Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii

As part of a study to identify novel lipids with immune adjuvant activity, a structural comparison was made between the polar lipids from two halophiles, an archaeon Haloferax volcanii and a eubacterium Planococcus H8. H. volcanii polar lipid extracts consisted of 44% archaetidylglycerol methylphosphate, 35% archaetidylglycerol, 4.7% of archaeal cardiolipin, 2.5% archaetidic acid, and 14% sulfated glycolipids 1 and 2. Nuclear magnetic resonance (NMR) and Fast atom bombardment mass spectrometry (FAB MS) data determined the glycolipids to be 6-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol] and a novel glycocardiolipin 6'-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol]. The polar lipids of Planococcus H8 consisted of 49% saturated phosphatidylglycerol and cardiolipin (9:1, w/w), and surprisingly 51% of the photosynthetic membrane lipid sulfoquinovosyldiacylglycerol (SQDG). This study documents archaeal cardiolipin and a novel glycocardiolipin in H. volcanii (lacking purple membrane), and is the first report of SQDG in a non-photosynthetic, halophilic bacterium.     

Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum

In the purple membrane (PM) of Halobacterium salinarum is present a phospholipid dimer consisting of sulfo-triglycosyl-diether (S-TGD-1) esterified to the phosphate group of phosphatidic acid (PA), i.e., S-TGD-1-PA, called glycocardiolipin (GlyC) (Corcelli, A., M. Colella, G. Mascolo, F. P. Fanizzi, and M. Kates. A novel glycolipid and phospholipid in the purple membrane. 2000. Biochemistry. 39: 3318-3326). The GlyC content of whole cells, PM, and other cell fractions of H. salinarum have been analyzed. GlyC is a nonabundant phospholipid in H. salinarum cells, and it represents one of the major phospholipids of isolated PM. 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.     

Crossing the lipid divide

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.     

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10-N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.     

The cardiolipin analogues of Archaea

The present article reviews studies of the structure and functional roles of the cardiolipin analogues of extremely halophilic prokaryotes belonging to the Archaea domain. Analogies and differences between the archaeal bisphosphatidylglycerol and the mitochondrial cardiolipin are presented. Furthermore the structure of archaeal glycophospholipid dimers is illustrated together with the available information on their function. The studies on the function of cardiolipin analogues in archaebacteria point out the tight interaction established by these phospholipids with membrane proteins and their role as bioactive lipids in the adaptation of microorganisms to osmotic stress.     

Geranylgeranyl Reductase and Ferredoxin from Methanosarcina acetivorans Are Required for the Synthesis of Fully Reduced Archaeal Membrane Lipid in Escherichia coli Cells

Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.     

A novel glycolipid and phospholipid in the purple membrane

A novel glycolipid of mass 1935 and a phospholipid of mass 1522 are the main residual lipids (along with traces of PGP-Me, S-TGD-1, and PG) specifically associated with "delipidated" bacteriorhodopsin fractions BR I and BR II, prepared by Triton X-100 treatment of purple membrane (PM), from a genetically engineered strain (L33) of Halobacterium salinarum, and chromatography on phenyl-Sepharose CL-4B. The novel glycolipid and phospholipid are components of the PM matrix not previously described. The TLC isolated and purified novel glycolipid and phospholipid were shown, by chemical degradation, mass spectrometry, and NMR analyses, to have the structure, respectively, of a phosphosulfoglycolipid, 3-HSO(3)-Galp-beta1,6Manp-alpha1,2Glcp-alpha1,1-[sn-2, 3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycero l], and of a glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2, 3-di-O-phytanylglycerol.     

Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in Escherichia coli

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Structural study of the β-hairpin marine antimicrobial peptide arenicin-2 in PC/PG lipid bilayers by fourier transform infrared spectroscopy

Arenicin-2 is a 21-residue β-hairpin antimicrobial peptide isolated from the marine lugworm Arenicola marina. The structure of this cationic peptide in partly charged lipid membrane made of PC/PG (7: 3) was studied by FTIR, CD, and Trp fluorescence spectroscopies. FTIR spectra of arenicin in amide I region were analyzed using curve-fitting and second derivative procedures. The FTIR data for the peptide in PC/PG liposomes were compared with the data obtained in anionic SDS micelles where arenicin forms a dimer stabilized by parallel association of two β-hairpins according to previous NMR spectroscopy studies [Ovchinnikova et al., Biopolymers, 2007, vol. 89, pp. 455–464; Shenkarev et al., Biochemistry, 2011, vol. 50, pp. 6255–6265]. The results obtained in present work indicate that arenicin forms the dimeric structure in partly charged PC/PG lipid membrane. This finding is discussed in relation to interpretation of low-conducting pores observed for arenicin in negatively charged membranes.     

Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.

The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8.The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.     

Archaebacterial lipid membranes as models to study the interaction of 10-N-nonyl acridine orange with phospholipids

The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms.     

Role of anionic lipid in bacterial membranes

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.     

Changing the phospholipid composition of Staphylococcus aureus causes distinct changes in membrane proteome and membrane‐sensory regulators

The dynamic lipid composition of bacterial cytoplasmic membranes has a profound impact on vital bacterial fitness and susceptibility to membrane‐damaging agents, temperature, or osmotic stress. However, it has remained largely unknown how changes in lipid patterns affect the abundance and expression of membrane proteins. Using recently developed gel‐free proteomics technology, we explored the membrane proteome of the important human pathogen Staphylococcus aureus in the presence or absence of the cationic phospholipid lysyl‐phosphatidylglycerol (Lys‐PG). We were able to detect almost half of all theoretical integral membrane proteins and could reliably quantify more than 35% of them. It is worth noting that the deletion of the Lys‐PG synthase MprF did not lead to a massive alteration but a very distinct up‐ or down‐regulation of only 1.5 or 3.5% of the quantified proteins. Lys‐PG deficiency had no major impact on the abundance of lipid‐biosynthetic enzymes but significantly affected the amounts of the cell envelope stress‐sensing regulatory proteins such as SaeS and MsrR, and of the SaeS‐regulated proteins Sbi, Efb, and SaeP. These data indicate very critical interactions of membrane‐sensory proteins with phospholipids and they demonstrate the power of membrane proteomics for the characterization of bacterial physiology and pathogenicity.     

Protein-Associated Lipid of Bacillus stearothermophilus

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.     

Treponemal phospholipids inhibit innate immune responses induced by pathogen-associated molecular patterns

Host innate immune responses to microbial components, known as pathogen-associated molecular patterns (PAMPs), are regulated and modified by cellular receptors and serum proteins, including Toll-like receptors (TLRs), CD14, and LPS-binding protein (LBP). We demonstrated that a treponemal membrane lipid inhibited PAMPs-induced immune responses. The chemical structure of the lipid was elucidated as a phosphatidylglycerol (PG) derivative, which is scarce in most mammalian tissues, but relatively abundant in treponemal membrane lipids. Natural and synthetic PG counterparts as well as related natural anionic phospholipids, phosphatidylinositol, phosphatidylserine, and cardiolipin, also demonstrated an inhibitory effect. Further, we noted that PG inhibited PAMPs-induced immune responses by blocking the binding of PAMPs with LBP and CD14. In addition, PG decreased proinflammatory cytokine production in serum of LPS-injected mice and depressed abscess formation in mice infected with treponemes. These results suggest that treponemal phospholipid interfere the function of LBP/CD14 and act as a modulator of innate immune responses.     

Interaction of phospholipids with the detergent-solubilized ADP/ATP carrier protein as studied by spin-label electron spin resonance

The interaction of spin-labeled phospholipids with the detergent-solubilized ADP/ATP carrier protein from the inner mitochondrial membrane has been investigated by electron spin resonance spectroscopy. The equilibrium binding of cardiolipin and phosphatidic acid was studied by titration of the protein with spin-labeled phospholipid analogues using a spectral subtraction protocol for the evaluation of the mobile and immobilized lipid portions. This analysis revealed the immobilization of two molecules of spin-labeled cardiolipin per protein dimer. Phosphatidic acid has a similar affinity for the protein surface as cardiolipin. The lipid-protein interaction was less pronounced with the neutral phospholipids and with phosphatidylglycerol. The importance of the electrostatic contribution to the phospholipid-protein interaction shows up with a strong dependence of the lipid binding on salt concentration. Cleavage by phospholipase A2 and spin reduction by ascorbate of the spin-labeled acidic phospholipids in contact with the protein surface suggest that these lipids are located on the outer perimeter of the protein. At reduced detergent concentration, the protein aggregated upon addition of small amounts of cardiolipin but remained solubilized when more cardiolipin was added. This result is discussed with respect to the aggregation state of the protein in the mitochondrial membrane. It is also tentatively concluded that binding of spin-labeled cardiolipin does not displace the tightly bound cardiolipin of mitochondrial origin, which was detected previously by 31P nuclear magnetic resonance spectroscopy.     

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Tightly associated cardiolipin in the bovine heart mitochondrial ATP synthase as analyzed by 31P nuclear magnetic resonance spectroscopy

The bovine heart F0F1-ATPase preparation (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) has been further delipidated. The lipid-deficient preparation contained 2.5 mol of cardiolipin, 1 mol of phosphatidylcholine (PC), and 1 mol of phosphatidylethanolamine (PE) per mol of F0F1. When reconstituted with asolectin the delipidated preparation exhibited an activity of 13 mumol of ATP hydrolyzed/min/mg of protein which was 88% oligomycin-sensitive. The phospholipids in this preparation were analyzed by 31P NMR spectroscopy to determine if they were immobilized by the enzyme (rendered NMR-invisible). The PC and PE were below the limits of detection under the conditions utilized and the cardiolipin was NMR-invisible until the enzyme was denatured by addition of either 1% sodium dodecyl sulfate or 8 M urea. Addition of cardiolipin to the delipidated preparation and subsequent analysis by NMR spectroscopy revealed that approximately 4 mol of cardiolipin were immobilized per mol of F0F1 ATPase. The enzyme appears to have high affinity for cardiolipin exclusively, since PC (a prominent inner membrane lipid), phosphatidyl serine (an acidic phospholipid), and phosphatidyl glycerol (the precursor to cardiolipin) were not immobilized (rendered NMR-invisible) when added to the delipidated preparation.