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A strategy has been developed for archaebacterial lipid analysis which provides three times the information to describe archaebacterial isolates and is compatible with simultaneous eubacterial/eukaryotic lipid analysis of environmental samples. Eubacterial and micro-eukaryotic biomass, community structure, and nutritional status have been routinely defined in environmental samples by lipid analysis. Lipid profiles are also useful in eubacterial identification and taxonomy. Polar lipid or whole cell ester-linked fatty acids are generally analyzed by gas chromatography-mass spectroscopy. Archaebacteria are characterized by their ether-linked membrane lipids. There is, however, less diversity in the side chains of archaebacterial membrane lipids as compared the eubacterial ester-linked membrane lipids. The information content of the archaebacterial lipid profile was increased by separately analyzing the polar lipid, glycolipid, and lipid-extracted residue fractions. Identification and quantification were performed by supercritical fluid chromatography. Results are presented for three species of methanogens and four thermoacidophile isolates, and compared with a literature review.

Fast atom bombardment mass spectrometry was used to analyse intact polar ether lipids present at microgram levels in crude lipid mixtures extracted from Halobacterium halobium, Natronococcus occultus and Halobacterium marismortui. Negative-ion spectra showed the intact deprotonated lipid molecules and in some instances their sodium salts. The simplicity of the mass spectra permits the rapid screening of polar lipid mixtures for the presence of novel lipids. Additional structural information of ions with selected masses was obtained after collisionally induced decomposition.

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 degrees C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C40 omega-omega' biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to "physiological" conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type alpha) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type beta'). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type alpha. (3) In all the phases with chains in the alpha conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 A. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the alpha conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains.(ABSTRACT TRUNCATED AT 400 WORDS)

A reproducible high performance liquid chromatography (HPLC) method for the separation of diethers and tetraethers isolated from archaebacterial phospholipids is reported. Fourier transform infrared spectroscopy was used for structural confirmation of these signature lipids. A mixture of tetraethers from a thermoacidophilic archaebacteria was resolved into three major components by the normal phase separation. These components were differentiated by Fourier self-deconvolution of infrared spectra. The application of the HPLC technique to environmental samples may provide an accurate assessment of archaebacterial biomass in various microbial communities.

A modified procedure for extraction and purification of hydrolyzed archaebacterial lipids is described. Lipids were extracted from Sulfolobus acidocaldarius using a Soxhlet extraction procedure followed by trichloroacetic acid solvent-extraction of the residue. The yield of total extractable material by this protocol was 14% which, after a two-phase wash, yielded 10% lipid. Modifications to the published steps for purifying the subsequently hydrolyzed lipids were developed to purify glycerol dialkyl nonitol tetraether (GDNT). The nearly colorless final macrocyclic product was characterized by TLC, IR, NMR, and mass spectrometry.

Cyclic AMP was found in species representative of the three major groups of the archaebacteria. In Methanobacterium thermoautotrophicum starvation for H2 led to a significant increase in cellular cAMP. The findings suggest that the occurrence of cAMP antedates the divergence of the major kingdoms of biology; the observations also imply that cAMP constitutes a very early regulatory molecule.

As a continuation of an X-ray scattering study of the tetraether lipids extracted from the thermophilic archaebacterium Sulfolobus solfataricus, the phase behaviour of four fractions of the complex polar lipid extract (PLE) is described. Each molecule of two of these fractions (P1 and GL) carries an unsubstituted glycerol headgroup, those of another (P2) no such group; the fourth fraction (WPLE) is obtained by water-washing PLE, thus reducing its P2 content from approximately 48% to approximately 24% and increasing the average number of molecules bearing an unsubstituted glycerol headgroup from approximately 0.4 to approximately 0.6. The main result is a striking correlation between the phase behaviour and the average ratio of unsubstituted glycerol headgroups to the total number of headgroups: the fractions P1, GL and WPLE, in which that number is respectively 0.5, 0.5 and 0.3, form rod-containing phases; the fraction P2, in which that number is zero, yields a lamellar phase throughout the phase diagram. An analysis of the dimensions of the structure elements confirms our previous conclusion that, in the presence of a sufficient amount of water, the unsubstituted glycerol headgroups partition preferentially in the hydrocarbon regions rather than at the polar/apolar interfaces. These results, moreover, corroborate our previous conjectures regarding the correlations between the structure of the plasma membrane, the phase behaviour of the lipid extract and life at high temperature.

A method for preparing glycerophosphoesters from ether phospholipids by dealkylation with boron trichloride (BCl3) is described. Treatment of ether phospholipids in chloroform with BCl3 for 30 min at room temperature yielded almost quantitatively the corresponding glycerophosphoesters retaining the intact polar head group of the ether phospholipids. Thus, glycerophosphocholine, glycerophosphoinositol, glycerophosphoglycerol, glycerophosphoserine, glycerophosphate, and glycerophosphoethanolamine were prepared from the diether analogs of phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, and phosphatidic acid, and the tetraether analog of phosphatidylethanolamine, respectively. BCl3 also cleaved diacyl, alkyl-acyl, and alk-1-enyl-acyl forms of phospholipids to yield corresponding glycerophosphoesters. The glycerophosphoesters were separated more rapidly by cellulose thin-layer chromatography with the same solvent system as in paper chromatography. This method is of great use for structure determination of glycerophosphoester backbones of ether phospholipids, analogous to the mild alkaline methanolysis of diacyl form of phospholipids, as well as for the analysis of alkyl chains. It is, however, not applicable to glycolipids because of cleavage of glycosidic bonds by BCl3.

The glycerol diether phospholipids of 25 monocultures of methanogenic bacteria were isolated and degraded with hydriodic acid. The resulting alkyl iodides were converted to acetate esters and alcohols which were examined using capillary gas-liquid chromatography. The presence of C20 phytanol was observed in accordance with previous studies. Soft fragmentation by chemical ionization mass spectrometry combined with selected ion monitoring enabled the detection, for the first time, of C15 and C25 isoprenologues as components of the diether phospholipids in several strains.