Liposome adjuvants prepared from the total polar lipids of Haloferax volcanii, Planococcus spp. and Bacillus firmus differ in ability to elicit and sustain immune responses.

Immune stimulating activity was compared for lipid vesicles consisting of the total polar lipids of an archaeon Haloferax volcanii, and the eubacteria Planococcus spp. and Bacillus firmus. Each total polar lipid extract readily formed liposomes of similar size, within which the protein antigen ovalbumin was entrapped, with comparable loading and internalization. Subcutaneous immunization of mice resulted in anti-ovalbumin antibody titers for all adjuvants, with memory recall responses that were significantly greater with the archaeal lipid (H. volcanii versus Planococcus). More striking, induction of cytotoxic T cell activity against the entrapped antigen, measured 10 days following a single vaccination (primary response) rapidly declined by week 7 (secondary response after injections on days 0 and 21) in mice immunized with Planococcus spp. liposomes, but was sustained in mice immunized with H. volcanii archaeosomes. Surprisingly, antigen free-Planococcus liposomes evoked potent non-specific inflammatory cytokine production (IL-12 and IL-6) by dendritic cells whereas archaeal H. volcanii vesicles evoked little inflammatory cytokines. This suggested that overt inflammatory response might not necessarily aid sustenance of immunity. B. firmus liposomes consisted of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin and was an ineffective CTL adjuvant, even for initiating a primary response. Considering that the polar lipids of H. volcanii and Planococcus spp. both consist of the same lipid classes (sulfoglycolipids, phosphoglycerols, and cardiolipins), the unique ability of archaeosomes to maintain antigen-specific T cell immunity may be attributable to a property of the archaeal 2,3-diphytanylglycerol lipid core.

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.

Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specific protective immunity against infection with the facultative intracellular pathogen, Listeria monocytogenes.

Protective immunity to intracellular bacterial pathogens usually requires the participation of specific CD8+ T cells. Natural exposure of the host to sublethal infection, or vaccination with attenuated live vaccines are the most effective means of eliciting prolonged protective cell-mediated immunity against this class of pathogens. The ability to replace these immunization strategies with defined sub-unit vaccines would represent a major advance for clinical vaccinology. The present study examines the ability of novel liposomes, termed archaeosomes, made from the polar lipids of various Archaeobacteria to act as self-adjuvanting vaccine delivery vehicles for such defined acellular antigens. Using infection of mice with Listeria monocytogenes as a model system, this study clearly demonstrates the ability of defined, archaeosome-entrapped antigens to elicit rapid and prolonged specific immunity against a prototypical intracellular pathogen. In this regard, all of the tested archaeosomes were superior to conventional liposomes.

Molecular mechanisms of water and solute transport across archaebacterial lipid membranes.

Archaebacteria thrive in environments characterized by anaeobiosis, saturated salt, and both high and low extremes of temperature and pH. The bulk of their membrane lipids are polar, characterized by the archaeal structural features typified by ether linkage of the glycerol backbone to isoprenoid chains of constant length, often fully saturated, and with sn-2,3 stereochemistry opposite that of glycerolipids of Bacteria and Eukarya. Also unique to these bacteria are macrocyclic archaeol and membrane spanning caldarchaeol lipids that are found in some extreme thermophiles and methanogens. To define the barrier function of archaebacterial membranes and to examine the effects of these unique structural features on permeabilities, we investigated the water, solute (urea and glycerol), proton, and ammonia permeability of liposomes formed by these lipids. Both the macrocyclic archaeol and caldarchaeol lipids reduced the water, ammonia, urea, and glycerol permeability of liposomes significantly (6-120-fold) compared with diphytanylphosphatidylcholine liposomes. The presence of the ether bond and phytanyl chains did not significantly affect these permeabilities. However, the apparent proton permeability was reduced 3-fold by the presence of an ether bond. The presence of macrocyclic archaeol and caldarchaeol structures further reduced apparent proton permeabilities by 10-17-fold. These results indicate that the limiting mobility of the midplane hydrocarbon region of the membranes formed by macrocyclic archaeol and caldarchaeol lipids play a significant role in reducing the permeability properties of the lipid membrane. In addition, it appears that substituting ether for ester bonds presents an additional barrier to proton flux.

The potent adjuvant activity of archaeosomes correlates to the recruitment and activation of macrophages and dendritic cells in vivo.

The unique glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with potent adjuvant activity. We studied the effect of archaeosomes on APCs to elucidate the mechanism(s) of adjuvant action. Exposure of J774A.1 macrophages to archaeosomes in vitro resulted in up-regulation of B7.1, B7.2, and MHC class II molecules to an extent comparable to that achieved with LPS. Similarly, incubation of bone marrow-derived DCs with archaeosomes resulted in enhanced expression of MHC class II and B7.2 molecules. In contrast, conventional liposomes made from ester phospholipids failed to modulate the expression of these activation markers. APCs treated with archaeosomes exhibited increased TNF production and functional ability to stimulate allogenic T cell proliferation. More interestingly, archaeosomes enhanced APC recruitment and activation in vivo. Intraperitoneal injection of archaeosomes into mice led to recruitment of Mac1alpha(+), F4/80(+) and CD11c(+) cells. The expression of MHC class II on the surface of peritoneal cells was also enhanced. Furthermore, peritoneal cells from archaeosome-injected mice strongly enhanced allo-T cell proliferation and cytokine production. The ability of archaeosome-treated APCs to stimulate T cells was restricted to Mac1alpha(high), B220(-) cells in the peritoneum. These Mac1alpha(high) cells in the presence of GM-CSF gave rise to both F4/80(+) (macrophage) and CD11c(+) (dendritic) populations. Overall, the activation of APCs correlated to the ability of archaeosomes to induce strong humoral, T helper, and CTL responses to entrapped Ag. Thus, the recruitment and activation of professional APCs by archaeosomes constitutes an efficient self-adjuvanting process for induction of Ag-specific responses to encapsulated Ags.

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help.

The unique ether glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.

Influence of coenzyme Q10 on tissue distribution of archaeosomes, and pegylated archaeosomes, administered to mice by oral and intravenous routes.

The biodistribution of orally and intravenously administered archaeosomes in mice was compared to that of archaeosomes containing either coenzyme Q10 (archaeosome-CoQ10), polyethylene glycol (archaeosome-PEG), or PEG plus CoQ10 (archaeosome-PEG-CoQ10). The archaeosome formulations were prepared by a reverse-phase evaporation method using the total polar lipids from the archaeobacterium Methanosarcina mazei. In the case of oral gavage, the most striking observation was that a significantly (p < 0.05) higher concentration (42.28+/-4.17%) of administered dose was found in the stomach content 3 h after administration of unmodified archaeosomes, as compared to that of archaeosome-CoQ10 (16.98+/-2.48%) and archaeosome-PEG-CoQ10 (5.8 +/-4.05"/ vesicles. This correlated with an increased uptake, notably of the archaeosome-PEG-CoQ 0 vesicles.,into liver and spleen; however, no more than 7% of the administered dose was found in liver, spleen and blood at any time point studied. In the case of intravenous administration, a significantly higher percentage of injected dose of unmodified archaeosomes was found in the liver (66.4 +/-.92%) and spleen (11.445+/-.68%) at 48 h, compared to archaeosome-CoQ10, archaeosome-PEG, and archaeosome-PEG-CoQ10 vesicles. The combination of PEG and CoQ10 significantly prolonged the circulation of archaeosomes in the blood, but after 48 h the amount of the vesicle marker in blood had declined to only about 0.5% of administered dose. These data indicate that the biodistribution of archaeosome formulations given orally or intravenously can be altered significantly by incorporating PEG or CoQ 10, alone or in combination, and these vesicles have the potential to act as a carrier for therapeutics and vaccines.

Archaeosome vaccine adjuvants induce strong humoral, cell-mediated, and memory responses: comparison to conventional liposomes and alum.

Ether glycerolipids extracted from various archaeobacteria were formulated into liposomes (archaeosomes) possessing strong adjuvant properties. Mice of varying genetic backgrounds, immunized by different parenteral routes with bovine serum albumin (BSA) entrapped in archaeosomes ( approximately 200-nm vesicles), demonstrated markedly enhanced serum anti-BSA antibody titers. These titers were often comparable to those achieved with Freund's adjuvant and considerably more than those with alum or conventional liposomes (phosphatidylcholine-phosphatidylglycerol-cholesterol, 1. 8:0.2:1.5 molar ratio). Furthermore, antigen-specific immunoglobulin G1 (IgG1), IgG2a, and IgG2b isotype antibodies were all induced. Association of BSA with the lipid vesicles was required for induction of a strong response, and >80% of the protein was internalized within most archaeosome types, suggesting efficient release of antigen in vivo. Encapsulation of ovalbumin and hen egg lysozyme within archaeosomes showed similar immune responses. Antigen-archaeosome immunizations also induced a strong cell-mediated immune response: antigen-dependent proliferation and substantial production of cytokines gamma interferon (Th1) and interleukin-4 (IL-4) (Th2) by spleen cells in vitro. In contrast, conventional liposomes induced little cell-mediated immunity, whereas alum stimulated only an IL-4 response. In contrast to alum and Freund's adjuvant, archaeosomes composed of Thermoplasma acidophilum lipids evoked a dramatic memory antibody response to the encapsulated protein (at approximately 300 days) after only two initial immunizations (days 0 and 14). This correlated with increased antigen-specific cell cycling of CD4(+) T cells: increase in synthetic (S) and mitotic (G(2)/M) and decrease in resting (G(1)) phases. Thus, archaeosomes may be potent vaccine carriers capable of facilitating strong primary and memory humoral, and cell-mediated immune responses to the entrapped antigen.

In vitro assessment of archaeosome stability for developing oral delivery systems.

The in vitro stability of archaeosomes made from the total polar lipids of Methanosarcina mazei, Methanobacterium espanolae or Thermoplasma acidophilum, was evaluated under conditions encountered in the human gastrointestinal tract. At acidic pH, multilamellar vesicles (MLV) prepared from T. acidophilum lipids were the most stable, releasing approximately 80, 20, 10 and 5% of encapsulated 14C-sucrose at pH 1.5, 2.0, 2.5 and 6.2, respectively, after 90 min at 37 degrees C. Archaeosomes from M. mazei lipids were the least stable. For each type of total polar lipid, unilamellar vesicles (ULV) were less stable than the corresponding MLV vesicles. Pancreatic lipase had relatively minor effect on the stability of archaeosomes made from either of the three types of total polar lipids, causing the release of 12-27% of the encapsulated 5(6)-carboxyfluorescein (CF) from ULV and MLV after 90 min at 37 degrees C. In simulated human bile at pH 6.2, MLV from M. mazei total polar lipids lost 100% of the encapsulated CF after 90 min at 37 degrees C, whereas those from the polar lipids of M. espanolae or T. acidophilum lost approximately 85% of the marker. Pancreatic lipase and simulated human bile had no synergistic effect on the release of carboxyfluorescein from ULV or MLV prepared from any of the total polar lipids. After 90 min in the combined presence of these two stressors at pH 6.2, the leakage of fluorescein conjugated bovine serum albumin from MLV prepared from T. acidophilum lipids was similar to that of CF, and 13% of the initially present vesicles appeared to be intact. These results indicate that archaeosomes show stability properties indicative of potential advantages in developing applications as an oral delivery system.

A structural comparison of the total polar lipids from the human archaea Methanobrevibacter smithii and Methanosphaera stadtmanae and its relevance to the adjuvant activities of their liposomes.

Mice were immunized with bovine serum albumin (BSA) entrapped within archaeosomes (i.e. liposomes) composed of the total polar lipids (TPL) from the two methanogenic archaea common to the human digestive tract. Methanobrevibacter smithii archaeosomes boosted serum anti-BSA antibody to titers comparable to those achieved with Freund's adjuvant, whereas Methanosphaera stadtmanae archaeosomes were relatively poor adjuvants. An explanation for this difference was sought by analysis of the polar lipid composition of each archaeobacterium. Fast atom bombardment mass spectrometry and NMR analyses of the purified lipids revealed a remarkable similarity in the ether lipid structures present in each TPL extract. However, the relative amounts of each lipid species varied dramatically. The phospholipid fraction in M. stadtmanae TPL was dominated by archaetidylinositol (50 mol% of TPL) and the glycolipid fraction by beta-Glcp-(1,6)-beta-Glcp-(1,1)-archaeol (36 mol%), whereas in M. smithii extracts, both caldarchaeol and archaeol lipids containing a phosphoserine head group were relatively abundant. Liposomes prepared from purified archaetidylinositol and from M. stadtmanae TPL supplemented with increasing amounts of phosphatidylserine elicited poor humoral responses to encapsulated BSA. A dramatic loss in the adjuvanticity of M. smithii archaeosomes was seen upon incorporation of 36 mol% of the uncharged lipid diglucosyl archaeol and, to a lesser extent, of 50 mol% of archaetidylinositol. Interestingly, the relative rates of uptake of M. smithii and M. stadtmanae archaeosomes by phagocytic cultures in vitro were similar. Thus, the lipid composition may influence archaeosome adjuvanticity, particularly a high diglucosyl archaeol and/or archaetidyl inositol content, resulting in a low adjuvant activity.

Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems.

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeobacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.

Coenzyme Q10 in vesicles composed of archaeal ether lipids or conventional lipids enhances the immuno-adjuvanticity to encapsulated protein.

Cellular accumulation, tissue distribution, and immuno-adjuvanticity were evaluated for liposomal CoQ10 prepared from either distearoylphosphatidylcholine:dicetylphosphate:cholesterol (4:1:5, mol. ratio) (conventional liposomes) or from the total polar lipids of the archaeon Methanosarcina mazei (archaeosomes). Liposomal CoQ10 vesicles of approximately 100 nm diameter, containing up to 179 mumol of CoQ10 per mg of lipid have been evaluated using J774A.1 macrophages and Balb/c mice. Archaeosomes uptake by J774A.1 macrophages was better than with the conventional liposome, and the incorporation of CoQ10 enhanced the uptake of both lipid vesicle types. All vesicle types were detected in the liver and spleen of mice (4-27% of injected dose) within 3 h of intraperitoneal injection. Moreover, incorporation of CoQ10 into lipid vesicles enhanced the immuno-adjuvanticity of both conventional liposomes and archaeosomes, to achieve approximately a doubling in the titres of BSA-specific antibody in sera to 169 and 430 micrograms ml-1, respectively. Increases in IgG1 and IgG2a/2b accounted for most of the CoQ10-induced increases in anti-BSA titres. These results are rationalized on the basis of surface hydrophobicity and opsonization changes induced by the presence of CoQ10 in vesicles. We suggest that liposomal CoQ10 has potential as a new generation of vaccine delivery system to enhance the immune response. Its use as a novel delivery system may be particularly effective under pathological conditions where the occurrence of an oxidative stress condition significantly impairs the immune system functions.

Lateral diffusion of the total polar lipids from Thermoplasma acidophilum in multilamellar liposomes.

31P NMR lineshapes of multilamellar liposomes composed mostly of a bilayer-spanning tetraether lipid are consistent with rapid axially symmetric motion about the bilayer normal. The residual chemical shift anisotropy of 36 ppm is comparable to that seen for diacylphosphatidylglycerol systems and suggests comparable headgroup motion. The lateral diffusion rates for Thermoplasma acidophilum total polar lipids in mutilamellar liposomes was measured by two dimensional exchange NMR as a function of temperature. At 55 degrees C, near the growth temperature, the rate of lateral diffusion, DL, is comparable to that of diester phospholipids in the Lalpha liquid crystalline phase, having a value of 2 x 10(-8) cm2/s. DL decreases with temperature reaching a value of 8-6 x 10(-9) cm2/s at 30 degrees C. The activation energy Ea for lateral diffusion is estimated to be 10 kcal/mol (approximately 42 kJ/mol). The lateral diffusion rates indicate that the tetraether liposomes have a membrane viscosity at 30 degrees C which is considerably higher than that of diester phospholipids in the liquid crystalline phase.

Archaeosomes as novel antigen delivery systems.

The humoral immune response mounted in BALB/c mice against bovine serum albumin or cholera toxin B subunit was compared when the antigens were associated with liposomes composed of either archaeal ether lipids or conventional lipids. Antibody titres in sera from mice immunised intraperitoneally were elevated to an extent comparable to those achieved with Freund's adjuvant by encapsulating bovine serum albumin in archaeal lipid vesicles (archaeosomes) of about 200 nm diameter. Comparison among six archaeosome and three conventional liposome compositions established that archaeosomes were generally much superior in potentiating an immune response. Further, only two immunisations, at the most, were needed to achieve close to the maximum antibody titre, as shown with archaeosomes composed of the polar lipids from Methanobrevibacter smithii, an inhabitant of the human colon. A similar positive response to presenting the more immunogenic cholera B subunit protein to the immune system of mice was shown for M. smithii archaeosomes. Encapsulation of the antigen in the archaeosome was necessary to achieve the full humoral response. This represents the first study to our knowledge where archaeosomes have been evaluated in vivo as possible antigen carriers.

Identification of beta-L-gulose as the sugar moiety of the main polar lipid Thermoplasma acidophilum.

The main polar lipid (MPL) of Thermoplasma acidophilum has been purified and its structure determined. NMR, mass spectrometry, and capillary gas chromatography-mass spectrometry experiments have shown that the previously unidentified sugar moiety of MPL is the rare sugar L-gulose. MPL is thus a tetraether lipid with cyclopentane rings and head groups of phosphoglycerol, as previously reported, and beta-L-gulopyranose. Further, MPL is also the dominant lipid found in lipid extracts from another species of the Thermoplasma genus, T. volcanium, suggesting that L-gulose may represent a dominant sugar moiety of the polar lipids biosynthesized by this archaeobacterial genus. Minor phospholipids were tentatively identified as diether and hydroxydiether analogs of phosphatidylglycerol, and phosphatidylinositol.

Stability of pressure-extruded liposomes made from archaeobacterial ether lipids.

Ether lipids were obtained from a wide range of archaeobacteria grown at extremes of pH, temperature, and salt concentration. With the exception of Sulfolobus acidocaldarius, unilamellar and/or multilamellar liposomes could be prepared from emulsions of total polar lipid extracts by pressure extrusion through filters of various pore sizes. Dynamic light scattering, and electron microscopy revealed homogeneous liposome populations with sizes varying from 40 to 230 nm, depending on both the lipid source and the pore size of the filters. Leakage rates of entrapped fluorescent or radioactive compounds established that those archaeobacterial liposomes that contained tetraether lipids were the most stable to high temperatures, alkaline pH, and serum proteins. Most ether liposomes were stable to phospholipase A2, phospholipase B and pancreatic lipase. These properties of archaeobacterial liposomes make them attractive for applications in biotechnology.

Tetraether lipids of Methanospirillum hungatei with head groups consisting of phospho-N,N-dimethylaminopentanetetrol, phospho-N,N,N-trimethylaminopentanetetrol, and carbohydrates.

Acyclic, standard tetraether and diether lipids each account for about 50% of the total ether lipids found in Methanospirillum hungatei. Sixteen ether lipids were purified and defined according to relative weight percentage and staining reactions on thin-layer plates. Structures were elucidated for six previously uncharacterized tetraether lipids. Four of these lipids had as one head group either alpha-glcp-(1-2)-beta-gal(f)-, or beta-gal(f)-(1-6)-beta-gal(f)-, in glycosidic linkage to the first glycerol of the lipid backbone, and either a N,N-dimethyl-aminopentanetetrol or a N,N,N-trimethylaminopentanetetrol moiety in phosphodiester linkage to the second glycerol of the backbone. A fifth lipid was a tetraether structure novel in having carbohydrate moieties at both head group positions; namely alpha-glcp-(1-2)-gal(f)- and beta-gal(f)-. Two other lipids, a diether and a tetraether, had a single head group consisting of alpha-glcp-(1-2)-beta-gal(f)- modified by O-acetylation of the gal(f) residue at C-6. In addition to the seven new lipids described above, diether and tetraether analogs of phosphatidylglycerol were found.

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts.

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per mug of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per mug of vector DNA. However, a 380-fold improvement (3,417 transformants per mug of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.

Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria.

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-C]-, [2-C]-, or [3-C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.

Hydroxydiether Lipid Structures in Methanosarcina spp. and Methanococcus voltae.

Hydroxylated diether lipids are the most abundant lipids in Methanosarcina acetivorans, Methanosarcina thermophila, and Methanosarcina barkeri MS and Fusaro, regardless of the substrate used for growth. Structural analysis of the lipid moiety freed of polar head groups revealed that the hydroxydiether lipids of all the Methanosarcina strains were hydroxylated at position 3 of sn-2 phytanyl chains. The finding that Methanosarcina strains synthesize the same hydroxydiether structure suggests that this is a taxonomic characteristic of the genus. Methanococcus voltae produced minor amounts of the 3-hydroxydiether characteristic of Methanosarcina spp. and also the 3'-hydroxydiether described previously for Methanosaeta concilii.