Osmotic shock stimulates de novo synthesis of two cardiolipins in an extreme halophilic archaeon

Abstract

The present report illustrates the response to osmotic stress of an extreme halophilic archaeon, Halorubrum sp., isolated from the saltern ponds of Margherita di Savoia in southern Italy. The hypotonic stress induces relevant changes in the membrane lipid composition: archaeal cardiolipin content markedly increases, whereas phosphatidylglycerol (PG) decreases. Membranes isolated from this archaeon after cell disruption by osmotic shock are highly enriched in archaeal cardiolipin and reveal the presence of a novel phospholipid. Electrospray ionization mass spectrometry and NMR analyses revealed that this novel lipid has the structure of a sulfo-diglyco-diether-phosphatidic acid, i.e., a phospholipid dimer or a novel cardiolipin analogue. As NMR analyses showed that the sugars in the novel phospholipid dimer are the same and in the same order of a sulfated diglycosyl diphytanylglycerol diether (S-DGD-5) present as a major lipid component in the archaeon membranes, the novel phospholipid dimer was named S-DGD-5-PA.We conclude that osmotic shock induces a specific increase in the membrane content of the two cardiolipins and suggest that PG and S-DGD-5 are intermediates for the de novo synthesis of archaeal cardiolipin and S-DGD-5-PA, respectively. The present report illustrates the response to osmotic stress of an extreme halophilic archaeon, Halorubrum sp., isolated from the saltern ponds of Margherita di Savoia in southern Italy. The hypotonic stress induces relevant changes in the membrane lipid composition: archaeal cardiolipin content markedly increases, whereas phosphatidylglycerol (PG) decreases. Membranes isolated from this archaeon after cell disruption by osmotic shock are highly enriched in archaeal cardiolipin and reveal the presence of a novel phospholipid. Electrospray ionization mass spectrometry and NMR analyses revealed that this novel lipid has the structure of a sulfo-diglyco-diether-phosphatidic acid, i.e., a phospholipid dimer or a novel cardiolipin analogue. As NMR analyses showed that the sugars in the novel phospholipid dimer are the same and in the same order of a sulfated diglycosyl diphytanylglycerol diether (S-DGD-5) present as a major lipid component in the archaeon membranes, the novel phospholipid dimer was named S-DGD-5-PA. We conclude that osmotic shock induces a specific increase in the membrane content of the two cardiolipins and suggest that PG and S-DGD-5 are intermediates for the de novo synthesis of archaeal cardiolipin and S-DGD-5-PA, respectively. Cardiolipin, a dimeric phospholipid discovered in heart mitochondria, plays an important role in the function and stability of many enzymatic complexes of the respiratory chain (1Schlame M. Rua D. Greenberg M.L. The biosynthesis and functional role of cardiolipin.Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (664) Google Scholar). A number of substituted cardiolipins or cardiolipin analogues have been described in bacteria (2Fischer W. Landgraf H.R. Glycerophosphoryl phosphatidyl kojiboisyl diacylglycerol, a novel phosphoglucolipid from Streptococcus faecalis.Biochim. Biophys. Acta. 1975; 380: 227-244Crossref PubMed Scopus (53) Google Scholar, 3Fischer W. Arneth-Seifert D. D-alanylcardiolipin, a major component of the unique lipid pattern of Vagococcus fluvialis.J. Bacteriol. 1998; 180: 2950-2957Crossref PubMed Google Scholar, 4Gutberlet T. Dietrich U. Bradaczek H. Pohlentz G. Leopold K. Fischer W. Cardiolipin, α-D-glucopyranosyl, and L-lysylcardiolipin from Gram-positive bacteria: FAB MS, monofilm and X-ray powder diffraction studies.Biochim. Biophys. Acta. 2000; 1463: 307-322Crossref PubMed Scopus (20) Google Scholar); the function of these cardiolipin analogues in bacteria is still unclear. Both authentic cardiolipin and cardiolipin analogues are composed of two diacylglycerol moieties and therefore possess four hydrophobic chains. Cardiolipins have also been found in the Archaea world (5Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar, 6Lattanzio V.M.T. Corcelli A. Mascolo G. Oren A. Presence of two novel cardiolipins in the halophilic archaeal community in the crystallizer brines from the salterns of Margherita di Savoia (Italy) and Eilat (Israel).Extremophiles. 2002; 6: 437-444Crossref PubMed Scopus (29) Google Scholar). The first evidence of the presence of archaeal cardiolipin was found in Halobacterium salinarum, in which a bisphosphatidylglycerol (BPG) and another phospholipid dimer composed of the glycolipid sulfated triglycosyl diphytanylglycerol diether-1 (S-TGD-1) esterified to phosphatidic acid (PA) have been described [S-TGD-1-PA, also called glycocardiolipin (GlyC)] (5Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar). GlyC, a major lipid component of the purple membrane (PM) of H. salinarum, plays an important role in the function and stability of bacteriorhodopsin (BR), the photoactivated proton pump (7Lopez F. Lobasso S. Colella M. Agostiano A. Corcelli A. Light-dependent and biochemical properties of two different bands of bacteriorhodopsin isolated on phenyl-sepharose CL-4B.Photochem. Photobiol. 1999; 69: 599-604Crossref Scopus (25) Google Scholar), whereas BPG is only a minor lipid of PM (5Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar, 8Corcelli A. Lattanzio V.M.T. Mascolo G. Papadia P. Fanizzi F. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.J. Lipid Res. 2002; 43: 132-140Abstract Full Text Full Text PDF PubMed Google Scholar). Interestingly, it has been shown that the amount of GlyC in the PM of H. salinarum increases when the cells are exposed to hypotonic shock and that the formation of GlyC occurs at the expense of S-TGD-1 (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In the present report, we show that a halo-archaeal isolate collected from the salterns of Margherita di Savoia in southern Italy, which is closely related to Halorubrum trapanicum, actively synthesizes high amounts of the archaeal cardiolipin BPG under osmotic shock. Membranes isolated after cell disruption by osmotic shock are highly enriched in this archaeal cardiolipin and reveal the presence of a novel phospholipid. The novel phospholipid has been isolated and identified as S-DGD-5-PA, i.e., a phospholipid dimer composed of the glycolipid S-DGD-5 (a sulfo-diglycosyl-diether) esterified to PA. We demonstrate that osmotic shock specifically induces an increase in the content of both archaeal cardiolipins and suggest that the archaeal cardiolipin and the novel cardiolipin analogue S-DGD-5-PA are synthesized at the expense of phosphatidylglycerol (PG) and the glycolipid S-DGD-5, respectively. DNase I was obtained from Sigma (St. Louis, MO); all organic solvents used were commercially distilled and of the highest available purity (Sigma-Aldrich). Plates for TLC (Silica gel 60A; 20 × 10 cm, layer thickness 0.2 mm) were obtained from Merck. The microorganism (MdS1 strain) used in this study was isolated from the salterns of Margherita di Savoia by enrichment culture on agar plates using a medium composed of 250 g/l NaCl, 20 g/l MgSO4·7H2O, 3 g/l tri-sodium citrate dihydrate, 2 g/l KCl, 0.2 g/l CaCl2·2H2O, and 10 g/l neutralized peptone (L34; Oxoid), pH 7.0, prepared as described previously (10Lanyi J.K. McDonald R.E. Light induced transport in Halobacterium halobium.Methods Enzymol. 1979; 88: 5-10Google Scholar). Routinely, the strain was grown aerobically in the light, in a gyratory shaker at 160 rpm at 37°C for ∼10 days, in a liquid medium of the same composition. Cells at the stationary growth phase were harvested by centrifugation and immediately used for lipid extraction or processed for membrane isolation. For preliminary identification of the isolate, the 16S rDNA of MdS1 was amplified and sequenced as described elsewhere (11Benlloch S. Martínez-Murcia A.J. Rodríguez-Valera F. Sequencing of bacterial and archaeal 16S rRNA genes directly amplified from a hypersaline environment.Syst. Appl. Microbiol. 1995; 18: 574-581Crossref Scopus (75) Google Scholar). The 513 nts determined were submitted to the Basic Local Alignment Search Tool to establish the relationships with known species of Haloarchaea. Cells from liquid cultures were collected by centrifugation (8,000 g for 15 min), washed, and resuspended in 4 M NaCl. A dialysis bag (cutoff 12,000–14,000) was filled with a concentrated suspension of cells in 4 M NaCl in the presence of DNase I and left in distilled water under stirring at 4°C overnight. The lysate was centrifugated at 30,000 g for 40 min, and the resulting pellet containing the membranes was washed twice with buffer A (0.15 M NaCl and 0.050 M Tris/Cl, pH 7.0). Isolated membranes were resuspended in the same buffer and stored at −20°C until further use. A suspension of cells (in 4 M NaCl) was diluted 5-fold with a hypoosmotic solution (0.1 M NaCl) and incubated at 25°C under stirring. At intervals (15, 30, 60, 90, and 180 min), equivalent aliquots of suspension were removed for the lipid extraction; the weights of the lipid extracts from these aliquots were not significantly different. A suspension of cells (in 4 M NaCl) was passed three times through a French pressure (FP) cell at 2,000 pounds per square inch. After disruption, the lysate was diluted twice with 4 M NaCl, and unbroken cells and large debris were removed by centrifugation at 4,000 g for 20 min. Membranes were collected by centrifuging the supernatant at 30,000 g for 40 min. Total lipids were extracted using the Bligh and Dyer method as modified for extreme halophiles (12Kates M. Techniques of lipidology.in: Burdon R.H. van Knippenberg P.H. Laboratory Techniques in Biochemistry and Molecular Biology. 2nd edition. Vol. 3. Elsevier, Amsterdam, The Netherlands1986: 100-110Google Scholar); the extracts were carefully dried under N2 before weighing. For the reextraction of whole cells, an aliquot of cell suspension (in 4 M NaCl) was extracted by standard procedure; then, organic solvents [chloroform-methanol-H2O (1:1:0.9; v/v/v)] were again added to the denatured cellular material and incubated at 80°C for 30 min to obtain a reextract. This procedure was repeated once more, and the two reextracts were combined. Plates for TLC were washed twice with chloroform-methanol (1:1; v/v) and activated at 120°C before use. Total lipid extracts were analyzed by TLC in solvent A (chloroform-methanol-90% acetic acid (65:4:35; v/v/v). All lipids were detected by spraying with 5% sulfuric acid, followed by charring at 120°C. The isolation of individual lipid components of the total extracts was performed by scraping the silica gel in each band from the TLC plate and extracting each lipid band from the silica as described previously (5Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar). If necessary, each component was further purified by rechromatography in neutral solvent B [chloroform-methanol-water (65:25:4; v/v/v)]. The determination of the archaeal cardiolipin and PG content of the lipid extracts was performed by densitometric analyses. For quantitative determination, peak data of the unknowns are correlated with data from calibration standards chromatographed on the same plate. In the was by using The standard of authentic standards for BPG and PG were in the For ionization mass spectrometry dried of lipid extracts were in chloroform-methanol (1:1; Electrospray mass were obtained with an mass with a mass spectrometry of band 3 after was performed with a of NMR of isolated lipids were in lipid All NMR analyses were performed on an and are to as an In this we have the lipid of an halophilic archaeon with a high archaeal cardiolipin content under osmotic The halophilic microorganism used in the present was isolated from the salterns of Margherita di Savoia in southern Italy. of the 16S rRNA the isolate used in the present study the Halorubrum in the 513 nts determined H. only nts from the H. a with two of the same or closely related it to to the isolate as Halorubrum In with the 16S rRNA the presence of a in the total cell lipid extract is with the of the MdS1 isolate in the Halorubrum we analyzed by TLC lipid of MdS1 cells in 4 M NaCl and cell lysate after dialysis water The individual lipid components of the extracts were identified by to of authentic standard by with specific and by of isolated individual The lipid components present in both lipid extracts were identified (in as diglycosyl and the archaeal cardiolipin the at the solvent The of purified individual lipids are also in As it is known that H. a glycolipid called S-DGD-5 A. A. The glycolipid of Halobacterium Biophys. Acta. PubMed Scopus Google Scholar), we to the major glycolipid of MdS1 cells as the two lipid it is that the lipid extract of cells a archaeal cardiolipin content and a PG content the lipid extract of whole lipid band from the is present only in the lipid of cells by As shown previously for the GlyC of the PM of H. salinarum, the data in suggest that archaeal cardiolipin is when MdS1 cells are exposed to hypotonic study in the response of MdS1 cells to osmotic we have in the osmotic of the cells exposed to hypotonic medium and the changes in membrane lipid composition. the of the of osmotic of The cells, resuspended in 4 M NaCl, were diluted 5-fold or with M NaCl. MdS1 cells to to an medium containing M not We have previously that cells of a strain of H. salinarum in M NaCl are (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). MdS1 cells when the medium only M it 15 min for the cells to and min for the cells to the changes in the lipid of MdS1 cells osmotic lipids were extracted from equivalent aliquots of cells, in hypotonic and at different times from the of osmotic The TLC in that changes in the lipid of cells after in M NaCl. cardiolipin in cells resuspended in hypotonic it is that archaeal cardiolipin at the expense of A pattern of changes in lipid was found in cells in M NaCl or in the dialysis bag We have by densitometric analyses the amount of individual lipids in the TLC The standard for the individual lipid components of MdS1 cells were in the the of changes in archaeal cardiolipin and the PG content of the osmotic to the shown in After 3 of of MdS1 cells in M NaCl, 3 of archaeal cardiolipin 40 of total lipid extract were to the of 3 of the plate shown in the lipid present in the extract of cells not This that the novel lipid only when the cells by osmotic shock and not in to the lipid we isolated a membrane containing this lipid from the cell by TLC lipid of cells in medium and isolated membranes it is that the presence of the named occurs membrane lipids The same lipid extracts were also analyzed by the of the lipid extracts of cells were identified as PG at S-DGD-5 at and at whereas at and the archaeal analogue of at as a peak to minor At the of the archaeal is also The of the lipid extracts of isolated membranes that archaeal cardiolipin is highly enriched in the lipid extract of membranes, with that of whole cells, whereas PG is significantly in with TLC peak in the lipid of the All of the individual lipids present in the lipid extract of archaeal membranes have been and analyzed by TLC and in solvent and ionization mass spectrometry of lipid components of MdS1 cells and band 3 was detected only in lipid extracts of lipids mass S-DGD-5, sulfated diglycosyl diphytanylglycerol Lipid band 3 was detected only in lipid extracts of in a mass S-DGD-5, sulfated diglycosyl diphytanylglycerol TLC from isolated and purified band 3 are shown in This lipid a on TLC plates for and that it is a analysis in of the purified glycolipid showed and as as The peak is the at and the is present at with at as a minor present at and are of a the peak at is for the of a the at and to the archaeal analogue of to S-DGD-5, and to a or respectively. The pattern of the novel to to that of or GlyC, previously in the PM isolated from H. salinarum (5Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar), that band 3 of a esterified to the of i.e., an S-DGD-5-PA. analysis of purified band 3 the the sugars present in the novel lipid with present in the major glycolipid of we have analyzed isolated from MdS1 membranes NMR in the by proton and band 3. The NMR of band 3 in the of was to that of S-DGD-5 In the two proton present in the of band 3 the same as in the of these at and in both to the of and data that the two have the same sugars in the same in the further to the structure in We conclude that the lipid called band 3 is a novel phospholipid dimer composed of S-DGD-5 and of acid and sulfated triglycosyl diphytanylglycerol of purple membrane of H. salinarum these data reveal that the lipid of cells after dialysis and of membrane isolated from these cells are different from of whole cells in medium of the archaeal cardiolipin content and the of the novel phospholipid dimer S-DGD-5-PA. to that it is the osmotic shock that induces de novo synthesis of the two cardiolipins in MdS1 cells, we analyzed total lipids extracted from cells by French and from membranes isolated from these The TLC shown in that the lipid of cells by French and isolated membrane M were to that of whole cells In the archaeal cardiolipin not significantly increase in the cell or the membrane and band 3 was present in the membrane isolated under these Osmotic stress is a by many The of the cell under different osmotic has not been of evidence suggest that cell plays an important role T. H. of the phospholipid of in medium containing Biophys. Acta. PubMed Scopus Google Scholar, D. M. changes in lipid and in PubMed Scopus Google Scholar). that in the of the cell the of the membrane A. H. in phospholipid of formation of Biophys. Acta. PubMed Scopus Google Scholar). also has been that important in membrane lipid in when the bacteria were grown in a in PG and an increase in cardiolipin content were H. of the of growth in Microbiol. 1998; PubMed Scopus Google Scholar). changes have been in as phospholipid in a of Bacteriol. PubMed Google and T. H. of the phospholipid of in medium containing Biophys. Acta. PubMed Scopus Google in hypersaline an increase in the amount of was in H. of the of growth in Microbiol. 1998; PubMed Scopus Google and in D. M. changes in lipid and in PubMed Scopus Google in NaCl in lipid also when in medium are to hypotonic The archaeal GlyC is in H. salinarum, relevant amounts of it were found in cells by osmotic shock (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). cardiolipin cell content also in the same cells osmotic this not in of the archaeal cardiolipin cell content (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). As archaeal membranes were found in an halophilic microorganism of the Halorubrum S. P. A. and S. in membranes isolated from an halophilic in Italy. Scholar), we that this microorganism a cellular in which to archaeal cardiolipin formation by osmotic shock. In this we found that osmotic the cells of the halophilic microorganism Halorubrum isolated from salterns of Margherita di Savoia (Italy) archaeal We were to the amount of synthesized archaeal cardiolipin by TLC and by the of lipid components in the lipid extracts of cells, we found that a of PG occurs that to the archaeal cardiolipin The that the archaeal cardiolipin increase not in the lipid extracts of cells by French and of membranes isolated from this cell lysate that the formation of cardiolipin dialysis is specifically to osmotic shock. The present report also the presence of a novel phospholipid dimer in Halorubrum and that a membrane isolated from this archaeon after cell disruption by osmotic shock is highly enriched in both archaeal cardiolipin and the novel phospholipid. and NMR analyses revealed that the novel phospholipid has the structure of a S-DGD-5-PA. In NMR analyses showed that the sugars in the novel phospholipid are the same and in the same order as of the glycolipid S-DGD-5 isolated from the Halorubrum membranes, in which the sulfated is to We not study the of S-DGD-5-PA synthesis of content in MdS1 on the the high amount of S-DGD-5 not the of decreases. We suggest that the in phospholipid induced by osmotic shock is to de novo synthesis of the two BPG and S-DGD-5-PA, at the expense of PG and S-DGD-5, respectively. S-DGD-5-PA formation detected only after cell disruption by osmotic whereas the archaeal cardiolipin increase in cells as the two synthesis of Halorubrum and by osmotic of different and the of synthesis of the two cardiolipins different. The changes in the lipid of the of the Halorubrum described in the present study are to in halophiles of the Halobacterium (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). we that the phospholipid dimer is present in amounts in the cells of H. salinarum under and is synthesized de novo cell by osmotic shock at the expense of the glycolipid the cardiolipin analogue specific it still not a cardiolipin in the archaeal to S-DGD-5-PA in Halorubrum and in are to these At we the that these phospholipid in study on the of the PM of H. salinarum (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), we that in the in which is after osmotic a specific to this lipid not is known that the only of the PM is and that are The Halorubrum cells used in the present study to osmotic a strain of H. salinarum cells previously (9Lobasso S. Lopalco P. Lattanzio V.M.T. Corcelli A. Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.J. Lipid Res. 2003; 44: 2120-2126Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). we that archaeal cardiolipin cell content the cell from The archaeal cardiolipin increase that occurs in cells osmotic shock the response of the to The in lipid the properties of the membrane as membrane and transport of have been identified in are in bacterial are activated by the lipid Molecular of in Scopus Google Scholar). Cardiolipins and Biophys. Acta. PubMed Scopus Google that the of two PG to cardiolipin increases the order on the membrane and that cardiolipin in the of by the it is known that the of the cardiolipin in the of through The increase in cardiolipin as a the high or to the transport T. H. of the phospholipid of in medium containing Biophys. Acta. PubMed Scopus Google Scholar). In the a to the of the cell to osmotic the on the transport of showed that this osmotic stress through in membrane The and the in the lipid are to the T. the osmotic and of an transport for PubMed Scopus Google Scholar). In this that the of cells to the membrane biochemical and changes in of cardiolipin We are further of the role of the two archaeal phospholipid in the In a preliminary we have shown that archaeal cardiolipin is to a S. P. A. and S. in membranes isolated from an halophilic in Italy. Scholar), whereas the function of S-DGD-5-PA is still The for M. for 16S rRNA and for microorganism and di di for NMR analyses. This was by of the and by the per bisphosphatidylglycerol or cardiolipin bacteriorhodopsin ionization mass spectrometry glycocardiolipin or or phosphatidic acid phosphatidylglycerol purple membrane