitivities in low pH medium. It was found that cells expressing the deletion mutant of slr1045 were more sensitive to acid stress than the wild-type cells. Moreover, slr1045 expression in the wild-type cells was increased under acid stress. These results indicate that slr1045 is an essential gene for survival under acid stress. The mutant displayed high osmotic stress resistance and high/low temperature stress sensitivity. Considering the temperature-sensitive phenotype and homology to the organic solvent-resistant ABC system, we subsequently compared the lipid profiles of slr1045 mutant and wild-type cells by thin-layer chromatography. In acid stress conditions, the phosphatidylglycerol (PG) content in the slr1045 mutant cells was approximately 40% of that in the wild-type cells. Moreover, the addition of PG to the medium compensated for the growth deficiency of the slr1045 mutant cells under acid stress conditions. These data suggest that slr1045 plays a role in the stabilization of cell membranes in challenging environmental conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II.

Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial-type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal-type extrinsic proteins (PsO, PsbQ', PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal-type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial-, red algal- and green algal-types during early phases of evolution after a primary endosymbiosis. This study also showed that a haptophyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal-type, whereas Euglena gracilis resulted from green algal secondary endosymbiosis contained the green algal-type extrinsic proteins, suggesting that the red algal- and green algal-type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immunological analyses, together with the current genome data, the evolution of photosynthetic oxygen-evolving PSII was discussed from a view of distribution of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed.

Identification of genes expressed in response to acid stress in Synechocystis sp. PCC 6803 using DNA microarrays.

Plant cells are always exposed to various environmental stresses such as high light, low temperature and acid rain, and thus have to respond in order to survive these stresses. Although some mechanisms of responses to high light and low temperature etc., have been clarified, there is little information about the acclimation process to acid stress. In this study, the gene expression changes of Synechocystis sp. PCC 6803 in response to acid stress were examined using DNA microarrays (CyanoCHIP). We compared gene expression profiles of the cells treated at pH 8 (control) and pH 3 for 0.5, 1, 2 or 4 h. As a result, we found that 32 genes were upregulated by more than 3-fold, and 29 genes were downregulated by at least 3-fold after the acid treatment. Among these upregulated genes, expressions of slr0967 and sll0939 kept-increasing until 4 h under the acid stress and increased by 7 to 16-fold after the 4 h treatment. This suggests that the products of these two genes play important roles in the acid acclimation process.

Isolation and characterization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis.

Oxygen-evolving photosystem II (PSII) complexes of Euglena gracilis were isolated and characterized. (1) The PSII complexes contained three extrinsic proteins of 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ), and showed oxygen-evolving activity of around 700 micromol O2 (mg Chl)(-1) h(-1) even in the absence of Cl- and Ca2+ ions. (2) NaCl-treatment removed not only PsbP and PsbQ but also a part of PsbO from Euglena PSII, indicating that PsbO binds to Euglena PSII more loosely than those of other organisms. Treatments by urea/NaCl, alkaline Tris or CaCl2 completely removed the three extrinsic proteins from Euglena PSII. (3) Each of the Euglena extrinsic proteins bound directly to PSII independent of the other extrinsic proteins, which is similar to the binding properties of the extrinsic proteins in a green alga, Chlamydomonas reinhardtii. (4) One of the significant features of Euglena PSII is that the oxygen evolution was not enhanced by Ca2+. When CaCl2-treated Euglena PSII was reconstituted with PsbO, the oxygen-evolving activity was stimulated by the addition of NaCl, but no further stimulation was observed by CaCl2. (5) Oxygen evolution of Euglena PSII reconstituted with PsbO from C. reinhardtii or spinach instead of that from Euglena also showed no enhancement by Ca2+, whereas a significant enhancement of oxygen evolution was observed by Ca2+ when the green algal or higher plant PSII was reconstituted with Euglena PsbO instead of their own PsbO. These results indicate that the PSII intrinsic proteins instead of the extrinsic PsbO protein, are responsible for the stimulation of oxygen evolution by Ca2+. Sequence comparison of major PSII intrinsic proteins revealed that PsbI of Euglena PSII is remarkably different from other organisms in that Euglena PsbI possesses extra 16-17 residues exposed to the luminal side. This may be related to the loss of enhancement of oxygen evolution by Ca2+ ion.

Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II.

To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N-succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodiimide, respectively. The N-succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester-modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N-succinimidyl propionate-modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11-Lys14, Lys27-Lys38, Lys40, Lys90-Lys96, Lys143-Lys152, Lys166-Lys174) were modified with N-succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056-2061].

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).

Comparison of binding and functional properties of two extrinsic components, Cyt c550 and a 12 kDa protein, in cyanobacterial PSII with those in red algal PSII.

Cyt c550 and 12 kDa protein are two extrinsic proteins of photosystem II (PSII) found in cyanobacteria and some eukaryotic algae. The binding patterns of these two extrinsic proteins are different between cyanobacterial (Thermosynechococcus vulcanus) and red algal (Cyanidium caldarium) PSIIs [Shen and Inoue (1993) Biochemistry 32: 1825; Enami et al. (1998) Biochemistry 39: 2787]. In order to elucidate the possible causes responsible for these differences, we first cloned the psbV gene encoding Cyt c550 from a red alga, Cyanidium caldarium, which was compared with the homologous sequences from other organisms. Cross-reconstitution experiments were then performed with different combinations of the extrinsic proteins and the cyanobacterial or red algal PSII. (1). Both the cyanobacterial and red algal Cyt c550 bound directly to the cyanobacterial PSII, whereas none of them bound directly to the red algal PSII, indicating that direct binding of Cyt c550 to PSII principally depends on the structure of PSII intrinsic proteins but not that of Cyt c550 itself. (2). Cyt c550 was functionally exchangeable between the red algal and the cyanobacterial PSII, and the red algal 12 kDa protein functionally bound to the cyanobacterial PSII, whereas the cyanobacterial 12 kDa protein did not bind to the red algal PSII. (3). The antibody against the cyanobacterial or red algal 12 kDa protein reacted with its original one but not with the homologous protein from the other organism, whereas the antibody against the red algal Cyt c550 reacted with both cyanobacterial and red algal Cyt c550. These results imply that the structure and function of Cyt c550 have been largely conserved, whereas those of the 12 kDa protein have been changed, in the two organisms studied here.

Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga, Chlamydomonas reinhardtii having his-tagged CP47.

Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) and Cl(-) when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B) was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca(2+). These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl(2). The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl(2)-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.

Comparison of the structure of the extrinsic 33 kDa protein from different organisms.

The psbO gene encoding the extrinsic 33 kDa protein of oxygen-evolving photosystem II (PSII) complex was cloned and sequenced from a red alga, Cyanidium caldarium. The gene encodes a polypeptide of 333 residues, of which the first 76 residues served as transit peptides for transfer across the chloroplast envelope and thylakoid membrane. The mature protein consists of 257 amino acids with a calculated molecular mass of 28,290 Da. The sequence homology of the mature 33 kDa protein was 42.9-50.8% between the red alga and cyanobacteria, and 44.7-48.6% between the red alga and higher plants. The cloned gene was expressed in Escherichia coli, and the recombinant protein was purified, subjected to protease-treatments. The cleavage sites of the 33 kDa protein by chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus), a euglena (Euglena gracilis), a green alga (Chlamydomonas reinhardtii) and two higher plants (Spinacia oleracea and Oryza sativa). The cleavage sites by chymotrypsin were at 156F and 190F for the cyanobacterium, 159M, 160F and 192L for red alga, 11Y and 151F for euglena, 10Yand 150F for green alga, and 16Y for spinach, respectively. The cleavage sites by V8 protease were at 181E (cyanobacterium), 182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E (euglena), 176E and 180E (green alga), and 18E or 19E (higher plants). Since most of the residues at these cleavage sites were conserved among the six organisms, the results indicate that the structure of the 33 kDa protein, at least the structure based on the accessibility by proteases, is different among these organisms. In terms of the cleavage sites, the structure of the 33 kDa protein can be divided into three major groups: cyanobacterial and red algal-type has cleavage sites at residues around 156-195, higher plant-type at residues 16-19, and euglena and green algal-type at residues of both cyanobacterial and higher plant-types.