Novel complex polar lipids from the methanogenic archaebacterium Methanospirillum hungatei.

The methanogenic archaebacterium Methanospirillum hungatei contains two unusual phosphoglycolipids that account for 64 percent of the total cellular lipids. These lipids are derivatives of the dibiphytanyl diglycerol tetraether, previously identified in methanogens. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid, and the other is linked glycosidically to a disaccharide. The two phosphoglycolipids may function as covalently bonded lipid bilayers to impart stability and rigidity to methanogen membranes.

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.

The bioenergetics of methanogenesis.

The reduction of CO2 or any other methanogenic substrate to methane serves the same function as the reduction of oxygen, nitrate or sulfate to more reduced products. These exergonic reactions are coupled to the production of usable energy generated through a charge separation and a protonmotive-force-driven ATPase. For the understanding of how methanogens derive energy from C-1 unit reduction one must study the biochemistry of the chemical reactions involved and how these are coupled to the production of a charge separation and subsequent electron transport phosphorylation. Data on methanogenesis by a variety of organisms indicates ubiquitous use of CH3-S-CoM as the final electron acceptor in the production of methane through the methyl CoM reductase and of 5-deazaflavin as a primary source of reducing equivalents. Three known enzymes serve as catalysts in the production of reduced 5-deazaflavin: hydrogenase, formate dehydrogenase and CO dehydrogenase. All three are potential candidates for proton pumps. In the organisms that must oxidize some of their substrate to obtain electrons for the reduction of another portion of the substrate to methane (e.g., those using formate, methanol or acetate), the latter two enzymes may operate in the oxidizing direction. CO2 is the most frequent substrate for methanogenesis but is the only substrate that obligately requires the presence of H2 and hydrogenase. Growth on methanol requires a B12-containing methanol-CoM methyl transferase and does not necessarily need any other methanogenic enzymes besides the methyl-CoM reductase system when hydrogenase is present. When bacteria grow on methanol alone it is not yet clear if they get their reducing equivalents from a reversal of methanogenic enzymes, thus oxidizing methyl groups to CO2. An alternative (since these and acetate-catabolizing methanogens possess cytochrome b) is electron transport and possible proton pumping via a cytochrome-containing electron transport chain. Several of the actual components of the methanogenic pathway from CO2 have been characterized. Methanofuran is apparently the first carbon-carrying cofactor in the pathway, forming carboxy-methanofuran. Formyl-FAF or formyl-methanopterin (YFC, a very rapidly labelled compound during 14C pulse labeling) has been implicated as an obligate intermediate in methanogenesis, since methanopterin or FAF is an essential component of the carbon dioxide reducing factor in dialyzed extract methanogenesis. FAF also carries the carbon at the methylene and methyl oxidation levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Energetics of sodium-dependent alpha-aminoisobutyric acid transport in the moderate halophile Vibrio costicola.

The energetics of alpha-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (delta psi) and alpha-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5-9.0. Cells specifically required Na+ ions to transport alpha-aminoisobutyric acid and to maintain the highest delta psi (150-160 mV). Sodium was not required to sustain high rates of O2-uptake. Delta psi (and alpha-aminoisobutyric acid transport) recovered fully upon addition of Na+ to Na+-deficient cells, showing that Na+ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of delta psi and alpha-aminoisobutyric acid transport. Also, dissipation of delta psi with triphenylmethylphosphonium cation abolished alpha-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in alpha-aminoisobutyric acid transport, without affecting delta psi. Similarly, N,N'-dicyclohexylcarbodiimide (10 microM) stimulated respiration and alpha-aminoisobutyric acid transport and did ot affect delta psi, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (delta psi and Na+ chemical potential), we conclude that delta psi is obligatory for alpha-aminoisobutyric acid transport, and that for maximum rates of transport an Na+ gradient is also required.

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.

Novel polar lipids from the methanogen Methanospirillum hungatei GP1.

The methanogenic bacterium Methanospirillum hungatei GP1 has been shown to contain two unusual phosphoglycolipids (phosphoglycolipid I and phosphoglycolipid II) that account for 64% (by wt.) of the total cellular lipids. These lipids are derivatives of the dibiphytanyldiglycerol tetraether. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid and the other is linked glycosidically to a disaccharide with structure alpha-Glcp-(1 leads to 2)-beta Gal phi in phosphoglycolipid I and beta-Gal phi-(1 leads to 6)-beta Gal phi in phosphoglycolipid II. Smaller amounts of the sn-2,3-diphytanylglycerol analog of phosphatidylglycerol and diglycosyldiphytanylglycerol ethers (DGD-I and DGD-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively, were identified, together with very small amounts of diglycosyldibiphytanyldiglycerol tetraethers (DGT-I and DGT-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively. A biosynthetic pathway involving head-to-head condensation of phosphatidylglycerol with DGD-I or DGD-II to form phosphoglycolipid I or phosphoglycolipid II, respectively, is proposed.

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.

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.

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.

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.

Crystalline order to high resolution in the sheath of Methanospirillum hungatei: a cross-beta structure.

Electron microscopy and electron diffraction indicate that the outer sheath of the cell wall of the archaebacterium Methanospirillum hungatei contains a two-dimensional crystalline lattice having, at least to low resolution, p2 symmetry in projection with a = 5.66 nm, b = 2.81 nm and gamma = 85.6 degrees. At a resolution of 2 nm, the unit cell contains two lobes, whereas high-angle electron diffraction shows the presence of a substantial quantity of beta structure, with the 0.47 nm spacing (between polypeptide chains within a sheet) oriented circumferentially. The sheath is unusual when compared to other regular surface arrays found on bacteria in that it is a compact structure with small subunits. It may have a structural role analogous to barrel hoops since it tends to fragment perpendicular to its axis to give rings or hoops.

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.

Application of time-resolved diffuse reflectance techniques in studies of reaction intermediates in suspensions of Bacillus subtilis.

Short lived reaction intermediates such as triplet states and free radicals can be detected in vivo using laser photolysis techniques with time-resolved diffuse reflectance detection. This novel approach is illustrated for bacterial suspensions of Bacillus subtilis.

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.

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.

Structures of archaebacterial membrane lipids.

Structural data on archaebacterial lipids is presented with emphasis on the ether lipids of the methanogens. These ether lipids normally account for 80-95% of the membrane lipids with the remaining 5-20% of neutral squalenes and other isoprenoids. Genus-specific combinations of various lipid core structures found in methanogens include diether-tetraether, dietherhydroxydiether, or diether-macrocyclic diether-tetraether lipid moieties. Some species have only the standard diether core lipid, but none are known with predominantly tetraether lipids as found in certain sulfur-dependent archaebacteria. The relative proportions of these lipid cores are known to vary in relation to growth conditions in Methanococcus jannaschii and Methanobacterium thermoautotrophicum. Polar headgroups in glycosidic or phosphodiester linkage to the sn-1 or sn-1' carbons of glycerol consist of polyols, carbohydrates, and amino compounds. The available structural data indicate a close similarity among the polar lipids synthesized within the species of the same genus. Detection of lipid molecular ions by mass spectrometry of total polar lipid extracts is a promising technique to provide valuable comparative data. Since these lipid structures are stable within the extreme environments that many archaebacteria inhabit, there may be specific applications for their use in biotechnology.

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii.

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little delta pH during growth) with an electrical potential of --127 mV in growth medium and --105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.

Solubilization and properties of a particulate hydrogenase from Methanobacterium strain G2R.

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-beta-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77 degrees C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5'-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

Composition and properties of the cell wall of Methanospirillum hungatii.

Dithiothreitol reacted, at pH 9.0, with the isolated cell walls of Methanospirillum hungatii, to release about 23% of the cell wall dry weight as a high molecular weight fraction (> 0.5 million daltons). Untreated walls consisted of 70% amino acids, 11% lipid, and 6.6% carbohydrate. Sugars were identified as rhamnose, ribose, glucose, galactose, and mannose. The wall material that was released contained only 47% amino acids and was enriched in lipid, glucose, and phosphate. These results support data from electron micrographs, showing the localized release of cell wall material by the disulfide bond-breaking reagent at alkaline pH. In amino acid composition the untreated walls did not differ greatly from the material released by dithiothreitol, but differed considerably from the walls of another strain of M. hungatii. The ratios of the amino acids found in the cell wall proteins of several archaebacteria and of Bacillus cereus spore coats were similar.