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Journal of Lipid Research, Vol. 44, 2120-2126, November 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology
Dipartimento di Fisiologia Generale ed Ambientale and Dipartimento di Chimica, Università di Bari, via Amendola 165/a, 70126 Bari, Italy
Published, JLR Papers in Press, August 16, 2003. DOI 10.1194/jlr.M300212-JLR200
1 To whom correspondence should be addressed. e-mail: a.corcelli{at}biologia.uniba.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this report, we show that a) GlyC is formed during the isolation of PM, b) GlyC increase in H. salinarum cells is specifically induced by osmotic shock, and c) in correspondence with GlyC increase, a decrease of S-TGD-1 levels occurs. The changes in membrane lipid composition observed during the isolation of PM are due to de novo synthesis of GlyC from S-TGD-1.
Abbreviations: BPG, archaeal analog of bisphosphatidylglycerol, or cardiolipin (diphytanylglycerol analog); BR, bacteriorhodopsin; ESI-MS, electrospray ionization-mass spectrometry; GlyC, glycocardiolipin, or 3-HSO3-Galp-ß1,6-Manp-
1,2-Glcp-1,1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol], or S-TGD-1-PA; PA, phosphatidic acid; PGP-Me, phosphatidylglycerophosphate methyl ester (diphytanylglycerol ether analog); PM, purple membrane; S-TeGD-1, 3-HSO3-Galp-ß1,6-Manp-3,1-galactofuranose-1,2-Glcp--sn-2,3-diphytanylglycerol; S-TGD-1, 3-HSO3-Galp-ß1,6- Manp-1,2-Glcp-1,1-sn-2,3-diphytanylglycerol
Supplementary key words bacteriorhodopsin • cardiolipin • archaeal lipids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By analyzing lipids of delipidated BR fractions by electrospray ionization-mass spectrometry (ESI-MS), it was found that the composition of the residual lipids in delipidated BR is different from that of the PM in that the delipidated fractions have much higher proportions of two bicharged peaks at 760 m/z and 966 m/z, corresponding to two novel membrane lipids. This suggested that these novel lipids are more resistant to detergents than the other lipid components of PM, or, alternatively, that the novel lipids are more strongly bound to BR and may be involved in stabilizing the BR trimer structure.
The two novel PM lipids were isolated, purified by thin-layer chromatography (TLC), and analyzed by conventional analytical and spectroscopic methods to determine their chemical structure. Combining ESI-MS data and proton and phosphorus nuclear magnetic resonance (NMR) data of the purified novel lipids, together with the identification of their acid degradation products, allowed the elucidation of the chemical structures of the novel PM phospholipids (2). Their structures are, respectively, of a phosphosulfoglycolipid, 3-HSO3-Galpß1-6Manp1-2Glcp-1-1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol] (called glycocardiolipin, or GlyC), and a glyceroldiether analog of bisphosphatidylglycerol, sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2,3-di-O-phytanylglycerol (archaeal BPG) (Fig. 1)
. Both novel lipids have two diphytanylglycerol moieties in their molecule, making their dimeric structures analogous to that of eukaryal cardiolipin.
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ManGlc. Also, the sulfate group is located on C-3 of the galactose residue, as in S-TGD-1 (3). We will refer to it as S-TGD-1-PA (see structure in Fig. 1A) or GlyC, adopting a previously introduced abbreviation (4). The cardiolipin/BR stoichiometries in the PM have also been estimated; one GlyC per BR is present in the PM, while BPG is only a minor component (4). Here, by analyzing the distribution of GlyC in the various fractions obtained in the course of PM isolation, we show that GlyC is present in very low amounts in the cells of H. salinarum under physiological conditions, and that it is de novo synthesized and accumulated mainly in the PM during cell lysis by osmotic shock.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microorganism cultures
The engineered high-producing BR strain of H. salinarum used in this study was kindly provided by Richard Needleman (5); the H. salinarum NRC-1 strain was kindly provided by Aharon Oren. The H. salinarum cells were grown in light at 37°C in liquid growth medium containing neutralized peptone (L34, Oxoid) prepared as previously described (6).
PM isolation
PMs were isolated from the high-producing BR strain as previously described (6). Briefly, a dialysis bag (cutoff 12,000–14,000) was filled with a concentrated suspension of halobacterial cells in 4 M NaCl in the presence of DNase I and left in water under stirring at 4°C overnight; the purple lysate was centrifuged at 28,000 g for 40 min, and the colorless supernatant decanted. The purple pellet was resuspended in 0.1 M NaCl and again centrifuged; this was repeated two more times, and the last pellet (resuspended in distilled water) was layered over a step gradient (60%, 35%, and 15% sucrose) and then centrifuged at 100,000 g for 18 h at 10°C. The purple band was collected, and sucrose was removed by dialysis. Finally, the collected PMs were suspended in water and frozen (-20°C). During PM isolation, samples of various cellular fractions were stored to be extracted (whole cells, lysed cells after dialysis, supernatant after the first centrifugation, and PM).
Cell disruption by French press
A suspension of cells (in 4 M NaCl) was passed through a French pressure cell at 2,000 psi three times and left overnight at 4°C. To remove salt from cells broken by French press, an aliquot of a previous sample was dialyzed in the presence of DNase I against water at 4°C overnight.
Time course of GlyC increase during the dialysis
Cells suspended in 4 M NaCl were dialyzed against water at 4°C for different incubation times (15, 30, and 60 min and 12 h). At intervals, lipids were extracted from equivalent aliquots of the lysate by the standard procedure (see below). The weights of the lipid extracts from these aliquots were not significantly different.
Time course of GlyC increase in halobacterial cells diluted in low-salt medium
A suspension of high-producing BR or NRC-1 cells in 4 M NaCl was diluted 5-fold with a hypo-osmotic solution (0.1 M NaCl) and incubated at 25°C under stirring. At intervals, equivalent aliquots of suspension were removed for determination of osmotic fragility (Abs700 nm) and the lipid extraction.
Lipid extraction
Total lipids were extracted using the Bligh and Dyer method, as modified for extreme halophiles (7); the extracts were carefully dried under N2 before weighing. Typically, before lipid extraction, whole cells were suspended in 4 M NaCl, while PMs were in water. In the case of the lipid extraction from PM high salt, 8 mg of PM in water was centrifuged (28,000 g for 40 min) and resuspended in 4 M NaCl (5 ml) before the extraction. For the experiment of lipid re-extract of cells, an aliquot of cell suspension (in 4 M NaCl) was extracted by the standard procedure; then, organic solvents (chloroform-methanol;
:3) were again added to the denatured cellular material and incubated at 80°C for 30 min to obtain a re-extract; this procedure was repeated once more, and the two re-extracts were combined.
Lipid analyses
Total lipid extracts were analyzed by TLC on silica gel (20 cm x 10 cm, layer thickness 0.2 mm). Lipids were eluted with Solvent A (chloroform-methanol-90% acetic acid; 65:4:35) and unless otherwise specified, detected by spraying with sulfuric acid in water, followed by charring at 120°C for 45 min.
The quantitative analyses of GlyC content were performed by video densitometry, using the software ImageJ (http://rsb.info.nih.gov/ij). The lipid standard curves were linear in the concentration range of 1–10 µg. For ESI-MS analyses, dried samples of lipid extract were dissolved in chloroform-methanol (1:1). Electrospray mass spectra were obtained with an API 165 mass spectrometer (Applied Biosystems/MSD Sciex, Concord, Ontario, Canada) equipped with a Turboion Spray interface. The samples were analyzed by loop injection introducing, by a 7125 Rheodyne valve, 5 µl of sample into a 25 µl/min flow of chloroform-methanol delivered by a Harvard model 11 syringe pump (South Natick, MA). The instrumental conditions were as follows: nebulizer gas flow (air), 1.2 l/min; curtain gas flow (nitrogen), 1.2 l/min; needle voltage, 5,600 V; interface temperature, ambient; orifice voltage, -150 V; ring voltage, -200 V; mass range, 50–2,000 amu; mass step, 0.1 amu; dwell time, 0.2 ms. With the orifice voltage used, CID-MS spectra were obtained showing [M-H]- and [M-2H]2-parent ions as well as some fragmentation ions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25% of lipid material was lost during the dialysis, b) 40% of lipids remained in the clear colorless supernatant of the first centrifugation, and c) PM lipids represented only 30% of total cell lipids; at the same time, 70% of starting BR was typically recovered in PM after sucrose gradient.
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Does efficiency of cardiolipin extraction depend on experimental conditions?
It has been previously reported that the efficiency of cardiolipin extraction from microorganisms may be significantly affected by the experimental conditions (8–10).
As cells of H. salinarum were usually suspended in an isotonic 4 M NaCl solution, while lysed cells after dialysis or PM were in medium containing very low or no salt, we examined the possibility that the low content of GlyC in the cell lipid extract could depend on a low efficiency of cardiolipin extraction from the cell membranes in the presence of high salt. Therefore, we have a) re-extracted and analyzed lipids from the denatured proteins remaining after the first lipid extraction of the cells resuspended in isotonic medium to check for the presence of GlyC among residual lipids, and b) checked if the lipid extraction of PM resuspended in 4 M NaCl yields a lipid extract having a lower amount of cardiolipins or is lacking them.
TLC analyses in Fig. 3 show that GlyC is absent in the re-extract (Fig. 3A) and present in the extract from PM in high salt (Fig. 3B); the methanol-chloroform phase resulting after the cell re-extraction was dried, and lipids were dissolved in chloroform and analyzed by TLC; no GlyC was found in this last residual lipid fraction (data not shown).
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GlyC is formed during the isolation of PM
Data in Fig. 2 indicate that GlyC is a nonabundant phospholipid in the whole cells and is formed during the dialysis, when the cells are disrupted by osmotic shock. To study the time course of the GlyC increase during the dialysis, we have analyzed by TLC lipids of equivalent cell aliquots taken at different times from the starting of dialysis and estimated the amount of newly formed GlyC by video densitometry (Fig. 4)
. As the standard curves for the determination by video densitometry of the amounts of individual phospholipids and glycolipids of PM are linear in the 1–10 µg range, to perform a quantitative analysis of the other major lipids present in the extracts before and after the dialysis, from 10 to 40 µg of lipid extracts were analyzed. By comparing the cell lipid profiles before and after the dialysis, it could be seen that in conjunction with the GlyC increase, S-TGD-1 and phosphatidylglycerophosphate methyl ester (PGP-Me) slightly decreased; data in Fig. 5
illustrate the S-TGD-1 and PGP-Me decrease at the end of dialysis in five different experiments. At the end of the dialysis, the S-TGD-1/GlyC molar ratio was found to be 3 and, therefore, identical to that in isolated PM determined by NMR analyses (4). Finally, video densitometric analysis showed that at the end of dialysis, the minor PM lipid component BPG also increases (Fig. 4A).
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In order to follow and quantitate the changes in GlyC and possibly other membrane lipids in samples of swelling and disrupting cells, lipids had been extracted from equivalent aliquots of cells taken at different time intervals from the start of the hypotonic shock. During the osmotic shock, the total cell lipid content and the lipid/BR ratio did not significantly change. Figure 6A
shows the time course of GlyC increase during the osmotic shock constructed by video densitometry, together with the plot of osmotic fragility of high-producing BR halobacterial cells. Three hours after the osmotic shock, 2 µg (i.e., 1 nmol) of GlyC over 40 µg of total lipids were newly formed in the cells (Fig. 6A), and the S-TGD-1/GlyC molar ratio was equal to 6 (data not shown).
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After 3 h of cell incubation in hypotonic medium, 3.8 µg of GlyC over 20 µg of total lipid extract were formed in NRC-1 cells; the final S-TGD-1/GlyC molar ratio was found to be 3. The amount of newly formed GlyC is, therefore, higher in NRC-1 cells than in high-producing BR cells under the same experimental conditions.
Finally, by reducing the amount of extracts loaded on the plate and the time of charring, we were able to find the experimental conditions to show, by video densitometric analyses, that during the osmotic shock, in conjunction with GlyC increase, a decrease in S-TGD-1 content occurred in NRC-1 cells exposed to osmotic shock (Fig. 6B). The decrease in S-TGD-1 could be detected only in the NRC-1 strain, as in these cells the amount of newly formed GlyC is higher, and the final S-TGD-1/GlyC ratio is lower than in the high-producing BR halobacterial cells. In addition, as previously found in the experiment shown in Fig. 5, video densitometric analysis revealed that PGP-Me also decreases during the osmotic shock (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An archaeal bisphosphatidilglycerol or archaeal cardiolipin (BPG) and a glycocardiolipin (GlyC) have been found among residual lipids associated with delipidated BR fractions isolated from an engineered strain of H. salinarum (1) in the natural halophilic community inhabiting the crystallizer brines of salterns (17), in a number of isolates from the saltern ponds of Margherita di Savoia (Italy), and in validated strains available from culture collections (17). While GlyC is only present in species producing PMs, BPG is ubiquitous in all examined members of Halobacteriaceae, suggesting additional roles for it in the extremely halophilic Archaea. Evidence for the association of archaeal BPG with cytochrome c oxidase in an archaeal microorganism has been recently shown (18); therefore, BPG might be only a contaminant of PM.
Very little is known about the biosynthetic pathways of cardiolipins in Archaea. Presumably, the route of archaeal cardiolipin biosynthesis passes through the common intermediates of other archaeal phospholipid pathways, while it is not known whether the final step involves a cardiolipin synthase of prokaryotic or eukaryal type. Present data show for the first time that the cardiolipin synthesis is stimulated by osmotic shock in the Archaea.
This study focuses on the GlyC of the PM of H. salinarum. As GlyC is highly enriched in PM compared with the whole cells, we previously suggested that GlyC is located only in the PM and not in other portions of the cellular membranes of H. salinarum (2). In this study, we have shown that the content of the archaeal cardiolipin GlyC increased in the extreme halophilic microorganism H. salinarum after osmotic shock, and that the newly formed GlyC is mainly accumulated in the PM. In other words, GlyC is normally absent in the PM patches of H. salinarum, and its presence in isolated PM is artificially induced by osmotic shock during the isolation procedure.
The fact that GlyC is not present in the lipid extract of cells disrupted by French press suggests that the formation of cardiolipin during the dialysis is specifically due to osmotic shock and that it is not the consequence of the cell disruption, independent of the method used to lyse the cells. Salt removal, after mechanical cell disruption, did not induce a significant increase of GlyC level, indicating that lowering salt does not per se increase GlyC levels, and that the integrity of the cell is required to efficiently synthesize GlyC. The formation of GlyC (and BPG) clearly occurs in cells undergoing osmotic shock before cell lysis and may represent the physiological response of the microorganisms to low external osmolarity. Whether the GlyC synthesis in H. salinarum requires a specific enzyme still is not clear; it cannot be excluded that GlyC might be chemically synthesized during PM isolation rather than by an enzymatic activity, also because it is well known that the sole protein of the large PM patches is BR, and no other enzymatic proteins are present in PM.
We have been able to quantitate the amount of newly synthesized GlyC in H. salinarum cells by TLC video densitometry. Furthermore, by comparing the quantities of various lipid components in the lipid extracts of swelling and disrupted cells, we have found that in conjunction with the GlyC increase, a decrease of S-TGD-1 occurs.
As the glycolipid S-TGD-1 has the same sugars in the same order as GlyC in the polar head, it can be suggested that the glycolipid S-TGD-1 is one of the precursors for the last step of the biosynthesis of GlyC. PGP-Me could represent a form of activated PA involved in the cardiolipin synthesis, but it remains to be ascertained whether its decrease during osmotic shock is correlated with GlyC synthesis.
Data reported in this study show that the other minor archaeal cardiolipin BPG also increased in H. salinarum cells after osmotic shock; we have not analyzed this phenomenon in detail, because the stimulation of BPG by osmotic shock is more evident and easier to study in other representatives of archaeal extreme halophiles having a higher content of BPG. The presence of BPG-enriched membranes in an extremely halophilic microorganism of the Halorubrum genus has been described in a preliminary report (18), and we are currently investigating the BPG formation in the presence of an osmotic shock.
The increase of cardiolipin content in microorganisms due to osmotic shock has already been reported in the literature (19, 20). In Staphylococcus aureus it has been demonstrated that the de novo synthesis of cardiolipin occurs during the preparation of autoplast by osmotic shock (19). It has been suggested that in Staphylococcus, the loss of cellular wall induced by osmotic shock could stimulate the cardiolipin synthesis to increase the buffer ability of the membrane (20). The present study shows that a specific link exists between the osmotic fragility and the de novo synthesis of cardiolipin in halobacterial cells.
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ACKNOWLEDGMENTS |
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Manuscript received May 22, 2003 and in revised form July 3, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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