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Membrane lipids of Rhodopseudomonas viridis
Biochimica et Biophysica Acta 1347 Ž1997. 151–163
Membrane lipids of Rhodopseudomonas Õiridis ¨ Michael Linscheid a , Bernd W.K. Diehl b, Monika Overmohle ¨ c , Iris Riedl c , Ernst Heinz
c,)
a
c
Institut fur ¨ Spektrochemie, Bunsen-Kirchhoffstr. 11, D-44139 Dortmund, Germany b Spectral SerÕice GmbH, Vogelsanger Str. 250, D-50825 Koln, ¨ Germany Institut fur ¨ Allgemeine Botanik, UniÕersitat ¨ Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany Received 21 March 1997; revised 22 April 1997; accepted 24 April 1997
Abstract In search of the precyanobacterial origin of the typical thylakoid lipids found in cyanobacteria and chloroplasts, we analyzed the polar lipids of the anaerobic phototrophic bacterium Rhodopseudomonas Õiridis. Glycolipids Žmonogalactosyl-, digalactosyl- and glucuronosyl diacylglycerol., phospholipids Žphosphatidyl choline, -ethanolamine, -glycerol and cardiolipin. and an ornithine lipid were isolated and identified by NMR Ž 1 H, 13 C, 31 P. and mass spectrometry. Positional distribution and pairing of fatty acids in molecular species show small, but significant differences between glyco- and phospholipids. In this context, a new enzymatic method is described for assigning the enantiomeric structure of the diacylglycerol moiety in glyco- and phospholipids. 14 C-Labelling studies suggest that monogalactosyl diacylglycerol is formed by galactosylation of diacylglycerol as in chloroplasts and not by glucosylation followed by epimerization as in cyanobacteria. The two 1,6-linked galactopyranose residues of digalactosyl diacylglycerol are both in b-linkage and thus differ from the corresponding chloroplast lipid with its a-b-sequence. R. Õiridis does not contain the sulfolipid, and even phosphate starvation does not induce the synthesis of this most characteristic thylakoid lipid, which on the other hand is present in other anaerobic phototrophic bacteria. q 1997 Elsevier Science B.V. Keywords: Monogalactosyl diacylglycerol; Digalactosyl diacylglycerol; Glucuronosyl diacylglycerol; Ornithine lipid; Phospholipid
1. Introduction A common feature of cyanobacteria, eukaryotic algae and plants is their ability to carry out oxygenic photosynthesis, which operates with two photosystems and water as abundant electron donor. Apart Abbreviations: MGD, DGD, GAD, monogalactosyl-, digalactosyl-, glucuronosyl diacylglycerol; PA, PC, PE, PG, phosphatidic acid, phosphatidyl choline, -ethanolamine, -glycerol; CL, cardiolipin, diphosphatidylglycerol; OL, ornithine lipid; TLC, thin-layer chromatography.; Fatty acids are characterized by carbon and double bond numbers separated by a double point. ) Corresponding author. Fax: q49 40 82282254. E-mail: [email protected]
from similarities in membrane proteins, the thylakoids of these organisms are characterized by a simple set of three glycolipids and one phospholipid w1x. In addition to this advanced type of photosynthesis, various forms of anoxygenic photosynthesis are realized in several groups of prokaryotes. They all use only a single photosystem with a reaction center, which may resemble either photosystem II or photosystem I of oxygenic organisms. It has been speculated that the cooperation of both systems as found in present-day organisms might be the result of a precyanobacterial symbiosis or gene transfer between two different photosynthetically active prokaryotes w2,3x. Comparable to the attempts to trace back the
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 6 5 - 9
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origin of the two reaction centers, similar questions can be asked regarding a precyanobacterial origin of the present-day thylakoid lipids, which likewise may comprise components from both ancestors. From this point of view, a detailed analysis of lipids from phototrophic bacteria other than cyanobacteria receives relevance in addition to the increasing taxonomical use of lipid and fatty acid patterns w4x. The most characteristic thylakoid lipids are two galactolipids and a sulfolipid w1x. In the course of our studies on the biosynthesis of these glycolipids we realized that also R. Õiridis contains two galactolipids. Since such components had not been observed before in this organism w4–6x, we carried out a more detailed analysis of its other membrane lipids as well. In this context, we were particularly interested in the labelling of monogalactosyl diacylglycerol, since cyanobacteria and higher plants show a small but significant difference in the biosynthesis of this compound w1x. This difference does not fit into the general picture, according to which chloroplasts of higher plants are the result of a second symbiosis involving cyanobacteria as domesticated guests in a finally eucaryotic cell w7x. A preliminary note of our results has been published w8x.
2. Materials and methods 2.1. Growth of organism and lipid extraction Rhodopseudomonas Õiridis DSM 134 was cultured anaerobically w9x in screw-cap bottles Ž1 l. in the modified DSM 27 medium under photosynthetic conditions Žtungsten lamp, 60 W, at a distance of 60 m Erm2rs. at 258C. This medium contains 3.7 mM phosphate, which in experiments on phosphate starvation was reduced to various degrees down to 50 m M. Before autoclaving Ž20 min at 1208C. the medium was freed from oxygen by bubbling for 5 min with Argon. A 1-l bottle was inoculated with 10 ml of culture and grown for 3 weeks before harvesting by centrifugation Ž15 min at 9500 = g . yielding 2–3 ml of packed cell volume per 1 l of culture. The sedimented cells were boiled for a few min in water, resedimented, resuspended in chloroformrmethanol Ž1:1, vrv, as throughout in the following. and kept overnight at 48C on a shaker. After addition of chlo-
roform to increase the chloroformrmethanol ratio to 2:1, the organic mixture was washed with 0.2 volumes of NaCl solution Ž0.45%, wrv.. The resultant subphase was withdrawn, freed from solvent and redissolved in a small volume of chloroform. The other strains available from this organism Ž R. Õiridis DSM 133 and DSM 136. were grown and extracted under the same conditions. They contained the same lipids as found in strain DSM 134. Occasionally, cell suspensions were spread on agar plates w9x for aerobic heterotrophic growth to ascertain the absence of microbial contamination. 2.2. Instrumental methods 1
H- and 13 C-NMR spectra were recorded using a JEOL GX 400 spectrometer. Electrospray mass spectra were acquired using a MAT 95 double focusing sector field mass spectrometer with a home-made electrospray ion source. For in-source collision induced dissociation Ž CID., the potential between the desolvation capillary and the skimmer was set to 200 V. GLCrMS for the analysis of the fatty acid pyrrolidides w10x was performed on a DB5 column using a VG 7070 E sector field mass spectrometer equipped with a Sichromat-1 GC ŽSiemens.. For recording 31 P-NMR spectra w11x, the sample of total, unfractionated lipid Žabout 100 mg. was dissolved in 0.6 ml of methanolrCDCl 3 Ž1:2.. 0.4 ml Cs-EDTA solution Ž see below. is added and the mixture shaken vigorously. Then 0.2 ml H 2 O is added and the sample shaken again followed by short centrifugation for phase separation. The complete sample Ž both phases. is poured Žnot pipetted. into the NMR tube. After clearing of the lower organic phase the measurement can be started. The pulse program used CPD proton decoupling, PW of 30 deg., 2.8 s acquisition time, 0.3 s relaxation delay, sweep with 5700 Hz and 32 k data points. The molar ratios of phospholipids are calculated from signals areas. The Cs-EDTA solution is prepared as follows: EDTA Ž0.2 M in D 2 O. is titrated to pH 8.5 with CsCO 3. This solution is diluted 1:4 Žvrv. with methanol. 2.3. Separation and isolation of lipid components The lipid extracts Žin portions of 50–100 mg. dissolved in chloroform were applied to a DE-Sep-
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hacel column Ž30 ml, precycled sequentially with 0.1 N NaOH, acetic acid, methanol and chloroform. and eluted with 200-ml portions of chloroformrmethanol mixtures of increasing methanol content Ž2, 10, 30, 100%. and in a final step with 200 ml of chloroformrmethanol Ž4:1. containing 10 mM ammonium acetate and 0.26 M ammonium hydroxide. The resulting fractions were freed from solvents Ž the last one after aqueous partitioning. and used for isolation of individual lipids by preparative TLC in the following solvent systems: Ža. chloroformrmethanolr25% ammonia Ž65:35:5, separates PE from OL and GAD from PGrCL.; Žb. acetonerbenzenerwater Ž91:30:8, separates MGD and OL from DGD . ; Ž c . chloroformrmethanolracetic acidrwater Ž91:30:4:4, separates PG from CL.. 2.4. Analysis and deriÕatization of lipids Analysis of fatty acids and their positional distribution was carried out as described before by preparation of p-bromophenacyl esters and their separation by HPLC w12x. Molecular species were analyzed after enzymatic or chemical release of diacylglycerols w13x, formation of dinitrobenzoyl derivatives and their separation by HPLC as described before w14x. Quantitation of individual components was based on fatty acid content, which was determined via internal standard after two-dimensional TLC Žsee above.. Spots were scraped off, mixed with margaric acid ŽC17:0, 120 nmol. and heated for 1 h at 908C in 0.2 N KOH in methanolrwater Ž7:3.. After acidification free fatty acids were extracted for preparation of pbromophenacyl esters. For NMR and mass spectrometry the two galactolipids were acetylated and hydrogenated as described before w15x. GAD was first converted to the methyl ester by treatment of the protonated lipid with diazomethane followed by acetylation w16x. The methyl ester formation resulted in an increased Rf-value Žfrom 0.4 to 0.85 in solvent a.. The derivatized lipid was purified by preparative TLC with chloroformrmethanol Ž100:1.. The 1 H-signals of the glucuronosyl Ž numbered u1-u5. and glycerol Žnumbered g1-g3. moieties Žrecorded in CDCl 3 , shifts given in ppm, coupling constants J in Hz. were assigned as follows: 5.49 Žu4, dd, 10r10.; 5.21 Žu1, d, 3.; 5.21 Žg2, m, 4.5r5.; 5.16 Žu3, dd, 10r10.; 4.87 Žu2, dd,
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3r10.; 4.36 Žg1, dd, 12r4.5 or 5.; 4.32 Žu5, d, 10.; 4.15 Ž g1X , dd, 12r5 or 4.5. ; 3.86 Ž g3, dd, 12r4.5 or 5.; 3.75 ŽOCH 3 of methyl ester at u6, s.; 3.67 Žg3X , dd, 12r5 or 4.5.. When OL was treated in its protonated form with diazomethane, the R f-value of the resultant methyl ester increased from 0.3 to 0.9 as compared to the underivatized lipid in solvent Ža.. 2.5. Acetate-labelling of lipids A batch of R. Õiridis was grown for 4 days in 1 l of medium before harvesting. The following manipulations were carried out under sterile conditions. The cells were washed three times in medium without carbon source and then resuspended in this medium. Aliquots of this cell suspension Ž0.3–0.5 ml, depending on the number of parallel experiments and their duration. were placed into Sovirel tubes and gassed with Argon. Following the addition of w1- 14 Cxacetate Ž2 m Ci, 52 m Cirm mol, aqueous solution of sodium salt., the tubes were capped and placed in nearly horizontal position in the fumehood under normal laboratory light Ž6 m Erm2rs.. After incubation for various times Ž15 min, 30 min, 1, 2, 5, 28, 72 h. the samples were heated for a few minutes to boiling after addition of hot water and subsequently washed 3 times with bicarbonate solution Ž0.1 M. to remove excess of acetate. Lipid extraction of the sedimented cells was carried out as described above. The incorporation of radioactivity into lipids varied from 1.5 to 6.1% depending on sampling time and incubation volume Žlarger at the shorter times.. To resolve glucosyl from galactosyl diacylglycerol by one-dimensional TLC w17x, the two solvent systems used were Žb. and Žd. diethyl etherrisopropanolrmethanol Ž100:4.5:2.5.. Developed plates were scanned and subjected to radioautography by exposure for one to two weeks Ž50 000 to 100 000 dpm. . The sample obtained after 28 h labelling was used for two-dimensional separation, in the first direction with solvent Že. chloroformrmethanolrwater Ž65:25:4. and in the second dimension with solvent Žf. chloroformracetonermethanolracetic acidrwater Ž50:20:10:10:5.. Sulfate labelling was carried out by incubating cells under identical conditions for 20 h in the presence of 19 m Ci of w 35 Sxsulfate Žabout 1000 mCirm mol. . For preparation of an authentic mixture of glucosyl and galactosyl diacylglycerol, a sample of a cyanobacte-
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rium Ž Anabaena sp.. was incubated under similar, but aerobic conditions for 10, 20, 40, 75 min and used for subsequent lipid extraction as described above. 2.6. Enantiomeric structure of 1,2-diacylglycerols in glycolipids Radioactively labelled glycolipids were isolated from cells incubated for 28 h with w 14 Cxacetate Žsee above.. The glycolipids were individually subjected to the sequence of periodate oxidationrhydrazinolysis, which results in release of 1,2-diacylglycerols w13x. They were isolated by preparative TLC in diethyl etherrpetroleum ether Ž3:1., aliquots Ž20 000 dpm. mixed with CL Žfrom Serva, 75 m g. and evaporated to dryness in Sovirel tubes. To the dried lipid the following solutions were added w18x: 50 m l buffer Ž100 mM PipesrNaOH, 100 mM NaCl, 25 mM MgCl 2 , 100 mM n-octyl b-D-glucopyranoside, pH 6.8., 10 m l dithiothreitol Ž20 mM., diacylglycerol kinase Žfrom Calbiochem, 5 m l., 25 m l water and 10 m l ATP Ž50 mM.. After short dispersion in a sonication bath the reaction was run for 90 min at room temperature. The lipids were extracted by phase partitioning w19x between chloroformrmethanol Ž1:1, 2.3 ml. and H 3 PO4 Ž0.2 M in 1 M KCl, 1 ml. . After solvent removal the labelled lipids were analyzed by TLC in solvent Žg. chloroformrmethanolr25% ammonia Ž60:40:10. and radioscanning. 2.7. Configuration of galactose To determine the absolute configuration ŽD or L. of galactose w20x from galactolipids, a sample of the bacterial DGD Ž 1 mg. was heated overnight at 808C in SŽq.-2-butanol containing 1 M HCl. Subsequently the sample was applied to a TLC plate and chromatographed in CHCl 3rMeOH Ž 6:2. . The major middle band of butyl galactosides was extracted, subjected to acetylation with acetic anhydride and analyzed by capillary GLC Ž15 m fused silica column, 0.32 mm internal diameter, coated with 0.2 m m Rt x-2330, from Amchro. applying a temperature program from 1708C to 2108C in 10 min. Reference samples were prepared in the same way from D- and L-galactose Ž from Sigma. as well as from spinach
DGD. Assignment of GLC peaks was confirmed by coinjection.
3. Results The chromatographic resolution of the lipid mixture extracted from R. Õiridis revealed eight major components ŽFig. 1A., from which five had been recognized before w4–6x. We isolated all the different components ŽFig. 1B. in quantities sufficient for a closer analysis. For this purpose we used prefractionation by column chromatography followed by preparative TLC, because the complex mixture could not be separated satisfactorily into individual components by a single one-dimensional chromatography. The separation on DE-Sephacel proved to be the most suitable w21x, since the resulting fractions contained mixtures of components, which did not overlap in the subsequent separation. Column chromatography on CMcellulose and silica gel were less satisfactory in this respect. We confirmed the presence of PG, CL, PC and PE by cochromatography and proved their identity by 31 P-NMR spectroscopy Ž see below. . In addition to these previously detected phospholipids, R. Õiridis contains three glycolipids. MGD was found to have the structure of 1,2-di-0-acyl-3-0-b-D-galactopyranosyl-sn-glycerol, which is identical with MGD from cyanobacteria and plants. The structural details were deduced from mass and NMR spectra of the hydrogenated and acetylated compound. Apart from fatty acid details, these spectra were identical with those recorded before for MGD from higher plants which will not be detailed again w22x. In particular, the major fragments in the mass spectrum are assigned to the acetylated hexopyranose Žmrz 331. and the 1,2-diacylglycerol moieties Žmrz 579 and 607. of the parent molecule. Molecular ions were observed at mrz 867 and 895 corresponding to 18:0r16:0 and 18:0r18:0 fatty acid combinations, respectively. In the 1 H-NMR spectrum the large coupling constant of 8 Hz indicates the b-linkage of the anomeric proton at d 4.47 ppm in CDCl 3 , and the pattern of splitting and coupling constants for the ring protons at C-2, C-3, C-4 and C-5 are characteristic for the bgalactopyranose structure w23x. The enantiomeric structure of the diacylglycerol portion was analyzed
M. Linscheid et al.r Biochimica et Biophysica Acta 1347 (1997) 151–163
by the use of diacylglycerol kinase from Escherichia coli. This enzyme has a pronounced enantiomeric selectivity for phosphorylating sn-1,2- and discriminating against sn-2,3-diacylglycerols w24x. The radioactive diacylglycerol released by a chemical reaction sequence w13x from the labelled DGD was completely converted to phosphatidic acid ŽFig. 2., which proves that it is the sn-1,2-diacyl enantiomer. The two other glycolipids were subjected to the same reaction sequence and yielded the same results. The mass spectrum of the acetylated DGD is very similar to that recorded for the corresponding galactolipid from plants Žnot to be detailed again. and confirms the overall structure w22x. The electrospray mass spectrum of the nonhydrogenated compound showed molecular ions ŽMNaqrMKq. of major abundance at mrz 939r955 Ž18:1r16:1., 941r957 Ž18:1r16:0. and 967r983 Ž18:1r18:1.. The fatty acid patterns will be discussed separately Žsee below.. The enantiomeric configuration of the constituent galactose ŽD or L. was determined by formation of ŽS.2-butyl galactosides followed by acetylation and
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GLC, which separates ŽS.2-butyl-L-galactosides from the diastereomeric D-galactosides w20x. The application of this reaction sequence revealed that DGD contains exclusively D-galactose. Assuming that MGD is the biosynthetic precursor of DGD, we conclude that also MGD contains only D-galactose. On the other hand, the NMR spectrum of the hydrogenated and acetylated compound shows a significant difference between DGD from R. Õiridis and the normal DGD from higher plants w22x. In DGD from R. Õiridis both sugar residues display a parallel series of identical, but slightly displaced signals for ring and anomeric protons in a spectrum, which in all details is identical with that published for a DGD fraction isolated from Adzuki beans w25x. Most significant are the clearly separated doublets of the anomeric protons centered at 4.44 and 4.50 ppm Ž in CDCl 3 . with their characteristic coupling constant of 8 Hz. In the 13 C-NMR spectrum the two C1 signals show up very close to each other at 103.19 and 103.78 ppm and not with the wide shift difference as characteristic for the normal DGD with C1-a at about 97 ppm and C1-b
Fig. 1. Chromatographic resolution of polar lipids from Rhodopseudomonas Õiridis. A: Two-dimensional TLC of 14C-labelled lipids Ždetails in Materials and methods. in solvent e Žfirst dimension. and f Žsecond dimension.. B: Purity of individual lipids obtained by a combination of column chromatography and preparative TLC; separation in solvent c. C: Separation of 14C-labelled glucosyl diacylglycerol Žmarked by an asterisk. from the slightly slower moving galactosyl diacylglycerol Žmarked by a triangle. from Anabaena Žlane 1, after 40 min labelling.; separation of lipids from R. Õiridis labelled for 15 min Žlane 2., 30 min Žlane 3. and 60 min Žlane 4. in solvent b, in which glucosyl diacylglycerol is missing.
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Fig. 2. Conversion of 14C-labelled 1,2-diacyl-sn-glycerol ŽDAG, released from DGD. into phosphatidic acid ŽPA. by incubation with diacylglycerol kinase Žfrom E. coli . in the presence of ATP Žc, for details see Section 2.. The separation of reference DAG and PA by TLC in solvent g is shown in a and b.
at about 102 ppm w22x. Therefore, the DGD from R. Õiridis has the structure of 1,2-di-0-acyl-3-0-Ž6-0-bD-galactopyranosyl-b-D-galactopyranosyl. -sn-glycerol. This all-b-DGD differs from that of cyanobacteria and most higher plants by the fact that both sugars have identical anomeric b-configuration, whereas in higher plants the terminal galactose has usually aand the internal b-configuration Ž arb-DGD.. The all-b-DGD has been detected in low proportions of the normal arb-DGD in various plants throughout the plant kingdom including eucaryotic algae, mosses, ferns, gymnosperms and angiosperms w25x. Somewhat higher percentages are present in the seeds of some beans with a maximum of 36% of total DGD in Adzuki beans Ž seeds of Vigna angularis .. In this context it should be mentioned that the biosynthesis
of DGD from MGD in chloroplasts is catalyzed by an intergalactolipid galactosyltransferase localized in the outer envelope membrane w26x. An equivalent location in cyanobacteria or R. Õiridis, i.e., not in the plasma membrane but in a membrane surrounding the plasma membrane on the outside, seems to be rather unlikely. Bacteria synthesize the lipids of their plasma membrane in this membrane system and do not rely on lipid import from the outer membrane, which in addition cannot be considered to be the immediate phylogenetic precursor of the outer envelope membrane of plastids. The acidic glycolipid was at first recognized as GAD by its conversion to a more apolar compound on treatment with diazomethane w27x. The mass spectrum of the resultant and acetylated methyl ester showed prominent fragments at mrz 317 Žrepresenting the acetylated glucuronopyranosyl methyl ester w28x. and at mrz 575r577 Ž representing diacylglycerol moieties with 18:1r16:1 and 18:1r16:0 pairings of about equal intensity.. The 1 H-NMR spectrum of the acetylated methyl ester of GAD displayed the series of ring protons with coupling constants characteristic for the a-glucopyranose structure w23,29x, from which H1 at 5.21 with a coupling constant of 3 Hz should be mentioned. Due to the absence of protons at C6 of the glucuronosyl residue, the spectral region between 3.5–4.5 ppm is less crowded than in spectra of normal sugars and results in clearly separated signals for the two pairs of nonequivalent protons at C1 and C3 of glycerol Ž see Section 2.. The OL is characterized on TLC plates as a ninhydrin-positive, phosphate-negative lipid. On treatment of the protonated lipid with diazomethane, a methyl ester is formed which has an increased R f-value. It may be recalled that alkali treatment Ž saponification. of OL removes the O-ester group and induces cyclization of the other part to a ninhydrin-negative 3-acylamino-piperidine-2-one with lower R f-value w30x. The proof for the structure of OL as 2-N-3Xacyloxyacyl-ornithine was provided by mass and NMR spectroscopy. The 1 H-signals Žobtained as COSY-spectrum in CDCl 3 . confirmed previous analyses w30,31x and will not be detailed again. Only the 13 C-assignments are given Ž Fig. 3. , which have not been published before. Our data do not allow a configurational assignment at the asymmetrical cen-
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Fig. 3. w 13 CxNMR signals Žgiven in ppm. assigned in the spectrum of the ornithine lipid OL as recorded in CDCl 3. Carbon atoms in the ornithine moiety are numbered O1-O5, in the amide-linked hydroxy fatty acid H1-H4 and in the O-ester linked fatty acyl group F1 and F2. O3 and O4 assignments are tentative, since these signals fall in the range of methylene carbons from fatty acids. Other carbons including those at olefinic double bonds Ž129.89 and 129.76 ppm. are not included.
ters of O2 and H3, but based on previous experiments, it may be justified to assume that ornithine is the L-enantiomer w30x and the 3-hydroxy fatty acid has D-configuration, if it results from fatty acid biosynthesis due to the stereochemical outcome of the b-keto-acyl-ACP reductase reaction w32x. The electrospray mass spectra and particularly the in-source fragmentation indicated that the compound was an ornithine amino acid with a hydroxy fatty acid bound as amide and a second fatty acid linked via an O-ester function to the amide-linked acyl group. The O-ester-linked second fatty acid was released by saponification and found to be mainly Ž93%. 18:1 Žvaccenic acid, see below.. Therefore, the CID experiments could be used to analyze the
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amide-linked hydroxy fatty acids under the assumption that all O-esters were 18:1. This assignment was supported Žfirst column of Table 1. by the intensities of the corresponding fragment ions resulting from loss of 18:1 Žsecond column of Table 1.. In contrast to the homogenous O-ester group the b-hydroxy fatty acids are very heterogenous and cover the range from C16-C21 including saturated and monounsaturated fatty acids with even and uneven carbon numbers, from which those with even carbon numbers predominate. An intense fragment at mrz 184 can be assigned to the loss of the O-ester group and a fission at the hydroxy function of the amide-linked hydroxy fatty acid. This confirms the position of the hydroxyl group at carbon 3 ŽH3 in Fig. 3. and the identity of ornithine as the constituent amino acid. Complete loss of the amide-linked acyl group Ž including the nitrogen. results in fragments with mrz 115r116, which again confirm the overall structure w30,31,33– 35x. Ornithine lipids of this structure have been detected in various bacteria, but different structures may occur as well w36x. Quantitation of the individual lipids ŽTable 2. was in general agreement with the labelling pattern shown in Fig. 1A. PC is predominating, whereas the other components are present in lower and varying proportions. The qualitative and quantitative analyses of phospholipids were confirmed by 31 P-NMR spectroscopy w11x. The spectrum recorded with the unfrac-
Table 1 Fatty acid pairings in the ornithine lipid OL as deduced from wM q Naxq ions Žfirst column. and fragment ions resulting from loss of the O-ester group, which is mainly vaccenic acid Žsecond column, see also Table 3. Fatty acid pairing
%
mrz wM q Naxq
Amide-linked hydroxy acid
%
mrz fragment
16:0r18:1 17:0r18:1 18:1r18:1 18:0r18:1 18:0r18:0 19:1r18:1 19:0r18:1 19:0r18:0 20:1r18:1
17 9 10 17 13 5 7 7 15
673 687 699 701 703 713 715 717 727
16:0 17:0 18:1
15 6 14
369 383 395
18:0 19:1 19:0
25 5 6
397 409 411
20:1 20:2 21:1
18 7 5
423 421 437
The fatty acid named first is the amide-linked 3-hydroxy fatty acid. Relative proportions are given in %, minor constituents have been omitted. A closer identification of 3-hydroxy 20:2 has not been attempted.
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Table 2 Lipid composition of R. Õiridis in mol% Lipid
PC
PE
PG
CL
OL
MGD
DGD
GAD
mole%
33.3
5.4
7.2
5.0
21.9
3.7
12.1
11.4
The results are means from two separate cultures, which did not deviate more than 10%. Data are based on fatty acid analysis with C17:0 as internal standard taking into account the different stoichiometries of fatty acids present in the different lipids or released by the experimental conditions.
tionated lipid mixture revealed four signals attributed to PC Žset to 0.0 ppm. , PE Ž 0.888 ppm. , CL Ž 1.032 ppm. and PG Ž1.347 ppm.. Their integration resulted in proportions confirming the predominance of PC and the data obtained by fatty acid analysis via internal standard ŽTable 2.. MGD amounts to only 30% of DGD, whereas in cyanobacteria and chloroplasts MGD predominates w1x. Phosphate limitation did not result in the appearance of SQD as might have been expected from similar experiments with Rhodobacter sphaeroides w37x. We incubated R. Õiridis cells for 20 h with w 35 Sxsulfate, but we could not detect any label in added SQD or in any other lipid, which excludes the presence of other sulfurcontaining lipids Ž such as taurine w38x or capnine w39x derivatives. in larger quantities. Also GAD proportions did not respond to phosphate starvation as has been observed in Pseudomonas w40x. The switching between PG and SQD or GAD has been interpreted as demonstrating a requirement of negatively charged lipids in the membrane surface irrespective of actual headgroups. A few microorganisms w30,41x including R. Õiridis w5x can also replace PE by OL when grown under phosphate limitation, which may indicate an additional requirement for zwitterionic charge pairings at the membrane interface. The major fatty acids present in all lipids were 16:0, 16:1, 18:0 and 18:1, from which 18:1 was predominating Ž Table 3. in accordance with previous data w4–6x. The location of the double bond was determined by GC-MS of the pyrrolidides w10x and found to be in the D9- Ž16:1. and the D11-position Ž18:1. as expected for an anaerobic biosynthesis w32x. Apart from these major fatty acids, minor ones not included in Table 3 covered the complete range of saturated and monounsaturated straight chain fatty acids from C14:0 up to C20:1 with even and uneven carbon numbers as observed before w4–6x. The double bond in fatty acids of adjacent chain lengths
Ž14r15, 16r17, 18r19. was always located at the same position, i.e., at D 7, D9 and D11, respectively, for these pairs. This indicates that during their biosynthesis the transrcis-isomerisation step is always carried out with the 2-trans-decenoyl- and -undecenoyl-ACP followed by several rounds of elongation w32x. The fatty acid profiles of the individual lipids are shown in Table 3. The group of phospholipids can be separated from glycolipids, which have an increased proportion of 16:0 and 16:1 Žwith the exception of DGD. at the expense of 18:1. The OL deviates by the predominance of 18:1. It should be pointed out that the conditions used for release of fatty acids from oxygen esters Ž0.2 M KOH in aqueous 70% methanol at 908C for 1 h. do not liberate the amide-linked hydroxy fatty acid present in OL as discussed above Žsee also w30x.. Positional analyses of fatty acids reveal that the two enzymes involved in the stepwise acylation of C1 and C2 of sn-glycerol-3-phosphate have different selectivities. The C1-position is enriched in 18:0 and 18:1 and depleted in 16:0 and Table 3 Fatty acid composition of individual lipids from R. Õiridis in mol% Fatty acid Lipid
16:0
16:1
18:0
18:1
PC PE PG CL OL MGD DGD GAD
7.6 10.0 11.4 9.2 3.8 15.2 9.6 18.3
10.4 8.6 4.9 5.5 0.8 15.5 20.0 16.5
4.6 5.0 5.3 4.8 1.8 4.3 2.8 3.5
77.5 76.4 78.3 80.4 92.7 64.9 67.6 61.7
Data are means from two independent determinations, which did not deviate more than 10%. For OL only the oxygen-linked fatty acids are included.
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18:1r16:1 and 18:1r16:0, which is supported by a recalculation of the total fatty acids and in agreement with the electrospray mass spectrum Žsee above.. This technique also demonstrates the difference in fatty acid make-up between glyco- and phospholipids, in which 18:1r16:1 and 18:1r18:1, respectively, predominate. A peculiar difference between chloroplasts and cyanobacteria is the way in which MGD is synthesized. In chloroplasts, diacylglycerol is converted to MGD in a reaction catalyzed by UDP-galactose:diacylglycerol galactosyltransferase w46x. This enzyme, which has recently been cloned w47x, requires UDPgalactose. In cyanobacteria, this enzyme is missing and replaced by a UDP-glucose:diacylglycerol glucosyltransferase w48x. It is the resultant glucosyl diacyl-
Fig. 4. Positional distribution of fatty acids between the sn-1- and sn-2-position of the diacylglycerol portion in Žfrom left to right. MGD, DGD, GAD, PC, PG and PE.
16:1, which in turn are directed into the C2-position ŽFig. 4.. Lipids synthesized by chloroplasts and cyanobacteria are usually characterized by the strict absence of C18-fatty acids from the sn-2 position w42x. Therefore, the selectivity of the first acyltransferase, i.e., the glycerol-3-phosphate acyltransferase, from R. Õiridis seems to reflect the properties of the corresponding selective enzyme from chloroplasts, whereas the second acyltransferase, the 1-acylglycerol-3-phosphate acyltransferase, does not show the high C16-selectivity characteristic for the envelope-bound enzyme from chloroplasts w43x. On the other hand, in R. Õiridis excess of C18:1 is available, and under similar conditions, i.e., when excess exogenous fatty acids were fed to cyanobacteria, C18 fatty acids were also found at the sn-2 position of cyanobacterial glycerolipids w44,45x. In contrast, the plastidial enzyme strictly prefers C16:0 and hardly accepts C18:1, which is usually present in excess in this organelle. The separation of molecular species results in the resolution of three dominant peaks for PC and DGD ŽFig. 5.. In combination with the positional distribution of fatty acids and fatty acid analysis of collected peaks, they may be characterized as 18:1r18:1,
Fig. 5. Reversed-phase HPLC separation of molecular species of DGD and PC in the form of dinitrobenzoyl derivatives of diacylglycerols. For release of diacylglycerols either a chemical reaction sequence ŽDGD. or phospholipase C hydrolysis ŽPC. was used. Peaks with assigned fatty acid combinations were identified by collection and subsequent analysis of the constituent fatty acids as p-bromophenacyl esters. s, solvent.
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glycerol, which is subsequently epimerized to MGD. The preservation of this epimerization step in cyanobacteria may indicate that the galactose residue with its pronounced hydrophilicrhydrophobic sidedness in the C3-C6 half of the pyranose ring seems to be required in a photosynthetically active membrane. The difference in galactolipid biosynthesis was detected by labelling experiments followed by TLC separation in solvents, which resolve glucosyl from galactosyl diacylglycerol w17x. After short-term labelling of cyanobacteria glucosyl diacylglycerol predominates, which is gradually converted into the corresponding galactolipid. In eucaryotic organisms with chloroplasts, MGD is the only glycosyl diacylglycerol present from the beginning. We carried out similar experiments with R. Õiridis, but as shown in Fig. 1C, we could not detect glucosyl diacylglycerol at times Ž15 min. when it was still predominating in cyanobacterial extracts prepared for comparison. Therefore, we assume that in R. Õiridis MGD is synthesized by direct galactosylation of diacylglycerol reflecting the situation in chloroplasts. On the other hand, definite proof for this assumption could not be obtained, since incubation of R. Õiridis membranes with radioactive UDP-galactose or UDP-glucose did not result in lipid labelling.
4. Discussion Cells of R. Õiridis contain three different types of membranes: the outer membrane, the plasma membrane and the internal stacks of thylakoids w49x. Lipid extraction yields a complex mixture of compounds, which have not yet been attributed to the individual membranes. We identified eight different membrane lipids, i.e., twice the number of lipid components present in thylakoid membranes of cyanobacteria and eukaryotic organisms, all of which contain MGD, DGD, SQD and PG. This fact became evident at the beginning of our investigation, when it had just been shown that phosphate starvation induced an increase of SQD in Rhodobacter sphaeroides w37x. Therefore, with regard to the phylogenetic origin of thylakoid lipids, R. Õiridis seemed to be a promising candidate, especially if phosphate starvation could also induce SQD biosynthesis in this organism. Our experiments have shown that this does not occur, and in the
meantime it was demonstrated that another response to phosphate starvation of Rhodobacter sphaeroides is the synthesis of MGD and its further conversion to the 4-O-a-glucosyl-MGD, a derivative structurally related to DGD w50x. In view of these new data, Rhodobacter sphaeroides Žor a related organism. is the more promising precyanobacterial source for providing genetic information required for the synthesis of thylakoid lipids. The only exception is DGD, but this is also not synthesized in its ‘correct’ arb-form in R. Õiridis. The apparent absence of the capacity to synthesize SQD in R. Õiridis is considered to be more significant, since in addition to the final sulfoquinovosyl transferase further enzymes are required for the synthesis of UDP-sulfoquinovose w51x. Compared to this rather specialized multi-step sequence, the conversion of MGD to DGD by attachment of a second sugar seems to be more easily accomplished from commonly available intermediates. When looking for the presence of thylakoid lipids in anaerobic photosynthetic bacteria, it is evident that only PG is generally present w4x, whereas thus far MGD has only been found in green sulfur bacteria Ž Chlorobiaceae. which have a PSI-like reaction center, and in species of Rhodobacter and Rhodopseudomonas, which as members of the purple non-sulfur bacteria Ž Rhodospirillaceae, a-subgroup of the Proteobacteria. have PSII-like reaction centers w2,3x. Even more rare is the occurrence of SQD w4x, which is also confined to this group Žexcluding R. Õiridis .. SQD is absent in primitive cyanobacteria w52x, which in phylogenetic trees based on 16S rRNA sequences diverge from the main branch leading to chloroplasts w7x. The recent cloning of a MGD synthase from a higher plant w47x has shown that sequence similarity exists only with a glycosyltransferase from E. coli involved in peptidoglycan biosynthesis. If genetic information from this part of metabolism in gramnegative bacteria is the source for deriving lipid glycosyltransferases, the ubiquitous distribution of peptidoglycans may indicate that a high degree of specialization may not have been required to evolve enzymes involved in glycolipid biosynthesis. Recent studies with Acholeplasma w53x and E. coli w54x suggest that cells keep biomembranes close to the limit of bilayer stability by appropriate mixing of bilayer-forming Ž e.g., DGD, SQD, PC, PG. and non-
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bilayer-forming lipids Že.g., MGD, glucosyldiacylglycerol, PE.. This allows membranes to respond immediately to various extra- and intracellular signals including insertion and functional accommodation of membrane proteins. Therefore, the invariant pattern of thylakoid lipids in cyanobacteria and eukaryotic organisms does not necessarily indicate that only those four lipid components are suitable to form this membrane and to accommodate its specific proteins. As mentioned above, SQD is not present in the oxygenic cyanobacterium Gloeobacter w52x, and SQD-free mutants of Chlamydomonas Ža eukaryotic alga w55x., Rhodobacter sphaeroides w37x and the cyanobacterium Synechococcus w56x grow photoautotrophically. Even DGD may be reduced to about 8% of its original level, although this reduction has phenotypic and functional consequences in the corresponding mutant of Arabidopsis w57x. Therefore, the uniqueness of the thylakoid lipid profile may be attributed less to structural necessity than to its phylogenetic history and the advantage of excluding as far as possible valuable elements such as phosphorus and nitrogen from bulk membrane lipids. Only carbon has to be assimilated for the synthesis of the predominating bilayer- Ž DGD. as well as nonbilayerforming components ŽMGD., and sulfate is present in high concentrations Ž28 mM. in sea water, where this evolution started. The negative charge at the membrane surface, guaranteed by SQD and PG with the possibility of mutual compensation, apparently cannot be provided by the N-, S- and P-free GAD. This may be ascribed to the comparatively high pK of its C6 carboxyl group ŽpK of about 3.0 w58x., which may become protonated and thus lose its charge in membranes, which produce at one surface low pH values to be used for ATP synthesis. From this point of view it is not surprising that in the heterogenous group of anoxygenic photosynthetic bacteria, various forms of bilayerrnonbilayer-forming lipid pairs are realized w4x. Particularly remarkable are Heliobacteria Žwith a PSI-like reaction center.. They synthesize as the predominating compound the nonbilayer-forming PE w59x, which is never found in oxygenic thylakoids, supplemented by smaller quantities of PG and CL. Others accumulate glycosyldiacylglycerols other than MGD and DGD. Chromatium Õinosum, a member of the purple sulfur bacteria Ž g-subgroup of the Proteobacteria having a
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PSII-like reaction center. contains glucosyldiacylglycerol w60x. The presence of this compound is interesting in view of the fact that cyanobacteria epimerize this lipid after its synthesis to MGD which then accumulates w1x. This could indicate that structural details of headgroups and not just their overall ability to induce nonbilayer structures may be important as well. Furthermore, it has to be pointed out that the light-harvesting systems of photosynthetically active pro- and eucaryotic organisms vary to a greater extent and usually are more abundant than the actual photosynthetic reaction centers w61,62x. Therefore, it is likely that the lipid composition of thylakoid membranes had to be optimized to provide the matrix for both antenna and reaction systems. From this point of view it is interesting to note that crystallization of the predominating light-harvesting protein of thylakoids from higher plants ŽLHCII., which is considered to be the most abundant membrane protein on earth, required addition of DGD w63x, whereas the reaction center from R. Õiridis w64x and the light-harvesting protein from R. acidophila w65x were crystallized without addition of galactolipids. However, interaction with LHCII cannot be the main function of DGD, since cyanobacterial thylakoids do not contain extended membrane-integrated light-harvesting proteins w66x and still have the same lipid profile as thylakoids from chloroplasts with a high proportion of DGD w1x. An unexpected localization of DGD was found in Amphidinium, a eucaryotic alga w67x with light-harvesting proteins bound to the inner surface of thylakoid membranes. Two molecules of DGD were localized in the crystalline light-harvesting complex sealing the hydrophobic interior packed with light-absorbing pigments. The increasing availability of cloned genes involved in lipid biosynthesis and their manipulation in different photosynthetically active organisms w37,56,57x will provide new strategies for a better understanding of the functions of thylakoid lipids in different organisms and may reveal roles other than bilayerrnonbilayer balancing.
Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is
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acknowledged. We thank T. Gehrke for GLC analysis of enantiomeric galactosides and Prof. Dr. J. Buddrus and H. Herzog for help with the spectroscopy.
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Membrane lipids of Rhodopseudomonas Õiridis ¨ Michael Linscheid a , Bernd W.K. Diehl b, Monika Overmohle ¨ c , Iris Riedl c , Ernst Heinz
c,)
a
c
Institut fur ¨ Spektrochemie, Bunsen-Kirchhoffstr. 11, D-44139 Dortmund, Germany b Spectral SerÕice GmbH, Vogelsanger Str. 250, D-50825 Koln, ¨ Germany Institut fur ¨ Allgemeine Botanik, UniÕersitat ¨ Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany Received 21 March 1997; revised 22 April 1997; accepted 24 April 1997
Abstract In search of the precyanobacterial origin of the typical thylakoid lipids found in cyanobacteria and chloroplasts, we analyzed the polar lipids of the anaerobic phototrophic bacterium Rhodopseudomonas Õiridis. Glycolipids Žmonogalactosyl-, digalactosyl- and glucuronosyl diacylglycerol., phospholipids Žphosphatidyl choline, -ethanolamine, -glycerol and cardiolipin. and an ornithine lipid were isolated and identified by NMR Ž 1 H, 13 C, 31 P. and mass spectrometry. Positional distribution and pairing of fatty acids in molecular species show small, but significant differences between glyco- and phospholipids. In this context, a new enzymatic method is described for assigning the enantiomeric structure of the diacylglycerol moiety in glyco- and phospholipids. 14 C-Labelling studies suggest that monogalactosyl diacylglycerol is formed by galactosylation of diacylglycerol as in chloroplasts and not by glucosylation followed by epimerization as in cyanobacteria. The two 1,6-linked galactopyranose residues of digalactosyl diacylglycerol are both in b-linkage and thus differ from the corresponding chloroplast lipid with its a-b-sequence. R. Õiridis does not contain the sulfolipid, and even phosphate starvation does not induce the synthesis of this most characteristic thylakoid lipid, which on the other hand is present in other anaerobic phototrophic bacteria. q 1997 Elsevier Science B.V. Keywords: Monogalactosyl diacylglycerol; Digalactosyl diacylglycerol; Glucuronosyl diacylglycerol; Ornithine lipid; Phospholipid
1. Introduction A common feature of cyanobacteria, eukaryotic algae and plants is their ability to carry out oxygenic photosynthesis, which operates with two photosystems and water as abundant electron donor. Apart Abbreviations: MGD, DGD, GAD, monogalactosyl-, digalactosyl-, glucuronosyl diacylglycerol; PA, PC, PE, PG, phosphatidic acid, phosphatidyl choline, -ethanolamine, -glycerol; CL, cardiolipin, diphosphatidylglycerol; OL, ornithine lipid; TLC, thin-layer chromatography.; Fatty acids are characterized by carbon and double bond numbers separated by a double point. ) Corresponding author. Fax: q49 40 82282254. E-mail: [email protected]
from similarities in membrane proteins, the thylakoids of these organisms are characterized by a simple set of three glycolipids and one phospholipid w1x. In addition to this advanced type of photosynthesis, various forms of anoxygenic photosynthesis are realized in several groups of prokaryotes. They all use only a single photosystem with a reaction center, which may resemble either photosystem II or photosystem I of oxygenic organisms. It has been speculated that the cooperation of both systems as found in present-day organisms might be the result of a precyanobacterial symbiosis or gene transfer between two different photosynthetically active prokaryotes w2,3x. Comparable to the attempts to trace back the
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 6 5 - 9
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origin of the two reaction centers, similar questions can be asked regarding a precyanobacterial origin of the present-day thylakoid lipids, which likewise may comprise components from both ancestors. From this point of view, a detailed analysis of lipids from phototrophic bacteria other than cyanobacteria receives relevance in addition to the increasing taxonomical use of lipid and fatty acid patterns w4x. The most characteristic thylakoid lipids are two galactolipids and a sulfolipid w1x. In the course of our studies on the biosynthesis of these glycolipids we realized that also R. Õiridis contains two galactolipids. Since such components had not been observed before in this organism w4–6x, we carried out a more detailed analysis of its other membrane lipids as well. In this context, we were particularly interested in the labelling of monogalactosyl diacylglycerol, since cyanobacteria and higher plants show a small but significant difference in the biosynthesis of this compound w1x. This difference does not fit into the general picture, according to which chloroplasts of higher plants are the result of a second symbiosis involving cyanobacteria as domesticated guests in a finally eucaryotic cell w7x. A preliminary note of our results has been published w8x.
2. Materials and methods 2.1. Growth of organism and lipid extraction Rhodopseudomonas Õiridis DSM 134 was cultured anaerobically w9x in screw-cap bottles Ž1 l. in the modified DSM 27 medium under photosynthetic conditions Žtungsten lamp, 60 W, at a distance of 60 m Erm2rs. at 258C. This medium contains 3.7 mM phosphate, which in experiments on phosphate starvation was reduced to various degrees down to 50 m M. Before autoclaving Ž20 min at 1208C. the medium was freed from oxygen by bubbling for 5 min with Argon. A 1-l bottle was inoculated with 10 ml of culture and grown for 3 weeks before harvesting by centrifugation Ž15 min at 9500 = g . yielding 2–3 ml of packed cell volume per 1 l of culture. The sedimented cells were boiled for a few min in water, resedimented, resuspended in chloroformrmethanol Ž1:1, vrv, as throughout in the following. and kept overnight at 48C on a shaker. After addition of chlo-
roform to increase the chloroformrmethanol ratio to 2:1, the organic mixture was washed with 0.2 volumes of NaCl solution Ž0.45%, wrv.. The resultant subphase was withdrawn, freed from solvent and redissolved in a small volume of chloroform. The other strains available from this organism Ž R. Õiridis DSM 133 and DSM 136. were grown and extracted under the same conditions. They contained the same lipids as found in strain DSM 134. Occasionally, cell suspensions were spread on agar plates w9x for aerobic heterotrophic growth to ascertain the absence of microbial contamination. 2.2. Instrumental methods 1
H- and 13 C-NMR spectra were recorded using a JEOL GX 400 spectrometer. Electrospray mass spectra were acquired using a MAT 95 double focusing sector field mass spectrometer with a home-made electrospray ion source. For in-source collision induced dissociation Ž CID., the potential between the desolvation capillary and the skimmer was set to 200 V. GLCrMS for the analysis of the fatty acid pyrrolidides w10x was performed on a DB5 column using a VG 7070 E sector field mass spectrometer equipped with a Sichromat-1 GC ŽSiemens.. For recording 31 P-NMR spectra w11x, the sample of total, unfractionated lipid Žabout 100 mg. was dissolved in 0.6 ml of methanolrCDCl 3 Ž1:2.. 0.4 ml Cs-EDTA solution Ž see below. is added and the mixture shaken vigorously. Then 0.2 ml H 2 O is added and the sample shaken again followed by short centrifugation for phase separation. The complete sample Ž both phases. is poured Žnot pipetted. into the NMR tube. After clearing of the lower organic phase the measurement can be started. The pulse program used CPD proton decoupling, PW of 30 deg., 2.8 s acquisition time, 0.3 s relaxation delay, sweep with 5700 Hz and 32 k data points. The molar ratios of phospholipids are calculated from signals areas. The Cs-EDTA solution is prepared as follows: EDTA Ž0.2 M in D 2 O. is titrated to pH 8.5 with CsCO 3. This solution is diluted 1:4 Žvrv. with methanol. 2.3. Separation and isolation of lipid components The lipid extracts Žin portions of 50–100 mg. dissolved in chloroform were applied to a DE-Sep-
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hacel column Ž30 ml, precycled sequentially with 0.1 N NaOH, acetic acid, methanol and chloroform. and eluted with 200-ml portions of chloroformrmethanol mixtures of increasing methanol content Ž2, 10, 30, 100%. and in a final step with 200 ml of chloroformrmethanol Ž4:1. containing 10 mM ammonium acetate and 0.26 M ammonium hydroxide. The resulting fractions were freed from solvents Ž the last one after aqueous partitioning. and used for isolation of individual lipids by preparative TLC in the following solvent systems: Ža. chloroformrmethanolr25% ammonia Ž65:35:5, separates PE from OL and GAD from PGrCL.; Žb. acetonerbenzenerwater Ž91:30:8, separates MGD and OL from DGD . ; Ž c . chloroformrmethanolracetic acidrwater Ž91:30:4:4, separates PG from CL.. 2.4. Analysis and deriÕatization of lipids Analysis of fatty acids and their positional distribution was carried out as described before by preparation of p-bromophenacyl esters and their separation by HPLC w12x. Molecular species were analyzed after enzymatic or chemical release of diacylglycerols w13x, formation of dinitrobenzoyl derivatives and their separation by HPLC as described before w14x. Quantitation of individual components was based on fatty acid content, which was determined via internal standard after two-dimensional TLC Žsee above.. Spots were scraped off, mixed with margaric acid ŽC17:0, 120 nmol. and heated for 1 h at 908C in 0.2 N KOH in methanolrwater Ž7:3.. After acidification free fatty acids were extracted for preparation of pbromophenacyl esters. For NMR and mass spectrometry the two galactolipids were acetylated and hydrogenated as described before w15x. GAD was first converted to the methyl ester by treatment of the protonated lipid with diazomethane followed by acetylation w16x. The methyl ester formation resulted in an increased Rf-value Žfrom 0.4 to 0.85 in solvent a.. The derivatized lipid was purified by preparative TLC with chloroformrmethanol Ž100:1.. The 1 H-signals of the glucuronosyl Ž numbered u1-u5. and glycerol Žnumbered g1-g3. moieties Žrecorded in CDCl 3 , shifts given in ppm, coupling constants J in Hz. were assigned as follows: 5.49 Žu4, dd, 10r10.; 5.21 Žu1, d, 3.; 5.21 Žg2, m, 4.5r5.; 5.16 Žu3, dd, 10r10.; 4.87 Žu2, dd,
153
3r10.; 4.36 Žg1, dd, 12r4.5 or 5.; 4.32 Žu5, d, 10.; 4.15 Ž g1X , dd, 12r5 or 4.5. ; 3.86 Ž g3, dd, 12r4.5 or 5.; 3.75 ŽOCH 3 of methyl ester at u6, s.; 3.67 Žg3X , dd, 12r5 or 4.5.. When OL was treated in its protonated form with diazomethane, the R f-value of the resultant methyl ester increased from 0.3 to 0.9 as compared to the underivatized lipid in solvent Ža.. 2.5. Acetate-labelling of lipids A batch of R. Õiridis was grown for 4 days in 1 l of medium before harvesting. The following manipulations were carried out under sterile conditions. The cells were washed three times in medium without carbon source and then resuspended in this medium. Aliquots of this cell suspension Ž0.3–0.5 ml, depending on the number of parallel experiments and their duration. were placed into Sovirel tubes and gassed with Argon. Following the addition of w1- 14 Cxacetate Ž2 m Ci, 52 m Cirm mol, aqueous solution of sodium salt., the tubes were capped and placed in nearly horizontal position in the fumehood under normal laboratory light Ž6 m Erm2rs.. After incubation for various times Ž15 min, 30 min, 1, 2, 5, 28, 72 h. the samples were heated for a few minutes to boiling after addition of hot water and subsequently washed 3 times with bicarbonate solution Ž0.1 M. to remove excess of acetate. Lipid extraction of the sedimented cells was carried out as described above. The incorporation of radioactivity into lipids varied from 1.5 to 6.1% depending on sampling time and incubation volume Žlarger at the shorter times.. To resolve glucosyl from galactosyl diacylglycerol by one-dimensional TLC w17x, the two solvent systems used were Žb. and Žd. diethyl etherrisopropanolrmethanol Ž100:4.5:2.5.. Developed plates were scanned and subjected to radioautography by exposure for one to two weeks Ž50 000 to 100 000 dpm. . The sample obtained after 28 h labelling was used for two-dimensional separation, in the first direction with solvent Že. chloroformrmethanolrwater Ž65:25:4. and in the second dimension with solvent Žf. chloroformracetonermethanolracetic acidrwater Ž50:20:10:10:5.. Sulfate labelling was carried out by incubating cells under identical conditions for 20 h in the presence of 19 m Ci of w 35 Sxsulfate Žabout 1000 mCirm mol. . For preparation of an authentic mixture of glucosyl and galactosyl diacylglycerol, a sample of a cyanobacte-
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rium Ž Anabaena sp.. was incubated under similar, but aerobic conditions for 10, 20, 40, 75 min and used for subsequent lipid extraction as described above. 2.6. Enantiomeric structure of 1,2-diacylglycerols in glycolipids Radioactively labelled glycolipids were isolated from cells incubated for 28 h with w 14 Cxacetate Žsee above.. The glycolipids were individually subjected to the sequence of periodate oxidationrhydrazinolysis, which results in release of 1,2-diacylglycerols w13x. They were isolated by preparative TLC in diethyl etherrpetroleum ether Ž3:1., aliquots Ž20 000 dpm. mixed with CL Žfrom Serva, 75 m g. and evaporated to dryness in Sovirel tubes. To the dried lipid the following solutions were added w18x: 50 m l buffer Ž100 mM PipesrNaOH, 100 mM NaCl, 25 mM MgCl 2 , 100 mM n-octyl b-D-glucopyranoside, pH 6.8., 10 m l dithiothreitol Ž20 mM., diacylglycerol kinase Žfrom Calbiochem, 5 m l., 25 m l water and 10 m l ATP Ž50 mM.. After short dispersion in a sonication bath the reaction was run for 90 min at room temperature. The lipids were extracted by phase partitioning w19x between chloroformrmethanol Ž1:1, 2.3 ml. and H 3 PO4 Ž0.2 M in 1 M KCl, 1 ml. . After solvent removal the labelled lipids were analyzed by TLC in solvent Žg. chloroformrmethanolr25% ammonia Ž60:40:10. and radioscanning. 2.7. Configuration of galactose To determine the absolute configuration ŽD or L. of galactose w20x from galactolipids, a sample of the bacterial DGD Ž 1 mg. was heated overnight at 808C in SŽq.-2-butanol containing 1 M HCl. Subsequently the sample was applied to a TLC plate and chromatographed in CHCl 3rMeOH Ž 6:2. . The major middle band of butyl galactosides was extracted, subjected to acetylation with acetic anhydride and analyzed by capillary GLC Ž15 m fused silica column, 0.32 mm internal diameter, coated with 0.2 m m Rt x-2330, from Amchro. applying a temperature program from 1708C to 2108C in 10 min. Reference samples were prepared in the same way from D- and L-galactose Ž from Sigma. as well as from spinach
DGD. Assignment of GLC peaks was confirmed by coinjection.
3. Results The chromatographic resolution of the lipid mixture extracted from R. Õiridis revealed eight major components ŽFig. 1A., from which five had been recognized before w4–6x. We isolated all the different components ŽFig. 1B. in quantities sufficient for a closer analysis. For this purpose we used prefractionation by column chromatography followed by preparative TLC, because the complex mixture could not be separated satisfactorily into individual components by a single one-dimensional chromatography. The separation on DE-Sephacel proved to be the most suitable w21x, since the resulting fractions contained mixtures of components, which did not overlap in the subsequent separation. Column chromatography on CMcellulose and silica gel were less satisfactory in this respect. We confirmed the presence of PG, CL, PC and PE by cochromatography and proved their identity by 31 P-NMR spectroscopy Ž see below. . In addition to these previously detected phospholipids, R. Õiridis contains three glycolipids. MGD was found to have the structure of 1,2-di-0-acyl-3-0-b-D-galactopyranosyl-sn-glycerol, which is identical with MGD from cyanobacteria and plants. The structural details were deduced from mass and NMR spectra of the hydrogenated and acetylated compound. Apart from fatty acid details, these spectra were identical with those recorded before for MGD from higher plants which will not be detailed again w22x. In particular, the major fragments in the mass spectrum are assigned to the acetylated hexopyranose Žmrz 331. and the 1,2-diacylglycerol moieties Žmrz 579 and 607. of the parent molecule. Molecular ions were observed at mrz 867 and 895 corresponding to 18:0r16:0 and 18:0r18:0 fatty acid combinations, respectively. In the 1 H-NMR spectrum the large coupling constant of 8 Hz indicates the b-linkage of the anomeric proton at d 4.47 ppm in CDCl 3 , and the pattern of splitting and coupling constants for the ring protons at C-2, C-3, C-4 and C-5 are characteristic for the bgalactopyranose structure w23x. The enantiomeric structure of the diacylglycerol portion was analyzed
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by the use of diacylglycerol kinase from Escherichia coli. This enzyme has a pronounced enantiomeric selectivity for phosphorylating sn-1,2- and discriminating against sn-2,3-diacylglycerols w24x. The radioactive diacylglycerol released by a chemical reaction sequence w13x from the labelled DGD was completely converted to phosphatidic acid ŽFig. 2., which proves that it is the sn-1,2-diacyl enantiomer. The two other glycolipids were subjected to the same reaction sequence and yielded the same results. The mass spectrum of the acetylated DGD is very similar to that recorded for the corresponding galactolipid from plants Žnot to be detailed again. and confirms the overall structure w22x. The electrospray mass spectrum of the nonhydrogenated compound showed molecular ions ŽMNaqrMKq. of major abundance at mrz 939r955 Ž18:1r16:1., 941r957 Ž18:1r16:0. and 967r983 Ž18:1r18:1.. The fatty acid patterns will be discussed separately Žsee below.. The enantiomeric configuration of the constituent galactose ŽD or L. was determined by formation of ŽS.2-butyl galactosides followed by acetylation and
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GLC, which separates ŽS.2-butyl-L-galactosides from the diastereomeric D-galactosides w20x. The application of this reaction sequence revealed that DGD contains exclusively D-galactose. Assuming that MGD is the biosynthetic precursor of DGD, we conclude that also MGD contains only D-galactose. On the other hand, the NMR spectrum of the hydrogenated and acetylated compound shows a significant difference between DGD from R. Õiridis and the normal DGD from higher plants w22x. In DGD from R. Õiridis both sugar residues display a parallel series of identical, but slightly displaced signals for ring and anomeric protons in a spectrum, which in all details is identical with that published for a DGD fraction isolated from Adzuki beans w25x. Most significant are the clearly separated doublets of the anomeric protons centered at 4.44 and 4.50 ppm Ž in CDCl 3 . with their characteristic coupling constant of 8 Hz. In the 13 C-NMR spectrum the two C1 signals show up very close to each other at 103.19 and 103.78 ppm and not with the wide shift difference as characteristic for the normal DGD with C1-a at about 97 ppm and C1-b
Fig. 1. Chromatographic resolution of polar lipids from Rhodopseudomonas Õiridis. A: Two-dimensional TLC of 14C-labelled lipids Ždetails in Materials and methods. in solvent e Žfirst dimension. and f Žsecond dimension.. B: Purity of individual lipids obtained by a combination of column chromatography and preparative TLC; separation in solvent c. C: Separation of 14C-labelled glucosyl diacylglycerol Žmarked by an asterisk. from the slightly slower moving galactosyl diacylglycerol Žmarked by a triangle. from Anabaena Žlane 1, after 40 min labelling.; separation of lipids from R. Õiridis labelled for 15 min Žlane 2., 30 min Žlane 3. and 60 min Žlane 4. in solvent b, in which glucosyl diacylglycerol is missing.
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Fig. 2. Conversion of 14C-labelled 1,2-diacyl-sn-glycerol ŽDAG, released from DGD. into phosphatidic acid ŽPA. by incubation with diacylglycerol kinase Žfrom E. coli . in the presence of ATP Žc, for details see Section 2.. The separation of reference DAG and PA by TLC in solvent g is shown in a and b.
at about 102 ppm w22x. Therefore, the DGD from R. Õiridis has the structure of 1,2-di-0-acyl-3-0-Ž6-0-bD-galactopyranosyl-b-D-galactopyranosyl. -sn-glycerol. This all-b-DGD differs from that of cyanobacteria and most higher plants by the fact that both sugars have identical anomeric b-configuration, whereas in higher plants the terminal galactose has usually aand the internal b-configuration Ž arb-DGD.. The all-b-DGD has been detected in low proportions of the normal arb-DGD in various plants throughout the plant kingdom including eucaryotic algae, mosses, ferns, gymnosperms and angiosperms w25x. Somewhat higher percentages are present in the seeds of some beans with a maximum of 36% of total DGD in Adzuki beans Ž seeds of Vigna angularis .. In this context it should be mentioned that the biosynthesis
of DGD from MGD in chloroplasts is catalyzed by an intergalactolipid galactosyltransferase localized in the outer envelope membrane w26x. An equivalent location in cyanobacteria or R. Õiridis, i.e., not in the plasma membrane but in a membrane surrounding the plasma membrane on the outside, seems to be rather unlikely. Bacteria synthesize the lipids of their plasma membrane in this membrane system and do not rely on lipid import from the outer membrane, which in addition cannot be considered to be the immediate phylogenetic precursor of the outer envelope membrane of plastids. The acidic glycolipid was at first recognized as GAD by its conversion to a more apolar compound on treatment with diazomethane w27x. The mass spectrum of the resultant and acetylated methyl ester showed prominent fragments at mrz 317 Žrepresenting the acetylated glucuronopyranosyl methyl ester w28x. and at mrz 575r577 Ž representing diacylglycerol moieties with 18:1r16:1 and 18:1r16:0 pairings of about equal intensity.. The 1 H-NMR spectrum of the acetylated methyl ester of GAD displayed the series of ring protons with coupling constants characteristic for the a-glucopyranose structure w23,29x, from which H1 at 5.21 with a coupling constant of 3 Hz should be mentioned. Due to the absence of protons at C6 of the glucuronosyl residue, the spectral region between 3.5–4.5 ppm is less crowded than in spectra of normal sugars and results in clearly separated signals for the two pairs of nonequivalent protons at C1 and C3 of glycerol Ž see Section 2.. The OL is characterized on TLC plates as a ninhydrin-positive, phosphate-negative lipid. On treatment of the protonated lipid with diazomethane, a methyl ester is formed which has an increased R f-value. It may be recalled that alkali treatment Ž saponification. of OL removes the O-ester group and induces cyclization of the other part to a ninhydrin-negative 3-acylamino-piperidine-2-one with lower R f-value w30x. The proof for the structure of OL as 2-N-3Xacyloxyacyl-ornithine was provided by mass and NMR spectroscopy. The 1 H-signals Žobtained as COSY-spectrum in CDCl 3 . confirmed previous analyses w30,31x and will not be detailed again. Only the 13 C-assignments are given Ž Fig. 3. , which have not been published before. Our data do not allow a configurational assignment at the asymmetrical cen-
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Fig. 3. w 13 CxNMR signals Žgiven in ppm. assigned in the spectrum of the ornithine lipid OL as recorded in CDCl 3. Carbon atoms in the ornithine moiety are numbered O1-O5, in the amide-linked hydroxy fatty acid H1-H4 and in the O-ester linked fatty acyl group F1 and F2. O3 and O4 assignments are tentative, since these signals fall in the range of methylene carbons from fatty acids. Other carbons including those at olefinic double bonds Ž129.89 and 129.76 ppm. are not included.
ters of O2 and H3, but based on previous experiments, it may be justified to assume that ornithine is the L-enantiomer w30x and the 3-hydroxy fatty acid has D-configuration, if it results from fatty acid biosynthesis due to the stereochemical outcome of the b-keto-acyl-ACP reductase reaction w32x. The electrospray mass spectra and particularly the in-source fragmentation indicated that the compound was an ornithine amino acid with a hydroxy fatty acid bound as amide and a second fatty acid linked via an O-ester function to the amide-linked acyl group. The O-ester-linked second fatty acid was released by saponification and found to be mainly Ž93%. 18:1 Žvaccenic acid, see below.. Therefore, the CID experiments could be used to analyze the
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amide-linked hydroxy fatty acids under the assumption that all O-esters were 18:1. This assignment was supported Žfirst column of Table 1. by the intensities of the corresponding fragment ions resulting from loss of 18:1 Žsecond column of Table 1.. In contrast to the homogenous O-ester group the b-hydroxy fatty acids are very heterogenous and cover the range from C16-C21 including saturated and monounsaturated fatty acids with even and uneven carbon numbers, from which those with even carbon numbers predominate. An intense fragment at mrz 184 can be assigned to the loss of the O-ester group and a fission at the hydroxy function of the amide-linked hydroxy fatty acid. This confirms the position of the hydroxyl group at carbon 3 ŽH3 in Fig. 3. and the identity of ornithine as the constituent amino acid. Complete loss of the amide-linked acyl group Ž including the nitrogen. results in fragments with mrz 115r116, which again confirm the overall structure w30,31,33– 35x. Ornithine lipids of this structure have been detected in various bacteria, but different structures may occur as well w36x. Quantitation of the individual lipids ŽTable 2. was in general agreement with the labelling pattern shown in Fig. 1A. PC is predominating, whereas the other components are present in lower and varying proportions. The qualitative and quantitative analyses of phospholipids were confirmed by 31 P-NMR spectroscopy w11x. The spectrum recorded with the unfrac-
Table 1 Fatty acid pairings in the ornithine lipid OL as deduced from wM q Naxq ions Žfirst column. and fragment ions resulting from loss of the O-ester group, which is mainly vaccenic acid Žsecond column, see also Table 3. Fatty acid pairing
%
mrz wM q Naxq
Amide-linked hydroxy acid
%
mrz fragment
16:0r18:1 17:0r18:1 18:1r18:1 18:0r18:1 18:0r18:0 19:1r18:1 19:0r18:1 19:0r18:0 20:1r18:1
17 9 10 17 13 5 7 7 15
673 687 699 701 703 713 715 717 727
16:0 17:0 18:1
15 6 14
369 383 395
18:0 19:1 19:0
25 5 6
397 409 411
20:1 20:2 21:1
18 7 5
423 421 437
The fatty acid named first is the amide-linked 3-hydroxy fatty acid. Relative proportions are given in %, minor constituents have been omitted. A closer identification of 3-hydroxy 20:2 has not been attempted.
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Table 2 Lipid composition of R. Õiridis in mol% Lipid
PC
PE
PG
CL
OL
MGD
DGD
GAD
mole%
33.3
5.4
7.2
5.0
21.9
3.7
12.1
11.4
The results are means from two separate cultures, which did not deviate more than 10%. Data are based on fatty acid analysis with C17:0 as internal standard taking into account the different stoichiometries of fatty acids present in the different lipids or released by the experimental conditions.
tionated lipid mixture revealed four signals attributed to PC Žset to 0.0 ppm. , PE Ž 0.888 ppm. , CL Ž 1.032 ppm. and PG Ž1.347 ppm.. Their integration resulted in proportions confirming the predominance of PC and the data obtained by fatty acid analysis via internal standard ŽTable 2.. MGD amounts to only 30% of DGD, whereas in cyanobacteria and chloroplasts MGD predominates w1x. Phosphate limitation did not result in the appearance of SQD as might have been expected from similar experiments with Rhodobacter sphaeroides w37x. We incubated R. Õiridis cells for 20 h with w 35 Sxsulfate, but we could not detect any label in added SQD or in any other lipid, which excludes the presence of other sulfurcontaining lipids Ž such as taurine w38x or capnine w39x derivatives. in larger quantities. Also GAD proportions did not respond to phosphate starvation as has been observed in Pseudomonas w40x. The switching between PG and SQD or GAD has been interpreted as demonstrating a requirement of negatively charged lipids in the membrane surface irrespective of actual headgroups. A few microorganisms w30,41x including R. Õiridis w5x can also replace PE by OL when grown under phosphate limitation, which may indicate an additional requirement for zwitterionic charge pairings at the membrane interface. The major fatty acids present in all lipids were 16:0, 16:1, 18:0 and 18:1, from which 18:1 was predominating Ž Table 3. in accordance with previous data w4–6x. The location of the double bond was determined by GC-MS of the pyrrolidides w10x and found to be in the D9- Ž16:1. and the D11-position Ž18:1. as expected for an anaerobic biosynthesis w32x. Apart from these major fatty acids, minor ones not included in Table 3 covered the complete range of saturated and monounsaturated straight chain fatty acids from C14:0 up to C20:1 with even and uneven carbon numbers as observed before w4–6x. The double bond in fatty acids of adjacent chain lengths
Ž14r15, 16r17, 18r19. was always located at the same position, i.e., at D 7, D9 and D11, respectively, for these pairs. This indicates that during their biosynthesis the transrcis-isomerisation step is always carried out with the 2-trans-decenoyl- and -undecenoyl-ACP followed by several rounds of elongation w32x. The fatty acid profiles of the individual lipids are shown in Table 3. The group of phospholipids can be separated from glycolipids, which have an increased proportion of 16:0 and 16:1 Žwith the exception of DGD. at the expense of 18:1. The OL deviates by the predominance of 18:1. It should be pointed out that the conditions used for release of fatty acids from oxygen esters Ž0.2 M KOH in aqueous 70% methanol at 908C for 1 h. do not liberate the amide-linked hydroxy fatty acid present in OL as discussed above Žsee also w30x.. Positional analyses of fatty acids reveal that the two enzymes involved in the stepwise acylation of C1 and C2 of sn-glycerol-3-phosphate have different selectivities. The C1-position is enriched in 18:0 and 18:1 and depleted in 16:0 and Table 3 Fatty acid composition of individual lipids from R. Õiridis in mol% Fatty acid Lipid
16:0
16:1
18:0
18:1
PC PE PG CL OL MGD DGD GAD
7.6 10.0 11.4 9.2 3.8 15.2 9.6 18.3
10.4 8.6 4.9 5.5 0.8 15.5 20.0 16.5
4.6 5.0 5.3 4.8 1.8 4.3 2.8 3.5
77.5 76.4 78.3 80.4 92.7 64.9 67.6 61.7
Data are means from two independent determinations, which did not deviate more than 10%. For OL only the oxygen-linked fatty acids are included.
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18:1r16:1 and 18:1r16:0, which is supported by a recalculation of the total fatty acids and in agreement with the electrospray mass spectrum Žsee above.. This technique also demonstrates the difference in fatty acid make-up between glyco- and phospholipids, in which 18:1r16:1 and 18:1r18:1, respectively, predominate. A peculiar difference between chloroplasts and cyanobacteria is the way in which MGD is synthesized. In chloroplasts, diacylglycerol is converted to MGD in a reaction catalyzed by UDP-galactose:diacylglycerol galactosyltransferase w46x. This enzyme, which has recently been cloned w47x, requires UDPgalactose. In cyanobacteria, this enzyme is missing and replaced by a UDP-glucose:diacylglycerol glucosyltransferase w48x. It is the resultant glucosyl diacyl-
Fig. 4. Positional distribution of fatty acids between the sn-1- and sn-2-position of the diacylglycerol portion in Žfrom left to right. MGD, DGD, GAD, PC, PG and PE.
16:1, which in turn are directed into the C2-position ŽFig. 4.. Lipids synthesized by chloroplasts and cyanobacteria are usually characterized by the strict absence of C18-fatty acids from the sn-2 position w42x. Therefore, the selectivity of the first acyltransferase, i.e., the glycerol-3-phosphate acyltransferase, from R. Õiridis seems to reflect the properties of the corresponding selective enzyme from chloroplasts, whereas the second acyltransferase, the 1-acylglycerol-3-phosphate acyltransferase, does not show the high C16-selectivity characteristic for the envelope-bound enzyme from chloroplasts w43x. On the other hand, in R. Õiridis excess of C18:1 is available, and under similar conditions, i.e., when excess exogenous fatty acids were fed to cyanobacteria, C18 fatty acids were also found at the sn-2 position of cyanobacterial glycerolipids w44,45x. In contrast, the plastidial enzyme strictly prefers C16:0 and hardly accepts C18:1, which is usually present in excess in this organelle. The separation of molecular species results in the resolution of three dominant peaks for PC and DGD ŽFig. 5.. In combination with the positional distribution of fatty acids and fatty acid analysis of collected peaks, they may be characterized as 18:1r18:1,
Fig. 5. Reversed-phase HPLC separation of molecular species of DGD and PC in the form of dinitrobenzoyl derivatives of diacylglycerols. For release of diacylglycerols either a chemical reaction sequence ŽDGD. or phospholipase C hydrolysis ŽPC. was used. Peaks with assigned fatty acid combinations were identified by collection and subsequent analysis of the constituent fatty acids as p-bromophenacyl esters. s, solvent.
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glycerol, which is subsequently epimerized to MGD. The preservation of this epimerization step in cyanobacteria may indicate that the galactose residue with its pronounced hydrophilicrhydrophobic sidedness in the C3-C6 half of the pyranose ring seems to be required in a photosynthetically active membrane. The difference in galactolipid biosynthesis was detected by labelling experiments followed by TLC separation in solvents, which resolve glucosyl from galactosyl diacylglycerol w17x. After short-term labelling of cyanobacteria glucosyl diacylglycerol predominates, which is gradually converted into the corresponding galactolipid. In eucaryotic organisms with chloroplasts, MGD is the only glycosyl diacylglycerol present from the beginning. We carried out similar experiments with R. Õiridis, but as shown in Fig. 1C, we could not detect glucosyl diacylglycerol at times Ž15 min. when it was still predominating in cyanobacterial extracts prepared for comparison. Therefore, we assume that in R. Õiridis MGD is synthesized by direct galactosylation of diacylglycerol reflecting the situation in chloroplasts. On the other hand, definite proof for this assumption could not be obtained, since incubation of R. Õiridis membranes with radioactive UDP-galactose or UDP-glucose did not result in lipid labelling.
4. Discussion Cells of R. Õiridis contain three different types of membranes: the outer membrane, the plasma membrane and the internal stacks of thylakoids w49x. Lipid extraction yields a complex mixture of compounds, which have not yet been attributed to the individual membranes. We identified eight different membrane lipids, i.e., twice the number of lipid components present in thylakoid membranes of cyanobacteria and eukaryotic organisms, all of which contain MGD, DGD, SQD and PG. This fact became evident at the beginning of our investigation, when it had just been shown that phosphate starvation induced an increase of SQD in Rhodobacter sphaeroides w37x. Therefore, with regard to the phylogenetic origin of thylakoid lipids, R. Õiridis seemed to be a promising candidate, especially if phosphate starvation could also induce SQD biosynthesis in this organism. Our experiments have shown that this does not occur, and in the
meantime it was demonstrated that another response to phosphate starvation of Rhodobacter sphaeroides is the synthesis of MGD and its further conversion to the 4-O-a-glucosyl-MGD, a derivative structurally related to DGD w50x. In view of these new data, Rhodobacter sphaeroides Žor a related organism. is the more promising precyanobacterial source for providing genetic information required for the synthesis of thylakoid lipids. The only exception is DGD, but this is also not synthesized in its ‘correct’ arb-form in R. Õiridis. The apparent absence of the capacity to synthesize SQD in R. Õiridis is considered to be more significant, since in addition to the final sulfoquinovosyl transferase further enzymes are required for the synthesis of UDP-sulfoquinovose w51x. Compared to this rather specialized multi-step sequence, the conversion of MGD to DGD by attachment of a second sugar seems to be more easily accomplished from commonly available intermediates. When looking for the presence of thylakoid lipids in anaerobic photosynthetic bacteria, it is evident that only PG is generally present w4x, whereas thus far MGD has only been found in green sulfur bacteria Ž Chlorobiaceae. which have a PSI-like reaction center, and in species of Rhodobacter and Rhodopseudomonas, which as members of the purple non-sulfur bacteria Ž Rhodospirillaceae, a-subgroup of the Proteobacteria. have PSII-like reaction centers w2,3x. Even more rare is the occurrence of SQD w4x, which is also confined to this group Žexcluding R. Õiridis .. SQD is absent in primitive cyanobacteria w52x, which in phylogenetic trees based on 16S rRNA sequences diverge from the main branch leading to chloroplasts w7x. The recent cloning of a MGD synthase from a higher plant w47x has shown that sequence similarity exists only with a glycosyltransferase from E. coli involved in peptidoglycan biosynthesis. If genetic information from this part of metabolism in gramnegative bacteria is the source for deriving lipid glycosyltransferases, the ubiquitous distribution of peptidoglycans may indicate that a high degree of specialization may not have been required to evolve enzymes involved in glycolipid biosynthesis. Recent studies with Acholeplasma w53x and E. coli w54x suggest that cells keep biomembranes close to the limit of bilayer stability by appropriate mixing of bilayer-forming Ž e.g., DGD, SQD, PC, PG. and non-
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bilayer-forming lipids Že.g., MGD, glucosyldiacylglycerol, PE.. This allows membranes to respond immediately to various extra- and intracellular signals including insertion and functional accommodation of membrane proteins. Therefore, the invariant pattern of thylakoid lipids in cyanobacteria and eukaryotic organisms does not necessarily indicate that only those four lipid components are suitable to form this membrane and to accommodate its specific proteins. As mentioned above, SQD is not present in the oxygenic cyanobacterium Gloeobacter w52x, and SQD-free mutants of Chlamydomonas Ža eukaryotic alga w55x., Rhodobacter sphaeroides w37x and the cyanobacterium Synechococcus w56x grow photoautotrophically. Even DGD may be reduced to about 8% of its original level, although this reduction has phenotypic and functional consequences in the corresponding mutant of Arabidopsis w57x. Therefore, the uniqueness of the thylakoid lipid profile may be attributed less to structural necessity than to its phylogenetic history and the advantage of excluding as far as possible valuable elements such as phosphorus and nitrogen from bulk membrane lipids. Only carbon has to be assimilated for the synthesis of the predominating bilayer- Ž DGD. as well as nonbilayerforming components ŽMGD., and sulfate is present in high concentrations Ž28 mM. in sea water, where this evolution started. The negative charge at the membrane surface, guaranteed by SQD and PG with the possibility of mutual compensation, apparently cannot be provided by the N-, S- and P-free GAD. This may be ascribed to the comparatively high pK of its C6 carboxyl group ŽpK of about 3.0 w58x., which may become protonated and thus lose its charge in membranes, which produce at one surface low pH values to be used for ATP synthesis. From this point of view it is not surprising that in the heterogenous group of anoxygenic photosynthetic bacteria, various forms of bilayerrnonbilayer-forming lipid pairs are realized w4x. Particularly remarkable are Heliobacteria Žwith a PSI-like reaction center.. They synthesize as the predominating compound the nonbilayer-forming PE w59x, which is never found in oxygenic thylakoids, supplemented by smaller quantities of PG and CL. Others accumulate glycosyldiacylglycerols other than MGD and DGD. Chromatium Õinosum, a member of the purple sulfur bacteria Ž g-subgroup of the Proteobacteria having a
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PSII-like reaction center. contains glucosyldiacylglycerol w60x. The presence of this compound is interesting in view of the fact that cyanobacteria epimerize this lipid after its synthesis to MGD which then accumulates w1x. This could indicate that structural details of headgroups and not just their overall ability to induce nonbilayer structures may be important as well. Furthermore, it has to be pointed out that the light-harvesting systems of photosynthetically active pro- and eucaryotic organisms vary to a greater extent and usually are more abundant than the actual photosynthetic reaction centers w61,62x. Therefore, it is likely that the lipid composition of thylakoid membranes had to be optimized to provide the matrix for both antenna and reaction systems. From this point of view it is interesting to note that crystallization of the predominating light-harvesting protein of thylakoids from higher plants ŽLHCII., which is considered to be the most abundant membrane protein on earth, required addition of DGD w63x, whereas the reaction center from R. Õiridis w64x and the light-harvesting protein from R. acidophila w65x were crystallized without addition of galactolipids. However, interaction with LHCII cannot be the main function of DGD, since cyanobacterial thylakoids do not contain extended membrane-integrated light-harvesting proteins w66x and still have the same lipid profile as thylakoids from chloroplasts with a high proportion of DGD w1x. An unexpected localization of DGD was found in Amphidinium, a eucaryotic alga w67x with light-harvesting proteins bound to the inner surface of thylakoid membranes. Two molecules of DGD were localized in the crystalline light-harvesting complex sealing the hydrophobic interior packed with light-absorbing pigments. The increasing availability of cloned genes involved in lipid biosynthesis and their manipulation in different photosynthetically active organisms w37,56,57x will provide new strategies for a better understanding of the functions of thylakoid lipids in different organisms and may reveal roles other than bilayerrnonbilayer balancing.
Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is
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acknowledged. We thank T. Gehrke for GLC analysis of enantiomeric galactosides and Prof. Dr. J. Buddrus and H. Herzog for help with the spectroscopy.
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