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First published online June 30, 2006; 10.1104/pp.106.083451 Plant Physiology 141:1274-1283 (2006) © 2006 American Society of Plant Biologists
Impaired Photosynthesis in Phosphatidylglycerol-Deficient Mutant of Cyanobacterium Anabaena sp. PCC7120 with a Disrupted Gene Encoding a Putative Phosphatidylglycerophosphatase1Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China (F.W., Z.Y., T.K.); and Graduate School of the Chinese Academy of Sciences, Beijing 100049, China (F.W.)
Phosphatidylglycerol (PG) is a ubiquitous phospholipid in thylakoid membranes of cyanobacteria and chloroplasts and plays an important role in the structure and function of photosynthetic membranes. The last step of the PG biosynthesis is dephosphorylation of phosphatidylglycerophosphate (PGP) catalyzed by PGP phosphatase. However, the gene-encoding PGP phosphatase has not been identified and cloned from cyanobacteria or higher plants. In this study, we constructed a PG-deficient mutant from cyanobacterium Anabaena sp. PCC7120 with a disrupted gene (alr1715, a gene for Alr1715 protein, GenBank accession no. BAB78081) encoding a putative PGP phosphatase. The obtained mutant showed an approximately 30% reduction in the cellular content of PG. Following the reduction in the PG content, the photoautotrophical growth of the mutant was restrained, and the cellular content of chlorophyll was decreased. The decreases in net photosynthetic and photosystem II (PSII) activities on a cell basis also occurred in this mutant. Simultaneously, the photochemical efficiency of PSII was considerably declined, and less excitation energy was transferred toward PSII. These findings demonstrate that the alr1715 gene of Anabaena sp. PCC7120 is involved in the biosynthesis of PG and essential for photosynthesis.
Thylakoid membranes of cyanobacteria and chloroplasts of plants and algae are the sites of the oxygenic photosynthesis, where highly organized pigment-protein complexes are embedded in the lipid matrix. Thylakoid membranes are characterized by its unique lipid composition, which is in contrast with animal, bacterial, and nonchloroplastic cell membranes dominated by phospholipids. The major lipids of thylakoid membranes are the uncharged galactolipids, namely, monogalacotsyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG). These galactolipids account for at least 50 mol% of the total thylakoid lipids. In addition, thylakoid membranes are specific in that there is a negatively charged sulfolipid, sulfoquinovosyl diacylglycerol (SQDG), which seems not to be present in nonphotosynthetic organisms. The only significant phospholipid is phosphatidylglycerol (PG), rather than phosphatidylcholine or phosphatidylethnolamine (Murata and Siegenthaler, 1998  ).
PG is an anionic phospholipid that is also extensively distributed in the functional domains of thylakoid membranes, suggesting that PG is critical for the structure and function of thylakoid membrane. Indeed, increasing biochemical and genetic studies provided evidence that PG plays an important role in photosynthesis of thylakoid membranes. It has shown that PG is indispensable for the dimerization of PSII (Kruse et al., 2000
In all organisms, PG is synthesized from phosphatidic acid (PA) in three steps. CDP-diacylglycerol (CDP-DG) is first synthesized by transfer of CMP from CTP to PA with the action of CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41; Carter and Kennedy, 1966 Although biochemical and genetic results have established that PG is indispensable for photosynthetic organisms and is necessary for photosynthesis process, it has not as yet been unambiguously demonstrated. There still exists the possibility that either the precursor of PG biosynthesis, PGP, or the metabolite of PG biosynthesis, cardiolipin, which remains to be clarified in thylakoid membranes, serves as an essential component for photosynthetic organisms.
It is thus of particular interest to clone the gene involved in the biosynthesis of PGP. Two genes encoding for PGP phosphatase, pgpA and pgpB, have been characterized in the nonphotosynthetic bacterial strain Escherichia coli (Icho, 1988a
Disruption of the Gene alr1715 in Anabaena sp. PCC7120
It was demonstrated that there are at least two genes, pgpA and pgpB, encoding for membrane-associated phospholipid phosphatases in E. coli (Icho, 1988a
In this work, we have investigated the mutation of the gene alr1715 of Anabaena sp. PCC7120 and characterized the photosynthetic properties of this mutant. Owing to remarkable sequence similarity to the pgpB gene of E. coli, this gene alr1715 is therefore also designated pgpB in this study. The gene alr1715 was disrupted by insertion of a kanamycin-resistant cassette into the coding region of alr1715, as described in "Materials and Methods." The transformation was conducted with the construct pPGP3 that was transformed into the wild type of Anabaena sp. PCC7120 through homologous recombination using the triparental mating method (Elhai and Wolk, 1988 ). To check whether or not all copies of this gene were disrupted in the genome of the mutant with the neomycin-resistant phenotype, we carried out PCR analysis of the wild type and the mutant, with primers for the 5'- and 3'-terminal regions of the putative pgpB gene. For the wild type, an approximately 1.0-kb DNA fragment corresponding to the native pgpB gene was amplified. In the case of the mutant, a 2.2-kb DNA fragment corresponding to the pgpB::Kanr gene owing to insertion of the 1.2-kb cassette was observed, but a 1.0-kb DNA fragment corresponding to the native pgpB gene was not amplified (Fig. 2A ). To determine if any transcript for the gene is still present in this mutant, reverse transcription (RT)-PCR was performed to examine pgpB expression in the wild type and pgpB mutant. Figure 2B shows RT-PCR results using total RNA isolated from the wild-type and pgpB mutant cells. No pgpB transcription product was detected in the pgpB mutant cells (lane 2). This was in contrast to the wild-type cells, where an approximately 1.0-kb transcription product was observed (lane 3). Thus, the pgpB gene was successfully knocked out.
Growth Phenotype of the Mutant To investigate the role of the putative pgpB gene in growth, the growth profiles of the wild type and the pgpB mutant under photoautotrophic growth condition were investigated. Figure 3 shows the growth of the wild-type and pgpB mutant cells in the liquid growth medium BG11. It can be clearly seen that although both the wild type and pgpB mutant could grow, a different growth existed between the wild type and the mutant. The mutant showed a slower growth than the wild type (Fig. 3A). Even after lengthy growth, the mutant was pale green in appearance, while the wild type remained normal blue green (Fig. 3B). However, similar growth curves were observed for the wild type and the pgpB mutant when the growth medium was supplemented with exogenous PG at concentrations above 2 µM (Fig. 3A). In this situation, the mutant appeared blue green (Fig. 3B).
Lipid Phenotype of the Mutant To explore the effect of the pgpB gene on the lipid composition of cyanobacterial cells, lipids from both the wild type and the mutant were extracted and analyzed. Table I shows the composition of lipid classes from intact cells of the wild type and the pgpB mutant cultured in the liquid medium BG11. In the wild type, MGDG was the most abundant lipid, making up 34% of the total lipids. DGDG and PG were second, each accounting for about 23% to approximately 24%. The amount of SQDG was only 20%. A major finding from the data is the clear changes in the contents of two acidic lipids, SQDG and PG. The relative amount of PG was reduced from 24 mol% in wild-type cells to 16% in the pgpB mutant. Concomitantly, the content of SQDG was increased in the mutant (29%) compared with the wild type (20%).The relative amount of MGDG and DGDG were slightly changed in the mutant with respect to the wild type, indicating that the pgpB mutant is little affected as to galactolipid metabolism in Anabaena sp. PCC7120.
The changes in the content of PG in the pgpB mutant cells with growth were also determined. Figure 4 shows changes in the content of PG in intact cells of the wild type and the pgpB mutant. During incubation with the culture medium BG11 for 10 d, the content of PG in the wild-type cells slightly decreased from 1.8 to 1.1 x 10–16 mol cells–1. By contrast, the content of PG in the pgpB mutant cells markedly decreased from 1.6 to 0.6 x 10–16 mol cells–1. However, the pgpB mutant cells with the addition of exogenous PG showed a near recovery of PG content to the level of PG in the wild type (Table I). These results suggest that the synthesis of PG in the pgpB mutant is affected and the pgpB gene is involved in the biosynthesis of PG.
Characteristics of Photosynthetic Pigments in the Mutant To check the effect of the pgpB mutation on the photosynthetic pigments, the cellular content of chlorophyll (Chl) a and phycobilisome pigments were investigated for the wild type and pgpB mutant. A reduction in Chl a levels was observed in the mutant. During the growth phase, the Chl a content on a per-cell basis increased slightly for the wild type, whereas it changed little for the pgpB mutant, being only about 54% of the level in the wild type (Fig. 5A ). In the meantime, the phycobilisome pigments such as phycocyanin and allophycocyanin in the mutant were little affected throughout growth and maintained at almost the same levels, similar to the wild type (data not shown). These findings indicate that the contents of Chl-protein complexes were reduced and the levels of phycobilisomes were nearly constant for the pgpB mutant cells following the decrease in the content of PG.
In accordance with the above results, the pgpB mutant cells showed the lower Chl absorbance, with a decrease in the ratio of Chl absorption at 680 nm to phycocyanin absorption at 630 nm, as compared to the wild type (Fig. 5B).
To characterize the effect of a decrease in the PG content on the oxygenic photosynthesis, the photosynthetic oxygen evolution activity (net photosynthesis, water to carbon dioxide) was determined for the pgpB mutant (Fig. 6 ). During the growth in liquid culture medium BG11, the photosynthetic activity on a per-cell basis of intact cells of the wild type increased gradually. By contrast, the activity of intact cells of the pgpB mutant remained almost constant. However, the CO2-dependent photosynthesis activity in the wild type was always higher than that in the pgpB mutant at the same culture time (Fig. 6A). The reduced photosynthetic activity of the mutant cells could, however, reach the similar level of the activity of the wild-type cells following addition of 6 µM PG to the growth medium for a further 4 d. These results indicate that the decrease in PG content brought about reduction in the cellular content of Chl a and, consequently, in the photosynthetic activity of a cell.
The effect of the reduced content of PG on PSII activity with an artificial electron acceptor, 2,6-dichloro-p-benzoquinone (DCBQ), was also determined (Table II ). As shown in Table II, the PSII activity in intact cells of the wild type and the pgpB mutant was essentially the same in the presence of PG. However, the pgpB mutant, in comparison with the wild type, showed about 73% of decrease in PSII activity when the cells were grown without the added PG. It is known that DCBQ shows an affinity for accepting electrons from the primary electron-accepting plastoquinone of PSII site of the D2 protein in the PSII reaction center (Schneider et al., 2004 ). These data suggest that the changes in the conformation of the PSII reaction center are induced by the decrease of the PG content in the pgpB mutant and the affinity of DCBQ to QB site is thereby changed. Consistent with the above results, the pgpB mutant showed decreased values in dark-level fluorescence yield (F0), the maximum fluorescence yield (Fm), and therefore a lower variable fluorescence yield (Fv). Consequently, the ratio of Fv/Fm, which is a measure of the photochemical efficiency of PSII in the dark-adapted state, was decreased in the mutant (Table III
).
To investigate further alterations of the pigment-protein complexes and the excitation energy transfer to the reaction centers, low temperature fluorescence spectra were measured for the wild type and the pgpB mutant. The 77 K fluorescence spectra of wild-type and mutant strains obtained after Chl a excitation at 436 nm are shown in Figure 7 . There are three distinct emission maxima at 684, 693, and 728 nm. The large emission peak at 728 nm is attributed to emanate primarily from PSI antenna, whereas the two peaks at 684 and 693 nm can be ascribed to emit mainly from the PSII core antenna complexes CP43 and CP47, respectively (Satoh et al., 1995 ). No position shift in the Chl fluorescence emission maxima was observed for both strains, indicating the complete assembly of PSII and PSI complexes in both wild type and mutant (Hartel et al., 1998). However, the ratio of PSII-associated fluorescence to PSI fluorescence was markedly reduced in the mutant cells as compared to the wild type. It reflects that there is a decrease in the number of PSII units, while the number of PSI units remains almost the same in the mutant (Krause and Weis, 1991). In addition, the magnitude of fluorescence emission at 684 nm slightly decreased relative to the 693 nm fluorescence emission in the pgpB mutant. These findings demonstrated that the molecular environment of the PSII core complexes and PSI is almost not affected, but more excitation energy is redistributed toward PSI in the mutant.
In this study, we isolated a mutant of Anabaena sp. PCC7120 through the disruption of a gene, alr1715. This gene shares considerable sequence identity with the pgpB genes of several nonphotosynthetical bacterial organisms (e.g. E. coli and B. cereus). A 320-amino acid sequence of this gene has a conserved domain at its C terminus with PgpB. In addition, there is very little similarity between the amino acid sequence of this gene and that of the PGP synthase (all4063) or the phosphatidate cytidylyltransferase (all3875) of Anabaena sp. PCC7120 (data not shown). Furthermore, inactivation of this gene could bring about a 30% reduction in PG content of intact cells, and supplementation of the alr1715 mutant with exogenous PG could restore a near wild-type state. Taken together, we believe that the alr1715 gene is involved in the pathway of PG biosynthesis in Anabaena sp. PCC7120 and most likely encodes a PGP phosphatase that catalyzes the formation of PGP.
The alr1715 mutant has the PG content reduced to only about 70% of that found in the wild type; the majority of PG was still present in this mutant. In addition, there is no transcript for this gene in the mutant as confirmed by RT-PCR, revealing that it is unlikely that the disrupted gene is still partially functional. These facts indicate that there is at least one other gene that encodes PGP phosphatase and thus accounts for the presence of residual PG in the pgpB mutant of Anabaena sp. PCC7120. Indeed, we found that one open reading frame, all7623, is highly homologous to the putative pgpB gene alr1715 through a similarity search of the genome of Anabaena sp. PCC7120 (37% identity and 53% similarity over 320 amino acids). The question of whether there is gene superfamily for PGP phosphatase in Anabaena sp. PCC7120 needs to be clarified and is currently under investigation in our group. In E. coli, at least two genes, pgpA and pgpB, and gene products responsible for the in vivo conversion of PGP to PG were identified (Icho, 1988a
Although only 30% of PG content in the pgpB mutant was reduced in comparison with the wild type, the visible phenotype and photosynthetic activity were considerably affected. The pgpB mutant showed a reduced photoautotrophic growth and a pale blue-green appearance. This is similar to the null PG-deficient strains of Synechocystis sp. PCC6803 (Hagio et al., 2000 The alteration of visible phenotype in the pgpB mutant could be related to the changes in pigment composition and photosynthetic light utilization. Indeed, as compared to the wild type, the pgpB mutant had a reduction in the content of Chl a on a per-cell basis, the sole Chl species of PSI and PSII complexes, with phycobilisome pigments being less affected, suggesting the decreased levels of Chl-protein complexes in mutant cells with decreasing PG content. Low temperature fluorescence measurements suggest that the PG deficiency in the mutant led to the structural modifications of the reaction center/core antenna complex of PSII. The mutant showed no change in the PSI and PSII fluorescence maxima positions following Chl a excitation at 436 nm, suggesting that the molecular environment or binding environment of the Chl a emitting from the core antenna proteins CP43 and CP47 of PSII core antenna complexes and the Chl a from PSI core antenna complexes are not affected by the PG reduction in the mutant. However, the mutant spectrum is characterized by a decrease in the magnitude of the fluorescence emission of PSII. This effect reflects that PG deficiency causes the structural changes (reduction) of the PSII antenna and/or redistribution of excitation energy more in favor of PSI.
The decrease in the low temperature Chl fluorescence ratio of PSII to PSI together with the decreased Chl content in the pgpB mutant indicated a decreased number of PSII (Vermaas et al., 1994
The pgpB mutant exhibited a decrease in PG content, which was accompanied concomitantly by an increase in the content of SQDG, the other anionic lipid of cyanobacteria and chloroplasts. It has been thought that photosynthetic organisms require constant anionic lipid content by keeping a certain total level of PG and SQDG; if necessary, the two anionic lipids could substitute for each other to some extent, such that the photosynthetic apparatus can function properly and normally. It was found that several photosynthetic organisms, depending on available sources of nutrition, could reciprocally adjust the PG and SQDG contents and maintain the level of the sum of these anionic lipids (Benning et al., 1993 In summary, we generated and characterized a PG-deficient mutant from Anabaena sp. PCC7120 with the disrupted alr1715 gene. The inactivation resulted in a reduction by 30% in the PG content and had a marked effect on growth, Chl levels, photosynthetic oxygen evolution, and photochemistry, particularly associated with PSII activity. The phenotype of the mutant was similar but less severe than those for other Synechocystis PG biosynthesis mutants that have reductions in PG by as much as 80% to 90% and require exogenous PG supplements for growth. These findings indicate that the alr1715 gene of Anabaena sp. PCC7120 is involved in the biosynthesis of PG and is essential for photosynthesis. Therefore, this mutant provides a promising opportunity toward understanding the biosynthesis and function of PG in photosynthetic organisms.
Cell Culture
Wild-type and mutant cells of Anabaena (Nostoc) sp. strain PCC 7120 (in this study referred to as Anabaena sp. PCC 7120) were grown photoautotrophically in BG11 medium (Allen, 1968
Cells of Anabaena sp. PCC7120 grown in the mid-log phase of growth were harvested by centrifugation (5,000g, 10 min, 4°C) and suspended in a buffer with 10 mM Tris and 0.1 mM EDTA, pH 7.5. DNA was isolated from these cells as described in Cai and Wolk (1990)
Two primers were designed for PCR to clone the putative pgpB gene on the genome of Anabaena sp. PCC7120, with upper primer CCGCCATGGTTTTACTCTTGTTTATCCTCTATC (NcoI) and lower primer CGTCTGCAGTTAGAGGCACAAATTCTAGAGGTA (PstI). Both primers have three extra bases and one NcoI or PstI restriction site (underlined) at the 5' end, respectively. PCR was performed according to standard procedures using Taq DNA polymerase with a DNA Thermal Cycler (Perkin-Elmer) under the following thermocycle conditions: 5 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 60 s at 72°C, and 10 min at 72°C at end to allow complete elongation of all product DNA.
A 4,822-bp DNA fragment containing the gene alr1715 with a 962-bp length was amplified by PCR with the genomic DNA of Anabaena sp. PCC7120 as a template and then ligated into pMD18-T vector at its EcoRV restriction site for production of the plasmid pPGP1. At one restriction site, XbaI of the alr1715 gene of the pPGP1 plasmid, we inserted the kanamycin phosphotransferase gene (1.2 kb) from pUC4K by digestion with SalI to yield a plasmid, pPGP2. The pPGP2 plasmid containing the disrupted putative pgpB gene was then cloned to the NcoI/SphI-digested conjugative plasmid pRL271, a cloning vector for sacB-mediated positive selection for targeted gene replacement and double recombinants in cyanobacteria (Black et al., 1993
RNA from Anabaena sp. PCC 7120 was prepared as described previously (Golden et al., 1987
The total lipids were extracted from the cells of the wild type and the mutant according to the method of Sato and Murata (1988)
Chl was extracted with 100% methanol from cell pellets obtained from 1 mL of the wild-type and mutant cell cultures, and the Chl content was determined according to Porra et al. (1989)
Photosynthetic O2 evolution coupled with CO2 fixation (net photosynthesis) was measured for intact cells equivalent to 2.5 µg Chl/mL in culture medium containing 10 mM NaHCO3 as the electron acceptor with a Clark-type oxygen electrode (DW 1 Liquid Clark electrode, Hansatech Institute). PSII activity was measured in BG11 medium containing intact cells equivalent to 2.5 µg Chl/mL, 0.25 mM DCBQ was used as the electron acceptor, and 0.25 mM ferricyanide added to keep the quinones in an oxidized form. For each measurement, an aliquot of cell culture was spun down and resuspended in 1 mL of fresh BG11 medium. The reaction mixture was kept at 30°C and illuminated under saturating white light (approximately 1,000 µmol photon m–2 s–1).
Low temperature (77 K) Chl emission fluorescence spectra were recorded in a spectrofluorimeter (F-4500, Hitachi) with 5 nm/2.5 nm (excitation/emission) slit widths. For fluorescence measurements, cells were diluted in BG11 medium containing 20% glycerol to a final Chl concentration of 5 µg/mL and frozen in liquid nitrogen (Schneider et al., 2001
Room temperature Chl fluorescence was determined with a pulse amplitude modulation fluorometer (PAM101, Walz) according to the procedure of Clarke et al. (1993) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers BAB78081/Q8YWA0.
We are grateful to Professor C. Peter Wolk (Michigan State University-U.S. Department of Energy Plant Research Laboratory, Michigan State University) for the generous gift of plasmid pRL271. We thank Dr. Zeneng Wang (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio) for critically reading the manuscript and giving useful suggestions. We also thank Professor Ding-Ji Shi (Institute of Botany, the Chinese Academy of Sciences, China) for his kind help throughout the experiments. Received May 11, 2006; returned for revision June 15, 2006; accepted June 15, 2006.
1 This work was supported by the Innovation Program of the Chinese Academy of Sciences.  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: Zhenle Yang (yangzhl{at}ibcas.ac.cn). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.083451. * Corresponding author; e-mail yangzhl{at}ibcas.ac.cn; fax 86–10–82594106.
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TOP
ABSTRACT
RESULTS
-positioned fatty acyl chains of the phospholipid molecules) treatment (Jordan et al., 1983
