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First published online May 19, 2006; 10.1104/pp.106.082859

Plant Physiology 141:1120-1127 (2006)
© 2006 American Society of Plant Biologists

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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Comparative Genomic Analysis Revealed a Gene for Monoglucosyldiacylglycerol Synthase, an Enzyme for Photosynthetic Membrane Lipid Synthesis in Cyanobacteria1

Koichiro Awai2,*, Takatoshi Kakimoto, Chie Awai, Takakazu Kaneko, Yuki Nakamura, Ken-ichiro Takamiya, Hajime Wada and Hiroyuki Ohta

Graduate School for Bioscience and Biotechnology (K.A., T. Kakimoto, C.A., Y.N., K.T., H.O.) and Research Center for the Evolving Earth and Planets (K.T., H.O.), Tokyo Institute of Technology, Midori-ku, Yokohama 226–8501, Japan; Kazusa DNA Research Institute, Kisarazu, Chiba 292–0818, Japan (T. Kaneko); and Graduate School of Arts and Sciences, University of Tokyo, Komaba, Tokyo 153–8902, Japan (H.W.)


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Cyanobacteria have a thylakoid lipid composition very similar to that of plant chloroplasts, yet cyanobacteria are proposed to synthesize monogalactosyldiacylglycerol (MGDG), a major membrane polar lipid in photosynthetic membranes, by a different pathway. In addition, plant MGDG synthase has been cloned, but no ortholog has been reported in cyanobacterial genomes. We report here identification of the gene for monoglucosyldiacylglycerol (MGlcDG) synthase, which catalyzes the first step of galactolipid synthesis in cyanobacteria. Using comparative genomic analysis, candidates for the gene were selected based on the criteria that the enzyme activity is conserved between two species of cyanobacteria (unicellular [Synechocystis sp. PCC 6803] and filamentous [Anabaena sp. PCC 7120]), and we assumed three characteristics of the enzyme; namely, it harbors a glycosyltransferase motif, falls into a category of genes with unknown function, and shares significant similarity in amino acid sequence between these two cyanobacteria. By a motif search of all genes of Synechocystis, BLAST searches, and similarity searches between these two cyanobacteria, we identified four candidates for the enzyme that have all the characteristics we predicted. When expressed in Escherichia coli, one of the Synechocystis candidate proteins showed MGlcDG synthase activity in a UDP-glucose-dependent manner. The ortholog in Anabaena also showed the same activity. The enzyme was predicted to require a divalent cation for its activity, and this was confirmed by biochemical analysis. The MGlcDG synthase and the plant MGDG synthase shared low similarity, supporting the presumption that cyanobacteria and plants utilize different pathways to synthesize MGDG.

Monogalactosyldiacylglycerol (MGDG) is a typical lipid in oxygen-evolving photosynthetic organisms and makes up over one-half of the thylakoid membrane of chloroplasts and cyanobacteria. Considering its abundance in the membrane, it is believed that MGDG plays a pivotal role in the development of photosynthetic membrane structure. Moreover, it is reported that, in cyanobacteria, one and six molecules of MGDG are found in PSI (Jordan et al., 2001Go) and PSII (Loll et al., 2005Go) complexes, respectively. Thus, MGDG is involved not only in maintaining membrane structure but also in comprising an integral structural component of the photosynthetic complex, and it is thought to be essential for oxygenic photosynthetic organisms.

Although lipid composition of the thylakoid membrane is very similar between chloroplasts and cyanobacteria (Joyard et al., 1998Go), the biosynthetic pathway of MGDG is thought to differ in the assembly of the head group. In plants, MGDG synthases utilize UDP-Gal and sn-1,2-diacylglycerol (DAG) as substrates and transfer the Gal moiety of UDP-Gal to DAG (Shimojima et al., 1997Go). On the other hand, it is presumed by biochemical analysis that cyanobacteria utilize UDP-Glc for the first step of MGDG synthesis (Sato and Murata 1982aGo; Fig. 1 ); namely, the Glc moiety of UDP-Glc is transferred to DAG and monoglucosyldiacylglycerol (MGlcDG) is synthesized as an intermediate. Then, an epimerase changes the Glc head to Gal to give rise to MGDG. The gene for plant MGDG synthase has been cloned (Shimojima et al., 1997Go), but no ortholog was found in cyanobacterial genomes even though many species have been sequenced. According to these facts, it is believed that plants and cyanobacteria have different genes encoding enzymes of MGDG synthesis, although cyanobacteria are thought to be the endosymbiotic ancestors of chloroplasts.


Figure 1
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  		Figure 1. Scheme for MGDG synthesis. A, MGDG synthesis in cyanobacteria. First, MGlcDG is synthesized from DAG and UDP-Glc by MGlcDG synthase. Then, an epimerase converts MGlcDG into MGDG. B, MGDG synthesis in photosynthetic eukaryotes. MGDG is synthesized from UDP-Gal and DAG by the one-step reaction of MGDG synthase. Note that UDP-Gal is also formed by epimerization of UDP-Glc.

 
MGlcDG is not a unique lipid of cyanobacteria. In the cell wall-less bacterium Acholeplasma laidlawii, MGlcDG is the most abundant lipid and occupies about one-half of its membrane (Foht et al., 1995Go). In Bacillus subtilis, MGlcDG is about 10% of the total lipid (Matsumoto et al., 1998Go). Although the genes for the MGlcDG synthases of these bacteria have been cloned (Jorasch et al., 1998Go; Berg et al., 2001Go), again there is no ortholog in any cyanobacterial genome. Thus, to date, the gene for MGlcDG synthase is unknown in cyanobacteria even though the first genome sequence of the bacteria was completed almost a decade ago (Kaneko et al., 1996Go).

To identify the genes responsible for galactolipid synthesis in cyanobacteria, we took advantage of available databases. According to a simple method described below, the gene for MGlcDG synthase was identified from two species of cyanobacteria and the enzyme activity was analyzed.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

MGlcDG Synthase Activity Is Conserved in Both Unicellular and Filamentous Cyanobacteria

To identify the gene for MGDG synthesis in cyanobacteria using a bioinformatics approach, we started by comparing two species of cyanobacteria, representative of unicellular and filamentous strains. Synechocystis sp. PCC 6803 (Synechocystis) and Anabaena sp. PCC 7120 (Anabaena) were chosen because their genomes had been fully sequenced (Kaneko et al., 1996Go, 2001Go). So far, four genes encoding enzymes for membrane-lipid assembly have been reported in cyanobacteria: UDP-sulfoquinovose synthase (Güler et al., 1996Go), sulfoquinovosyldiacylglycerol (SQDG) synthase (Güler et al., 2000Go), cytidine diphosphate-diacylglycerol synthase (Sato et al., 2000Go), and phosphatidylglycerol phosphate synthase (Hagio et al., 2000Go). In all cases, proteins are highly conserved between Synechocystis and Anabaena, being 84%, 72%, 62%, and 61% identical, respectively, suggesting that the enzymes for MGDG synthesis are also conserved between these two bacteria.

First, MGDG biosynthetic activity was analyzed to determine whether the conservation could be observed at the enzyme activity level between these bacteria. We used radiolabeled UDP-Glc as a substrate because the proposed pathway utilizes UDP-Glc to synthesize MGDG in the filamentous cyanobacterium Anabaena variabilis (Sato and Murata, 1982bGo). Using isolated membranes, we detected accumulation of a radioactive compound from both Synechocystis and Anabaena cochromatographing with cyanobacterial MGlcDG (Fig. 2 ). When we used UDP-Gal, we could not detect any accumulation of MGDG, which suggests that the accumulated lipid was synthesized via a UDP-Glc-dependent pathway, namely, by an MGlcDG synthase in both types of cyanobacteria.


Figure 2
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  		Figure 2. UDP-Glc-dependent glycolipid synthesis activity in both unicellular and filamentous cyanobacteria. Sugar transferase activities were measured using radiolabeled UDP-Glc or UDP-Gal. Lipids were chromatographed by a solvent system (acetone:toluene:water = 90:30:7 [v/v]) and visualized by autoradiography. Crude extract of Arabidopsis leaves was used as a control for UDP-Gal-dependent MGDG synthesis. Lipids of Synechocystis sp. PCC 6803 were visualized by {alpha}-naphthol for lipid standard. The identities of the minor radiolabeled bands were not determined.

 

Comparative Genomic Analysis Found Candidates for MGlcDG Synthase

We used a bioinformatics approach to identify the MGlcDG synthase gene by comparing the genome sequence of the two bacteria. We expected that MGlcDG synthase would have three characteristics. (1) Glycosyltransferase motifs should be present in the predicted primary structure of the enzyme. So far, numerous glycosyltransferase genes have been reported and categorized into 78 glycosyltransferase families (Coutinho et al., 2003Go). MGlcDG synthase should have similarity to one of these families. (2) The enzyme should not have a high overall similarity to proteins that have a well-known function. There are two reports of glucosyltransferase genes encoding for the enzymes that synthesize mono- or polyglucosyldiacylglycerol (Jorasch et al., 1998Go; Berg et al., 2001Go). However, no homolog has been found in any cyanobacterial genome other than SQDG synthase from Synechocystis (Berg et al., 2001Go). Thus, the MGlcDG synthase of cyanobacteria would be categorized as a protein of unknown function. (3) The enzyme would be conserved among oxygen-evolving photosynthetic organisms, where MGDG is mostly found. Thus, the protein was expected to have orthologs in both Synechocystis and Anabaena because of conservation of the enzyme activity described above.

Over 3,100 and 5,300 genes have been annotated in the genomes of Synechocystis and Anabaena, respectively. According to the above criteria, we first performed a motif search of all genes on the Synechocystis genome using the Pfam database (http://pfam.wustl.edu) because Synechocystis has fewer genes than Anabaena. We then picked 67 genes that have a glycosyltransferase motif and analyzed them by a BLAST search. Twenty-one genes turned out to be genes encoding protein of unknown function, and, among them, four (sll0071, sll1004, sll1377, and slr1508) were highly conserved only in cyanobacterial species, except slr1508. Table I shows the list of candidate genes. All of them have over 50% identity in amino acid sequence with the presumed ortholog of Anabaena, and two of them were predicted to have membrane-spanning domains.


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  		Table I. Candidate genes for MGDG synthesis

 

MGlcDG Synthase Is a UDP-Glc-Dependent Enzyme

We expressed these four candidate genes in Escherichia coli and measured MGlcDG synthase activity. When radiolabeled UDP-Glc was used as a substrate, the sll1377-encoded protein synthesized a lipid with label from UDP-Glc (Fig. 3 ). On the other hand, when UDP-Gal was used for any of the tested proteins, we could not find any accumulation of radiolabeled lipid, whereas the positive control cucumber (Cucumis sativus) MGDG synthase was able to utilize UDP-Gal for MGDG synthesis (Fig. 3). It was also found that E. coli expressing the protein encoded by sll1377 accumulates a glycolipid in its cell membranes, which cochromatographs with MGlcDG of Synechocystis (Fig. 4 ). This suggests that the protein encoded by sll1377 has an MGlcDG synthase activity both in vitro and in vivo.


Figure 3
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  		Figure 3. Sugar transferase activity of the candidate genes. Open reading frames of the genes were expressed in E. coli and the activity of crude extracts was measured using radiolabeled UDP-Glc (A) or UDP-Gal (B). Cucumber MGDG synthase (csMGD1) was used as a positive control for UDP-Gal-dependent sugar transfer. Small dots at the bottom indicate origin of TLC. TLC was developed with a solvent system (chloroform:methanol:water = 65:20:2). Incubation of the sll1377 enzyme with UDP-Glc resulted in the production of MGlcDG, whereas MGDG was formed in assays of the csMGD1 enzyme with UDP-Gal.

 

Figure 4
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  		Figure 4. Glycolipid accumulated in E. coli membrane. Glycolipids were isolated from transformed E. coli and analyzed by TLC. Lipids of Synechocystis sp. PCC 6803 and E. coli-expressing cucumber MGDG synthase were used as a control. TLC was developed by a solvent system (acetone:toluene:water = 90:30:7 [v/v]) and visualized by {alpha}-naphthol. pET24a, Vector control; sll1377, E. coli-expressing sll1377 gene; all4933, E. coli-expressing all4933 gene; csMGD1, E. coli-expressing cucumber MGDG synthase gene.

 
To confirm whether the protein encoded by sll1377 synthesizes MGlcDG, the glycolipid accumulating in the E. coli membrane was purified by thin-layer chromatography (TLC) and analyzed by proton-NMR spectroscopy (Fig. 5 ). Typically, a signal around 3.4 ppm is originated from the proton attached to carbon 3 (C3) of the hexose moiety of the glycolipid. In Glc, this proton gives rise to a triplet signal, whereas in Gal two doublets are derived from this proton. As shown in Figure 5B, a glycolipid from the transformant showed a triplet in that region, whereas MGDG from Synechocystis showed two doublets, indicating that the glycolipid has a Glc moiety on the head group. The spectrum also shows a doublet at 4.26 ppm annotated with the proton at the anomeric carbon 1. This chemical shift is characteristic of a beta-linkage between the sugar head group and the glycerol backbone at the C1 position. A doublet is also observed at 4.22 ppm in Figure 5A, suggesting a beta-linkage of cyanobacterial MGDG (Xu et al., 2003Go). According to these results, we conclude that sll1377 encodes the MGlcDG synthase of cyanobacteria, which utilizes UDP-Glc and DAG to synthesize 1,2-diacyl-3-O-beta-D-glucopyranosyl-sn-glycerol. We also expressed the ortholog of sll1377 from Anabaena (all3944) in E. coli and confirmed that the protein encoded by the gene has the same activity in vitro (data not shown) and in vivo (Fig. 4).


Figure 5
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  		Figure 5. Anomeric and epimeric portion of cyanobacterial MGDG (A) and glycolipids (B) accumulated in E. coli-expressing sll1377 protein. Glycolipids were isolated from Synechocystis or transformed E. coli and analyzed by 1H-NMR. Right arrow in the top column (3.48 ppm) points to the two doublet peaks characteristic of Gal, whereas the arrow in the bottom column (3.42 ppm) points to the triplet peak of Glc. Double peaks (around 4.2 ppm) indicated by left arrow are the peaks from a beta-glycosidic carbon atom of the hexose moiety in the head group of the glycolipids.

 

MGlcDG Synthase Belongs to the GT2 Family

Figure 6 shows an amino acid sequence alignment of MGlcDG synthase as predicted in Synechocystis (sll1377) and Anabaena (all4933). We found that every cyanobacterium species whose genome sequence had been completed has an ortholog in its genome. However, we could not find any ortholog of the cyanobacterial MGlcDG synthase gene in plant genomes. The enzyme shared more than 40% identity in amino acid sequence among cyanobacteria species, suggesting that the transferase is only conserved among them. There are one and three membrane-spanning domains predicted in the N and C termini of the protein, respectively. We also found D...DxD and QxxRW motifs, which are common to GT2 family transferases (Charnock et al., 2001Go). Based on this fact and the fact that the GT2 family is known to belong to the inverting glycosyltransferase family (e.g. cellulose synthase, chitin synthase) and the MGlcDG synthase makes a beta-linkage between Glc and glycerol backbone, it is reasonable to place MGlcDG synthase in the GT2 family. Some of the transferases of the GT2 family are also known to require divalent cations for their activity. In fact, crystallographic structure analysis of one GT2 family protein, SpsA in B. subtilis, revealed that a manganese ion is required for binding to the phosphate residue of UDP via the D...DxD motif (Charnock and Davies, 1999Go). Because the protein encoded by sll1377 protein not only has this motif but also has a similar secondary structure as SpsA (data not shown), the protein should also have a similar three-dimensional structure and bind UDP using a divalent cation.


Figure 6
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  		Figure 6. A, Amino acid alignment of MGlcDG synthase from Synechocystis (sll1377) and Anabaena (all4933). Black boxes, Identical amino acids; gray boxes, similar amino acids. Asterisks on the top of the sequences indicate position of D...DxD and QxxRW motifs. Predicted secondary structure is also shown at the bottom of the alignment. E, beta-Strand. H, {alpha}-Helix. B, Structural feature of cyanobacterial MGlcDG synthase with other GT2 family transferases. TMs, Transmembrane domains; SpsA, SpsA protein; CesA, cellulose synthase protein.

 

MGlcDG Synthase Requires Magnesium for Its Activity

Using recombinant MGlcDG synthase, the effect of divalent cations on its activity was analyzed (Fig. 7 ) because the native enzyme is known to require magnesium for activity (Sato and Murata, 1982bGo). Compared to the control, the enzyme activity was reduced to 12.4% when magnesium ions were omitted from the reaction buffer. This residual activity is probably due to carryover of divalent cations from the E. coli lysate. To test this possibility, we added EDTA to chelate divalent cations and observed a further decrease in activity (to 5.3%). Thus, MGlcDG synthase is a divalent cation-dependent glycosyltransferase. We also analyzed whether the enzyme can utilize manganese instead of magnesium because secondary structure analysis showed significant similarity to SpsA protein, which utilizes manganese for UDP binding. As shown in Figure 7, manganese as well as calcium was not effective in producing activity. Thus, magnesium is the divalent cation that is required for the activity, probably for binding of a UDP molecule.


Figure 7
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  		Figure 7. Enzymatic property of MGlcDG synthase. Relative activity to control is described. MgCl2 (control): OPDAG, 25 mM MgCl2; –MgCl2: OPDAG, 0 mM MgCl2; EDTA: OPDAG, 5 mM EDTA; CaCl2: OPDAG, 25 mM CaCl2; MnCl2: OPDAG, 25 mM MnCl2; DPDAG: DPDAG, 25 mM MgCl2. SD is based on three independent experiments.

 
Substrate preference of the enzyme was also analyzed. Because we already found that the enzyme is UDP-Glc dependent (Fig. 3), the other substrate, DAG, was analyzed. Cyanobacteria lipids are known to have 16-carbon fatty acids at the sn-2 position of the glycerol backbone. Thus, we compared two types of DAG, namely, di-palmitoyl (DP) or 1-oleoyl-2-palmitoyl (OP) DAG, which have 16- or 18-carbon fatty acids at the sn-1 position, respectively. As shown in Figure 7, when DPDAG was used as a substrate, the activity was less than 20% of OPDAG. Thus, the MGlcDG synthase preferred OPDAG rather than DPDAG. This agrees with the fact that most MGDG from Synechocystis has 18-carbon fatty acids at the sn-1 position, whereas SQDG has more 16-carbon fatty acids at its sn-1 position (Murata et al., 1992Go). Moreover, it is reported that a native enzyme of MGlcDG synthase from A. variabilis also preferred to utilize OPDAG (Sato and Murata, 1982bGo). Because MGlcDG synthase and SQDG synthase share their substrate, DAG, this substrate specificity might affect fatty acid composition of those lipids in vivo.


   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
The key finding of this research is the identification of the MGlcDG synthase gene of cyanobacteria. The gene was identified by a combined approach of comparative genomics, molecular biology, and enzymology. Hundreds of genome sequences are available now; however, among the annotated genes in those genomes, almost one-half are still categorized as a protein with unknown function. Our method described here is simply based on conservation of enzyme activity between two types of cyanobacteria and predicted characteristics of the enzyme sequence. Thus, we believe that this method is applicable to other enzymes in which activity is specifically conserved among certain groups of organisms and whose genome sequence has been completed. For example, MGlcDG epimerase and DGDG synthase of cyanobacteria are good candidates for future analysis.

To date, identification of two glucosyldiacylglycerol synthases has been reported, namely, ypfP of B. subtilis and MGlcDAG synthase of A. laidlawii (Jorasch et al., 1998Go; Berg et al., 2001Go). However, neither of these have significant sequence similarity to MGlcDG synthase of cyanobacteria. The ypfP gene encodes a UDP-Glc-dependent glucosyltransferase and synthesizes mono-, di-, tri-, and tetra-glucosyldiacylglycerol. This enzyme synthesizes beta-linked glucolipids; thus, the anomeric portion of the head group is the same as cyanobacterial MGlcDG. However, this enzyme has processive activity and most of the product lipid is polyglucosylated. Therefore, the mechanism of transfer is likely different from MGlcDG synthase of cyanobacteria. MGlcDAG synthase of A. laidlawii transfers only one Glc moiety from UDP-Glc and synthesizes MGlcDG just as cyanobacterial MGlcDG synthase does. However, this enzyme is a retaining transferase that results in an {alpha}-linkage between Glc and DAG, which is different from cyanobacterial MGlcDG. Therefore, it is reasonable that these proteins do not have significant similarity to each other. Recently, the gene for a sugar transferase that can catalyze beta-linked MGlcDG synthesis from the green nonsulfur bacteria Chloroflexus aurantiacus was reported (Hölzl et al., 2005Go). This protein has higher similarity to the plant-type MGDG synthase than to cyanobacterial MGlcDG synthase even though the protein transfers Glc to acceptors. However, by using cyanobacteria, it was shown that the enzyme catalyzes transfer of Glc to MGDG rather than to DAG in vivo, similar to diglucosyldiacylglycerol synthase of A. laidlawii (Edman et al., 2003Go). Thus, the enzyme is a diglycosyldiacylglycerol synthase, probably not an MGlcDG synthase.

Based on structural analysis of the glycolipid accumulating in the E. coli transformant membrane, cyanobacterial MGlcDG synthase was found to catalyze only MGlcDG synthesis and does not have an epimerase activity to change the head group of MGlcDG to give rise to MGDG. Thus, there must be another gene encoding the epimerase to produce MGDG from MGlcDG in the cyanobacterial genome. We expected that the transferase and epimerase are present in an operon, at least in some cyanobacterial species, because these enzymes are involved in sequential reactions. But, so far, we could not find any candidate for the epimerase from available genomic sequences. NMR analysis also revealed that the MGlcDG synthesized by the cyanobacterial enzyme has a beta-linkage between Glc and DAG. This matches the finding that the MGDG of cyanobacteria has a beta-linkage between Gal and glycerol, which was confirmed by NMR analysis and reported by glycolytic analyses (Feige et al., 1980Go). Thus, one function of the epimerase is epimerization of C4 of Glc in MGlcDG, although Hölzl et al. (2005)Go showed that the epimerase can epimerize Glc attached to DAG, whether the linkage is {alpha} or beta. Our results also suggest that E. coli does not have epimerase activity because we did not see any accumulation of MGDG in the transformant's membrane. The mechanism of epimerization is still unknown.

We attempted to knock out the MGlcDG synthase gene, but no null mutant could be segregated. Although so far proposed as just an intermediate for MGDG synthesis, it is possible that MGlcDG is an essential lipid for cyanobacteria and this is the reason why cyanobacteria have a distinct pathway from plants for MGDG synthesis. Actually, in media replete with Glc, Synechocystis is known to accumulate MGlcDG to more than 12% of total membrane lipids (Sato, 1994Go), indicating that the lipid is not a minor component in certain conditions. Moreover, MGlcDG was recently suggested as a heat shock lipid that is involved in acquired heat thermotolerance of heat/light-primed cyanobacterial thylakoids (Balogi et al., 2005Go). Currently, we are trying to feed MGlcDG in the medium, as reported for knockout mutants of the other membrane lipid synthases (Hagio et al., 2000Go; Sato et al., 2000Go; Aoki et al., 2004Go), to determine whether the lipid can complement the mutation and help to isolate a null mutant.

In the Arabidopsis (Arabidopsis thaliana) genome, we have identified three genes for MGDG synthases, which fall into two classes (Awai et al., 2001Go; Kobayashi et al., 2004Go). However, no ortholog has been found on cyanobacterial genomes. Similarly, no ortholog of the MGlcDG synthase was encoded in the Arabidopsis genome. Plant-type MGDG synthase is conserved from higher plants like Arabidopsis, rice (Oryza sativa), maize (Zea mays), soybean (Glycine max), tobacco (Nicotiana tabacum), and cucumber, to moss (Physcomitrella patens), green algae (Chlamydomonas reinhardtii), and red algae (Cyanidioschyzon merolae), whereas the MGlcDG synthase genes are common throughout cyanobacterial genomes instead of the plant-type MGDG synthases. This observation completely agrees with the fact that accumulation of MGlcDG is only seen in cyanobacteria species and not in the eukaryotes described above (Feige et al., 1980Go). Thus, our data described here support the presumption that cyanobacteria have a distinct system to synthesize MGDG from that of the chloroplast, even though the bacteria are thought to be evolutionary ancestors of chloroplasts. The other galactolipid, digalactosyldiacylglycerol (DGDG), is synthesized from MGDG. In plants, the gene for bulk synthesis of DGDG and its paralog have been identified (Dörmann et al., 1999Go). These enzymes are reported to utilize UDP-Gal as the substrate (Kelly and Dörmann, 2002Go; Kelly et al., 2003Go). Cyanobacteria DGDG synthase is also presumed to utilize a nucleotide sugar (probably UDP-Gal; Sato and Murata, 1982aGo); however, no ortholog of plant DGDG synthase has been found in any cyanobacteria genome. We still do not know which genes correspond to cyanobacterial DGDG synthase, although, together with MGlcDG synthase, identification of the genes will shed light on the evolutionary origin of galactolipid synthesis of oxygen-evolving photosynthetic organisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Materials

UDP-Gal and UDP-Glc were purchased from Sigma, radioactive UDP-[14C]Glc from American Radiolabeled Chemicals, and UDP-[14C]Gal from Perkin-Elmer Life Sciences.


Cell Culture

Cyanobacteria (Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120) were grown in BG11 medium as described (Hagio et al., 2000Go). For enzyme assays, 7-d-old cyanobacteria cells (OD750 approximately 1.0) were collected, dissolved in buffer A (50 mM TES-KOH, pH 7.0, 25 mM MgCl2), and sonicated, followed by brief centrifugation to eliminate unbroken cell contamination. Then cell-free extracts were ultracentrifuged twice at 150,000g (Beckman Optra TL) with the same volume of buffer A. The precipitate (membrane fraction) was resuspended in buffer A and used for enzyme assay. The supernatant did not have glycolipid biosynthetic activity (data not shown).


Functional Expression of Candidate Genes in Escherichia coli

The open reading frame of each candidate gene was amplified by PCR, cloned into the pPICT2 vector (Kawaguchi et al., 2001Go), and sequenced. Then they were ligated into the NdeI site of the pET24a vector (Novagen) or the SalI site of the pQE31 vector (Qiagen) and transformed into Escherichia coli, BL21 (DE3), or XL1-Blue MRF, respectively. Protein production was induced by addition of IPTG as described (Gad et al., 2001Go). Then the cells were collected, washed, and resuspended with buffer A. After sonication, cell-free extracts were used to measure the enzyme activities.


In Vitro Assay for Glycolipid Biosynthetic Activity

Glycolipid biosynthetic activity was measured based on a previous method for MGDG synthase (Yamaryo et al., 2003Go). Fifty microliters of cell-free extract from E. coli or cyanobacteria membrane fraction were mixed with 110 µL of buffer A and 30 µL of DAG (4 mg/mL; 1-oleoyl-2-palmitoyl glycerol unless otherwise specified), which was dispersed in 0.01% Tween 20 (v/v). Then the mixture was preincubated for 10 min at 30°C and reaction was started by addition of 10 µL of UDP-[14C]Glc or Gal (8 mM; 308.3 Bq/nmol). For analysis of the effect of divalent cations, magnesium in buffer A was replaced by other cations. After 40-min incubation at 30°C, reactions were stopped by vigorous vortexing with 1 mL of ethyl acetate. The organic phase was washed twice with 0.45% (w/v) NaCl and 900 µL of the upper layer were applied to one-dimensional TLC analysis by solvent systems (chloroform:methanol:water = 65:20:2 or acetone:toluene:water = 90:30:7). Radioactive spots were detected by Image Plate (Fuji Photofilm) and quantified by Image Analyzer (Storm; Amersham Biosciences).


Glycolipid Structure Analysis

Membrane lipids of Synechocystis and the E. coli-expressing sll1377 protein were extracted according to Bligh and Dyer (1959)Go. Then, glycolipids were isolated by TLC and dissolved in CDCl3/CD3OD/CD3OOD (10:10:1 by volume) as described (Xu et al., 2003Go). Data were collected on a Varian Inova-600 spectrometer at 600 MHz for protons and analyzed with the Varian software package.


Database Analysis

For the motif search, the Pfam database (http://pfam.wustl.edu) was used. By using a whole open reading frame sequence of Synechocystis sp. PCC 6803, approximately 33,000 motifs were listed. Among them, we found 270 genes that have glycosyltransferase motifs. Then the genes, which have E values >1, were eliminated. Sixty-seven genes were applied to further BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) and 21 genes were found encoding proteins of unknown function or less than 30% similarity to the other proteins with well-known function. Secondary structure prediction was done using the Meta Server (http://bioinfo.pl).

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers BAA18254 and BAB76632.


   ACKNOWLEDGMENTS
 
We wish to thank Christoph Benning, who allowed portions of this research to be performed in his laboratory at the Department of Biochemistry and Molecular Biology, Michigan State University. We are also grateful to John Ohlrogge and Carl Andre for reading the manuscript and to Wayne Riekhof for helping with NMR analysis.

Received May 3, 2006; returned for revision May 9, 2006; accepted May 9, 2006.


   FOOTNOTES
 
1 This work was supported by the 21st Century Center of Excellence Program ("How to build habitable planets"), Tokyo Institute of Technology, sponsored by the Ministry of Education, Culture, Sports, Technology and Science, Japan, and in part by Grants-in-Aid for Scientific Research on Priority Areas (grant nos. 15380049 and 17051009). Back

2 Present address: Graduate School of Science and Engineering, Saitama University, Saitama 338–8570, Japan. Back

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: Koichiro Awai (awai{at}molbiol.saitama-u.ac.jp).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.082859.

* Corresponding author; e-mail awai{at}molbiol.saitama-u.ac.jp; fax 81–48–858–3384.


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