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First published online August 20, 2008; 10.1104/pp.108.123117 Plant Physiology 148:1068-1081 (2008) © 2008 American Society of Plant Biologists
slr1923 of Synechocystis sp. PCC6803 Is Essential for Conversion of 3,8-Divinyl(proto)chlorophyll(ide) to 3-Monovinyl(proto)chlorophyll(ide)1Department of Life Science, Graduate School of Life Science, University of Hyogo, Ako, Hyogo 678–1297, Japan (M.R.I., S.A., Y.K., K.S., H.K.); and Department of Life Sciences (Biology), University of Tokyo, Meguro, Tokyo 153–8902, Japan (T.M.)
The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaean F420H2 dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex I. In this study, the gene was inactivated and characteristics of the mutant were analyzed. The mutant grew slower than the wild type under 100 µE m–2 s–1 but did not grow under high light intensity (300 µE m–2 s–1). The cellular content of chlorophyll was lower in the mutant, and the absorption spectrum showed a shift in the absorption peak of the Soret band to a longer wavelength by about 10 nm compared with the wild type. It was found, by high-performance liquid chromatography analysis, that the retention time of chlorophyll of the mutant is shorter than that of the wild type and that the peak wavelength of the Soret band was also shifted to a longer wavelength by 11 nm. Proton nuclear magnetic resonance analysis of the chlorophyll of the mutant revealed that the ethyl group of position 8 of ring B is replaced with a vinyl group. The spectrum indicates that the chlorophyll of the mutant is not a normal (3-vinyl)chlorophyll a but a 3,8-divinylchlorophyll a. These results strongly suggest that the Slr1923 protein is essential for the conversion from divinylchlorophyll(ide) to normal chlorophyll(ide). We thus designate this gene cvrA (a gene indispensable for cyanobacterial vinyl reductase).
Land plants, algae, cyanobacteria, and photosynthetic bacteria use various types of chlorophyll molecules (chlorophyll [chl] a, b, c, and d and bacteriochlorophyll a, b, c, etc.) as inevitable photon-capturing pigments in photosynthesis. Extensive studies with various organisms by biochemical and genetic approaches have disclosed many aspects related to chlorophyll metabolism (for review, see Beale, 1993  , 1999; Senge and Smith, 1995). However, the functional or regulatory genes related to various chlorophyll biosynthetic pathways have not yet been fully elucidated. In land plants, chlorophyll precursors are sometimes accumulated as both 3-monovinyl (MV-) or 3,8-divinyl (DV-) intermediates, and the ratio between the two forms changes depending on the species, tissues, and growth conditions (Kim and Rebeiz, 1996). Chlorophyll biosynthetic heterogeneity is assumed to originate mainly in parallel DV- and MV-chl biosynthetic routes interconnected by 8-vinyl reductases (8 VRs) that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group. So far, five 8-VR activities have been detected at the levels of DV-Mg-protophorphyrin IX, Mg-protomonomethyl ester, protochlorophyllide (Pchlide) a, chlorophyllide (chlide) a, and chl a (Kolossov et al., 2006). It has not been clear whether the various 8-VR activities are catalyzed by a single enzyme with broad specificities encoded by a single gene or by a family of enzymes with narrow specificity encoded by multiple genes, as is the case of NADPH Pchlide oxidoreductases (Rebeiz et al., 2003).
Mutants that accumulate DV-chl(ide) a can be a potential model system. A mutant that accumulates DV-chl was reported by Bazzaz and Govindjee (1974)
An ortholog of the DVR gene, however, is not found in the complete genome sequences of cyanobacteria, Synechocystis sp. PCC6803 (Synechocystis 6803; Kaneko et al., 1996
The model photosynthetic organism Synechocystis 6803 has a unique gene, slr1923, which encodes a hypothetical protein with unknown function. The deduced amino acid sequence of the gene has a similarity with FpoF, which functions as a F420H2 dehydrogenase subunit F. The protein acts as an electron input device to incomplete NADH dehydrogenase complex 1 (NDH1) of the methanogenic archaean species Methenosarcina mazei (Prommeenate et al., 2004 In this study, we created a mutant of slr1923 of Synechocystis 6803 by insertional inactivation, and the characteristics of the mutant were analyzed. It was revealed that all of the molecular species of chl a of the mutant were DV-chl a but not MV-chl a (normal chl a). The molecular identity was confirmed by absorption spectra, HPLC, and 1H-NMR analysis. These results show that slr1923 is inevitable for the conversion of 3,8-DV-(proto)chl(ide) a to 3-MV-(proto)chl(ide) a in Synechocystis 6803. We thus designate the gene cvrA (a gene indispensable for cyanobacterial vinyl reductase). The distribution and evolution of vinyl reductases are discussed.
Recently, Ito et al. (2008)
Construction of the Mutant In order to elucidate the role of the protein Slr1923, we have constructed an slr1923-inactivated mutant of Synechocystis 6803. Wild-type cells were transformed by the plasmid pGEM-Nf-SpR-Cf, in which a spectinomycin resistance cassette with a reverse orientation with respect to the direction of slr1923 transcription was inserted (Fig. 1A ). After transformation by homologous recombination, colonies grown on a spectinomycin-containing BG11 agar medium were picked up and successive transplantations were performed to segregate the mutant lines. Segregation was confirmed by PCR amplification of the corresponding DNA region by genomic DNA as a template. Figure 1B shows that the amplified band of the mutant was totally replaced by the mutated DNA fragment, indicating total segregation in the mutant.
Based on the genomic sequence of Synechocystis 6803 (Kaneko et al., 1996 ), two other genes are located downstream of slr1923 (Fig. 1A). slr1924 is only 25 bp apart from slr1923, and slr1924 and slr1925 overlap by 1 bp. It is possible that these three genes are cotranscribed as a single mRNA. Inactivation of slr1923, the most upstream located among the three genes (Fig. 1A), may affect expression of the downstream genes. Figure 1C shows northern analysis of transcripts of the three genes. The signal intensity of the mutant was about half or lower compared with the wild type. However, the signal intensities of the transcripts were not much different between the three genes. This suggests that expression of slr1924 and slr1925 is not inhibited by disruption of slr1923. Although higher molecular mass signals that correspond to the length equivalent of two genes were observed (Fig. 1C, arrow), further analysis is necessary to determine whether the three open reading frames form operons or not.
Ito et al. (2008)
When wild-type and mutant cells were grown under 100 µE m–2 s–1 and aerated by 3% CO2-containing air (normal condition hereafter), they showed similar growth patterns except that the slr1923 inactivation mutant (slr1923M) grew at a slower rate (Fig. 2A ). The cells grew exponentially up to about 36 h of cultivation, with doubling times of 10.6 and 12.1 h for the wild type and the mutant, respectively, followed by a gradual decrease in the growth rates, finally reaching a stationary phase after about 80 h.
The growth rates of the wild type under 30, 100, and 300 µE m–2 s–1 were very similar to those reported previously (Ohtsuka et al., 2004 ). Under moderate light intensities, the mutant cells grew significantly, although the rates were slower compared with those of the wild type (Table I
). When slr1923M was grown under strong light (300 µE m–2 s–1), there was almost no considerable growth from the beginning and the cells started to die at about 34 h of inoculation (Fig. 2B), indicating a high sensitivity to strong illumination. High sensitivity to strong illumination is reported in an Arabidopsis (Arabidopsis thaliana) mutant (Nagata et al., 2005; Nakanishi et al., 2005) and also in a Synechocystis 6803 mutant (Ito et al., 2008). Although the SD is large, slr1923M showed a tendency to grow slower than the wild type under low CO2 (0.03%). The growth rates under high-salt (0.5 M NaCl) plus low-CO2 conditions were not much different between the wild type and the mutant. The doubling times of wild-type and mutant cells grown under various conditions are summarized in Table I.
Spectral Characteristics
An absorption spectrum of wild-type cells showed absorption maxima at 678 and 630 nm in the red region by absorption of chl a and phycobilisomes and at 435 nm in the Soret region (Fig. 3A
, solid line). However, the absorption spectrum of slr1923M showed a rather low absorbance in the chlorophyll red region, while absorption by phycobilisome was practically unchanged at any stage of growth on the basis of cell number (Fig. 3A, broken line). The peak wavelengths of the red band region due to chl a and phycobilisomes were the same as those of wild-type cells. It was found that the absorption peak of the Soret band shifted to a longer wavelength by about 10 nm. The absorption spectrum of the same mutant reported by Ito et al. (2008)
Figure 3B shows changes of absorption spectra of the mutant cells grown under the high-light condition (300 µE m–2 s–1). The cells show low chlorophyll absorbance relative to phycobilisome at the initial stage (9.5 h) of cultivation (Fig. 3B, line a). After 17 h, the mutant still retained the spectral characteristics of the initial inoculum (Fig. 3B, line b). However, the color of the culture turned to bluish after 24 h. The spectrum showed a decreased amount of chlorophyll, while phycobilisome content did not change so much based on the number of the cells (Fig. 3B, line c). Finally, the cells lost almost all of the chlorophyll and carotenoids, but a significant amount of phycobilisomes remained. This resulted in total blue color after 33.5 h of growth (Fig. 3B, line d). This is rather different from the responses to high-light illumination of wild-type cells. The wild-type cells show increased carotenoids and decreased phycobilisomes, giving rise to yellowish-green color when grown under strong illumination. Decreased absorption of the chlorophyll red band relative to phycobilisome could result in higher fluorescence from phycobilisomes and reduced energy transfer efficiency from phycobilisomes to PSII core complexes. This possibility was addressed by measuring fluorescence emission spectra at 77 K. When wild-type cells were excited by a broad-band blue light, three emission bands peaking at 650 to 665, 685 to 695, and 725 nm emitted from phycobiliproteins, phycobilisome anchor (LCM)/PSII, and PSI, respectively, were observed (Fig. 4A , solid line). Fluorescence intensity at 725 nm was about two times higher than that at 695 nm. On the other hand, a very high intensity of fluorescence at around 685 nm relative to that at 725 nm was observed in slr1923M. The intensity of fluorescence at 685 to 695 nm was higher than that emitting from PSI (725 nm), reflecting a higher content of phycobilisome in the cells and less efficiency of energy transfer to the PSII core (Fig. 4A, dotted line). The emission peak corresponding to allophycocyanin was shifted to longer wavelengths in the mutant, indicating that the energy trapped by phycobilisome could not be fully transferred to anchor and/or PSII core complexes. These results indicate that the efficiency of energy transfer from phycobilisomes to PSII is decreased in the mutant.
When cells were excited by violet light to excite mainly the Soret band of chl a, small but distinct emissions at 685 and 695 nm were observed in addition to a large emission peaking at 725 nm in wild-type cells (Fig. 4B, solid line). The emission spectrum of the mutant was practically the same as that of wild-type cells, although emission at 685 nm was higher (Fig. 4B, dotted line), suggesting that the relative content of PSII is little increased in the mutant (see Table IV below). These results indicate that the microenvironment of chlorophylls responsible for emitting those bands is not much altered in the mutant.
Pigment Analysis
The mutant showed a characteristic absorption spectrum. The peak wavelength of chl a of the Soret band is shifted by about 10 nm to a longer wavelength (Fig. 3A, broken line). This suggests that components and/or the composition of the pigments are changed in the mutant. Pigments were extracted from wild-type and mutant cells and their compositions were analyzed by HPLC. On the pigment analysis of the mutant, we recognized that the retention time of chl a (39.4 min) was always a little shorter than that of the wild type (39.9 min; Fig. 5A
). The absorption spectra of separated chlorophyll were different between the wild type and the mutant (Fig. 5B). The spectra showed an absorption maximum at 660 nm in both the wild type and the mutant. The peak wavelength of the Soret band was 431 nm in the wild type; on the other hand, it was shifted to a longer wavelength by 11 nm in the mutant. It is also of note that chl a species were totally replaced by the new chl a species in the mutant. The spectral characteristics and the retention time on HPLC of the modified chlorophyll coincided with those reported by Shedbalkar and Rebeiz (1992)
Table II shows the cellular contents of chlorophyll and carotenoids obtained by HPLC analysis. The cellular content of β-carotene was reduced by about 30%, while that of myxozanthophyll was not much different in the mutant compared with the wild type. Zeaxanthin was increased by about 50% in the mutant, suggesting that light intensity under the "normal" culture condition is strong for the mutant (Schäfer et al., 2006 ). On the other hand, chlorophyll content was decreased by about 45%. Accordingly, the ratios of myxoxanthophyll and zeaxanthin to chlorophyll were increased by about 1.5 and 2.7 times in the mutant compared with the wild type.
The results obtained by HPLC analysis indicate that inactivation of the slr1923 gene will result in the total replacement of the chlorophyll species from MV type to DV type. The mutant cannot accomplish the reduction of the vinyl group of position 8 of ring B to an ethyl group.
In order to confirm the molecular species and determine the structure, 1H-NMR of the chlorophyll purified from the mutant was measured and compared with that of wild-type chlorophyll. Figure 6, A and B , shows 1H-NMR spectra of chlorophyll from the wild type and the mutant, respectively. In the 7.3 to 7.4 ppm region, the spectrum of wild-type chlorophyll shows signals of a doublet of doublets that is normally observed in MV-chl a (Fig. 6A, inset a). Vinyl proton signals are also exhibited at 5.25 and 5.45 ppm (Fig. 6A, inset b). A resonance signal of the ethyl group connected to position 8 of ring B was also observed in the 0.9 to 0.95 ppm region (Fig. 6A, inset c).
An NMR spectrum of the chlorophyll purified from the mutant showed differences from the wild type at the above characteristic signal regions. The signal appearing in the 7.3 to 7.5 ppm region showed two sets of double doublets, instead of a single doublet of doublets (Fig. 6B, inset a), while their coupling constants were similar to each other. A difference was also observed in the 5.2 to 5.3 ppm region (Fig. 6B, inset b). It exhibited two well-separated sets of double doublets. In addition, a signal originating from the ethyl group is missing in the 0.90 to 0.95 ppm region (Fig. 6B, inset c). These results confirm the presence of two vinyl groups, one at position 3 of ring A and the other at position 8 of ring B, of the chlorophyll molecule in the mutant (Wu and Rebeiz, 1985 ).
Judging from the results obtained from the absorption and 1H-NMR spectra, it was concluded that the molecular identity of the chlorophyll of the mutant is a 3,8-DV-chl a, not 3-vinyl 8-ethyl (MV-)chl a, which is present in wild-type cells. A mutant that accumulates DV-chl was recently reported independently in Arabidopsis by two groups (Nagata et al., 2005
Ito et al. (2008)
The photosynthetic activities or reaction center contents were compared between the wild type and the mutant. The CO2 fixation rates on the basis of MV-chl a for the wild type and DV-chl a for the mutant were not much different. However, in the mutant, whole chain electron transport activity and segmented electron transport of PSII measured by a Clark-type O2 electrode were reduced (Table III ). The reduced activities could be due to the reduced amount of reaction center or the decreased efficiency of energy transfer from phycobilisome or chlorophyll to the reaction centers. In fact, the light saturation curve of CO2 fixation activity indicated that the efficiency of utilization of light energy is reduced in the mutant, although the activity is the same at the saturating light intensities (data not shown). It should be noted that we observed a strong dark oxygen consumption activity in the presence of ascorbate and 2,6-dichlorophenolindophenol in the mutant for unknown reasons. This resulted in low activity of PSI electron transport in the mutant.
Relative contents of PSI and PSII in slr1923M were determined and compared with those in the wild type (Table IV ). It was found that PSI and PSII contents on the basis of chlorophyll were not very much different (80%–110% of wild-type values). On a cellular basis, however, both PSI and PSII contents were reduced in the mutant by about 40% and 20%, respectively, compared with the wild type. These results suggest that decreased chlorophyll content on the basis of the cell comes from decreased numbers of active reaction centers but that the number of chlorophylls bound to active reaction center complexes is not changed. These results strongly suggest that the amounts of DV-chl a synthesized are reduced and/or that degradation of the chlorophyll molecule is faster in the mutant.
The results obtained in this study strongly suggest that the slr1923 gene product of Synechocystis 6803 is essential for the conversion of 3,8-DV-chl(ide) a to 3-MV-chl(ide) a. The Slr1923 protein has a molecular mass of 45 kD, deduced from its gene sequence, and is a soluble protein, as analyzed by the SOSUI program (http://sosui.proteome.bio.tuat.ac.jp). The homolog of Synechocystis 6803 is also found in Anabaena sp. PCC7120 (all1601; Kaneko et al., 2001
It was found that the homolog is present not only in cyanobacteria but also in eukaryotic algae such as the red alga C. merolae 10D (CMJ076C; Matsuzaki et al., 2004
In addition to the oxygenic photosynthetic organisms, the slr1923 homologues are also found in the genomes of the purple bacteria Rhodopseudomonas palstris CGA009 (RPA1501) and Rhodobacter rubrum (Rru_A0937). Multiple sequence alignment from photosynthetic bacteria to higher plants constructed by ClustalW is shown in Figure 7
. The aligned sequences indicate that the homologues have a common sequence cluster showing CXXCXXCX[12]C in the 30th to 50th amino acids, except R. palstris. This sequence motif is found in the N4 iron-sulfur cluster of Nqo3 protein, which is one of the subunits of NADH dehydrogenase of Thermus thermophilus (Sazanov and Hinchliffe, 2006
A phylogenetic tree of Slr1923 homologues was constructed by the neighbor-joining method (Saitou and Nei, 1987 ) using F420H2 dehydrogenase subunits as an outgroup. It indicates that the Slr1923 homologues are separated into two large groups: a purple bacterial group and other photosynthetic organisms (Fig. 8
). Oxygenic photosynthetic organisms form a subcluster closely related to each other. It is of note that among them, a cyanobacterial group and green lineages were clearly separated. A red algal homolog, CMJ076C of C. merolae, was classified into the cyanobacterial group as reported by Ito et al. (2008). Interestingly, a homolog of the diatom T. pseudonana was categorized into a group of green lineages, although diatoms originated by second symbiosis of red algae. This is in sharp contrast with other genes, such as 16S rDNA, PsbA, or PsaA proteins, in which eukaryotic groups branched out from the cyanobacterial group. This suggests that slr1923 homologues of eukaryotic algae did not originate from some cyanobacterial strain. It is also noted that one of the homologues present in G. violaceus, gll0878, is classified into the cyanobacterial group, while glr2543 has highest affinity with the green bacterium Chlorobium phaerobacteroides homolog.
Recently, Nagata et al. (2005) and Nakanishi et al. (2005) independently identified DVR in the land plant Arabidopsis. Chew and Bryant (2007) confirmed the presence of the corresponding enzyme in a green sulfur bacterium, C. tepidum TL3 (CT1063). On the other hand, Nagata et al. (2005) could not identify DVR homologues in most cyanobacteria. Only a small number of strains, such as Synechococcus WH8102 and Synechococcus spp. CC9605, CC9902, and CC9311, have DVR homologues. Slr1923 and its homologues found in other cyanobacteria could be important factors of the second type of vinyl reductase they predicted. Recently, Ito et al. (2008) identified the same gene by means of bioinformatics analysis, which is totally different from our approach. Interestingly, an slr1923 homolog was found in the green sulfur bacterium C. phaeobacteroides DSM266 (Cpha266_0188). This indicated that the slr1923 homolog is widely distributed among photosynthetic organisms irrespective of oxygen evolution. Neither the Slr1923 nor DVR homologous gene is found in divinylchlorophyll-harboring cyanobacteria, Prochlorococcaceae. It is also of note that photosynthetic organisms possess either DVR or slr1923 orthologues, except green lineages. The distribution of the slr1923 homologues has no relationship with the acquisition of the gene and taxonomy (Table V ).
We have created the slr1923 inactivation mutant and analyzed its phenotypes. slr1923 is located within a cluster comprising slr1924 and slr1925, separated by only 25 bp from slr1923 (Fig. 1A). Northern analysis indicated that inactivation of slr1923, which is located upstream of the cluster, does not affect the transcription of other genes located downstream. The created mutant cells, slr1923M, exhibited a characteristic absorption spectrum. It has an absorption maximum at 678 nm in the red band, which is the same as that of the wild type. However, the Soret band was shifted to a longer wavelength by about 10 nm (Fig. 3A). Absorption and 1H-NMR spectral analyses of the extracted and purified chl a (Figs. 5B and 6) revealed that the chlorophyll species of the mutant is DV-chl a, not MV-chl a (normal chl a). It is also found that all of the chlorophyll species present in the mutant were replaced with DV-chl a (Fig. 5A). According to the pathway of chlorophyll biosynthesis (Kim and Rebeiz, 1996 ), DV-(P)chl(ide) a is a precursor of MV-(P)chl(ide) a. Judged from the accumulation of DV-chl a in the mutant, it is strongly suggested that Slr1923 is essential to convert 3,8-divinyl(proto)chlorophyll(ide) a to 3-vinyl(proto)chlorophyll(ide) a of Synechocystis 6803. It is highly possible that Slr1923 of Synechocystis 6803 is a divinylchlorophyll(ide) reductase or a part of the complex. Thus, we designate this gene cvrA (a gene indispensable for cyanobacterial vinyl reductase).
The cellular content of DV-chl a of the slr1923M cells was reduced by about half compared with the content of MV-chl a of the wild type. Electron microscopic analysis has shown that the number of thylakoid membranes was reduced in the mutant (Ito et al., 2008
It is of note that DV-chl a is incorporated into PSI and PSII complexes and that they seem to be somehow functioning. However, the complexes do not seem to be fully functional and/or they are less stable. The inactivated mutant was sensitive to high-light illumination and started to die under such conditions (Fig. 2B). A similar phenotype was also observed with the dvr (AT5G18660) mutant of Arabidopsis (Nagata et al., 2005 Incomplete matching of DV-chl a to the binding niche of PSI and PSII chlorophyll-protein complexes would lead to reduced efficiency of energy transfer within and between the complexes. This was found to be the case. The mutant shows a high fluorescence peak at 663 nm emitting from phycobiliproteins at room temperature (data not shown) and at 660 and 685 nm at 77 K emitting from phycobilisomes and the core linker polypeptide, LCM (Fig. 4A). This reflects the reduced efficiency of energy transfer from phycobilisomes to chlorophyll-protein complexes and the requirement of higher light intensity for the saturation of CO2 fixation (data not shown). It is also possible that the redox potential of DV-chl a is different from that of MV-chl a and, thus, that the efficiency of the photochemical reaction might be reduced. Determination of the redox potential of the DV-chl a molecule and reaction center is now in progress.
The reduced contents of PSI and PSII on a cell basis (Table IV) can also be accounted for by the replacement of MV-chl a (normal chl a) with DV-chl a. Insufficient spatial fitting of DV-chl a to the binding site will bring about instability of the chlorophyll-protein complexes. Due to their instability, the degradation rate of the complex would be higher than in the wild type. When the mutant cells are grown under strong illumination, the degradation rate becomes further increased due to photodamage of the complex and finally exceeds the synthesis rate of the complex, leading to a decrease in chlorophyll protein complexes under strong illumination. This was indeed the case depicted by the absorption spectral changes shown in Figure 3B. Similar phenomena were also observed by Nagata et al. (2005)
The deduced amino acid sequence of slr1923 has homology to that of FpoF protein, which functions as an electron input device of archaean NDH1 as an F420H2 oxidoreductase (Prommeenate et al., 2004
Another type of vinyl reductase (DVR) has been found in the land plants Arabidopsis and rice and in the marine cyanobacterium Synechococcus WH8102 (Nagata et al., 2005
The distribution of the two genes indicated that the cyanobacteria possess either a dvr or cvrA homolog, except Prochlorococcaceae. This clade does not have either gene. On the other hand, green lineages harbor both genes in their nuclei. There seems to be no relationship between the distribution of both proteins and taxonomy (Table V). Taking into account these facts, it is very likely that dvr and cvrA homologous genes were acquired independently, as pointed out by Ito et al. (2008)
In green lineages, both dvr and cvrA homologues are present. If both enzymes are functioning in the chloroplast, inactivation of dvr alone will not suffer from incapability of the conversion from DV-chlide to MV-chlide. However, the DVR-inactivated mutant accumulated only DV-chl and no MV-chl (Nagata et al., 2005
Experimental Organism and Growth Conditions A Glc-tolerant Synechocystis 6803 strain was used as the wild-type organism. The cells were grown photoautotrophically in a liquid BG11 medium at 30°C. Cultures were grown under white light at a light intensity of 100 µE m–2 s–1 under 3% CO2-containing air, except where indicated otherwise. For measurements of growth rates, cells were grown in liquid BG11 medium and aliquots of the culture solution were sampled at various time points. The cell density was determined by monitoring the A750.
The nucleotide sequence of slr1923 was obtained from Cyanobase (http://www.kazusa.or.jp/cyano/cyano.html). Two DNA fragments consisting of 549 bp of the N-terminal side (N-fragment, Nf) and 557 bp of the C-terminal side (C-fragment, Cf) of the slr1923 gene were amplified by PCR separately. For N-fragment amplification, forward (N-5) and reverse (N-3) primers with the sequences 5'-CATGACCGTTCCTGCCC-3' and 5'-GGGGAATTCCCACACAGGGCGTTCCC-3', respectively, and for the C-fragment, forward (C-5) and reverse (C-3) primers with the sequences 5'-GGGGAATTCATGTTTCCCGGGCTGGG-3' and 5'-GAGGGACGTGGTCAGCC-3', respectively, were used. The EcoRI recognition site was introduced into the 5' end of reverse (N-3) and forward (C-5) primers for the N- and C-fragments, respectively. The amplified N- and C-fragments were cloned separately into the pGEM-T vector (Promega) and introduced into competent Escherichia coli cells (XL-1 blue). N- and C-fragment-containing recombinant plasmids were cut out by double digestion by EcoRI and SpeI. The C-fragment, which was purified by gel electrophoresis, was ligated into gel-purified N-fragment containing pGEM-T vector (pGEM-Nf), giving rise to pGEM-Nf-Cf plasmid. A spectinomycin resistance cassette (SpR) cut out from pHSG398-SpR by EcoRI was inserted between N- and C-fragment-containing recombinant plasmid (pGEM-Nf-Cf), which was linearized by EcoRI digestion. The constructed recombinant vector (pGEM-Nf-SpR-Cf) was amplified in E. coli and purified for transformation. The wild-type Synechocystis 6803 cells were transformed by the resulting plasmid pGEM-Nf-SpR-Cf vector. The transformed cells, which have a disrupted slr1923 gene, were segregated by successive streaks (about five to six times) on BG11 agar plates that contained 20 µg mL–1 spectinomycin. The segregation was confirmed by PCR using N-fragment forward (N-5) and C-fragment reverse (C-3) primers, with genomic DNA prepared from mutant cells as a template.
Total RNA from wild-type and mutant cells at mid-log phase was extracted according to Hihara et al. (1998)
Absorption spectra of the intact cells were measured with a Shimadzu MPS-2000 spectrophotometer (Shimadzu). Fluorescence emission spectra were recorded by a laboratory-constructed setup at 77 K (Yamane et al., 1997
A cell suspension (1.3 mL) in which the A750 was adjusted to 0.2 was precipitated, and then 10 µL of pure water and 190 µL of dimethyl formamide were added. After a short vortexing, the solution was transferred to a brown plastic bottle and kept at –20°C overnight. After a short vortexing, an aliquot of 50 µL was withdrawn and centrifuged. Ten microliters of the supernatant was injected onto an HPLC column (Phenomenex). Other chemicals and instrumental settings were as described by Kashino and Kudoh (2003)
Cells were harvested by centrifugation (3,200g, 5 min) and washed in buffer A (25% [w/v] glycerol, 10 mM CaCl2, 10 mM MgCl2, and 50 mM MES-NaOH [pH 6.5]). The precipitated cells were resuspended in buffer A. The cells were ruptured by zirconia beads (
Chlorophylls were extracted by sonicating the mixture of 1.5 mL of thylakoid membrane preparation and 6.0 mL of acetone-Na2HPO4 for 5 min. The resulting solution was filtered by a filter paper and then a glass filter. Filtrate volume was adjusted to 10 mL with acetone, and 1.33 mL of dioxane was added with stirring on ice. Then, water was slowly added until green aggregates were formed. Samples were kept at –20°C for 1 h and centrifuged at 2,300g for 15 min at 4°C. The green pellet was dissolved in 3 mL of ethanol and again filtered through a glass filter. Chlorophyll purification was repeated to remove contaminating carotenoids. Finally, purified chlorophyll was lyophilized overnight. Dried samples were dissolved in 700 µL of NMR-grade deuterated acetone. Two hundred microliters of the sample was loaded in the NMR machine. 1H-NMR was measured on a JEOL ECP-600 at 600 MHz using a solvent peak as a reference (J = 2.00).
Cells were suspended in liquid BG11 medium, and chlorophyll concentration was adjusted to 20 µg mL–1. Oxygen evolution or consumption activities were measured by a Clark-type oxygen electrode (Rank Brothers) as described previously (Inoue et al., 2001
For the determination of PSI content, light-induced absorption changes of P700 were measured at 703 nm by a Shimadzu MPS-2000 spectrophotometer by thylakoid membranes at 15 µg chl mL–1 as described by Nakayama et al. (1979) Intact cell suspension was used for the measurement of PSII content. Oxygen evolution under repetitive flash excitation (10-µs duration) was recorded by a Clark-type O2 electrode (Rank Brothers) at 30°C in the presence of 5 mM NaHCO3 and 1 mM K3Fe(CN)6. The PSII content was determined from an average yield of oxygen per flash on the basis of chlorophyll and then on the basis of cells.
The deduced amino acid sequences of slr1923 homologues were trimmed to the predicted mature forms by TargetP (http://www.cbs.dtu.dk/services/TargetP/) for nucleus-encoded genes. The amino acid sequences were aligned by the ClustalW algorithm. A neighbor-joining tree was constructed based on multiple sequence alignment (Saitou and Nei, 1987 Sequence data from this article can be found in the GenBank/EMBL data libraries. Accession numbers of cvrA homologues are as follows: Rhodopseudomonas palstris CAE26943, Q6N9N9; Roseobacter denitrificans, Q161A4; Jannaschia sp. CCS1, Q28VP5; Halorhodospira halophila, A1WXJ2; Chlorobium phaeobacteroides DSM266, A1BCX8; Synechocystis sp. PCC6803, P74473; Thermosynechococcus elongatus BP1, Q8DHV0; Anabaena sp. PCC7120, P46015; Gloeobacter violaceus PCC7421 gll0878, Q7NM89; Gloeobacter violaceus PCC7421 glr2543, Q7NHJ2; Trichodesmium erythraeum, Q118A1; Synechococcus sp. PCC7942, Q31NI0; Cyanobacteria Yellowstone B-Prime (Cyanobacteria CYB), 3318347HEL; Synechococcus sp. RCC307, A5GS18; Synechococcus sp. WH7803, A5GM11; Ostreococcus lucimarinus, A4RSL2; Chlamydomonas reinhardtii, A8JC46; Arabidopsis thaliana, Q8GS60; Oryza sativa, O23023; Methenosarcina berkeri FpoF, 3223320LC; Methenosarcina mazei FpoF, Q8PZ67; Methenosarcina berkeri beta, 3223320VV; Methenosarcina mazei beta, Q46A78. Accession numbers of DVR are as follows: Rhodobacter sphaeroides 2.4.1, Q3IXP7; Chlorobium tepidum TLS, Q8KDI7; Synechococcus WH8102, Q7U7L8; Chlamydomonas reinhardtii, A8HMQ3; Arabidopsis thaliana, Q1H537; Oryza sativa, Q10LH0.
We are especially thankful to Prof. Takashi Sugimura and Ms. Aya Inoue (Department of Material Science, Graduate School of Material Science, University of Hyogo) for their great help with 1H-NMR measurements and analysis. We are also grateful to Yohei Ikeda and Takeshi Takahashi (Graduate School of Life Science) for helping with HPLC manipulation and data analysis and to M. Ikeuchi (Department of Life Sciences [Biology], University of Tokyo, Komaba) for helpful discussions. Received May 18, 2008; accepted August 14, 2008; published August 20, 2008.
1 This work was supported by a grant from the 21st Century Center of Excellence Program to K.S. 
2 Present address: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo, Tokyo 112–8551, Japan. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hiroyuki Koike (hkoike{at}bio.chuo-u.ac.jp). www.plantphysiol.org/cgi/doi/10.1104/pp.108.123117 * Corresponding author; e-mail hkoike{at}bio.chuo-u.ac.jp.
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ABSTRACT
RESULTS
slr1923, was estimated as 
CO2, 5 mM NaHCO3; H2O 
= 0.1 mm, 1.6 g mL–1 cell suspension) with the Mini Bead Beater (Cole-Parmer). Agitation was performed for 20 s followed by cooling on ice for 1 min to minimize heat denaturation. This cycle was repeated three times. The supernatant after precipitation of zirconia beads was recovered and centrifuged at 6,000g for 10 min at 4°C to remove unbroken cells. Thylakoid membranes and soluble fractions were separated by centrifugation at 300,000g for 30 min. The thylakoid membranes recovered as a precipitate were resuspended in buffer A and stored at –80°C until use.