Archaebacterial ether lipid diversity analyzed by supercritical fluid chromatography: integration with a bacterial lipid protocol.

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.

Archaeal rRNA operons.

Ribosomal RNA (rRNA) operons of the archaea reflect both the unity and the diversity of this third primary taxon. They have proven to be a rich source of both molecular biological and phylogenetic information.

Structural characteristics of methanogenic cofactors in the non-methanogenic archaebacterium Archaeoglobus fulgidus.

Archaeoglobus fulgidus is an extremely thermophilic, sulphate reducing archaebacterium thought to represent a biochemical missing-link between sulphur-metabolizing bacteria and methanogenic bacteria. Whereas the phylogenetic position of A.fulgidus is closer to the sulphur-metabolizing bacteria, there is a partial overlap in the biochemical machinery of A.fulgidus with both groups of bacteria. In particular, the presence of a number of aberrant cofactors up to now thought to be involved exclusively in the process of methanogenesis in methanogenic archaebacteria, i.e. coenzyme F420, methanofuran and methanopterin, has been indicated by previous studies. Here we present evidence for the structural identity of the methanopterin cofactor of A.fulgidus with the methanopterin isolated from Methanobacterium thermoautotrophicum and show that this non-methanogenic bacterium contains two as yet unknown analogues of coenzyme F420. The levels of the various cofactors were determined in cultures grown either on formate or lactate as the carbon source and sulphate or thiosulphate as the sulphur source.

Fast atom bombardment mass spectrometry as a rapid means of screening mixtures of ether-linked polar lipids from extremely halophilic archaebacteria for the presence of novel chemical structures.

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.

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).

Occurrence in the archaebacterium Sulfolobus solfataricus of a ribosomal protein complex corresponding to Escherichia coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)2.P0.

Two-dimensional electrophoresis of total protein from 50 S ribosomal subunits of the archaebacterium Sulfolobus solfataricus demonstrated a complex between two proteins that was stable in 6 M urea, but dissociable in detergent or below pH 5.5. The proteins, numbered L1 and L10 according to their electrophoretic mobilities, corresponded to Escherichia coli ribosomal proteins L10 and L7/L12, respectively. The members of the complex were therefore designated Sso L10e and Sso L12e. Sso L12e had other properties in common with E. coli L7/L12: low molecular weight, relative acidity, selective release from the ribosome by high salt/ethanol, and dimeric structure. The Sso L12e.Sso L10e complex was isolated by gel filtration of total 50 S proteins in 4 M urea. The stoichiometry of the components was approximately four copies of Sso L12e to one copy of Sso L10e. The occurrence in an archaebacterium of a complex of acidic ribosomal proteins similar to E. coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)/.P0 strongly supports the concept that this element of quaternary structure is a major conserved feature of the ribosome and reaffirms its importance in the translocation step of protein synthesis.

Conformational study of the chromosomal protein MC1 from the archaebacterium Methanosarcina barkeri.

Methanogen chromosomal protein MC1 is a polypeptide of 93 amino acid residues (Mr 10,757) which represents the major protein associated with the DNA of the archaebacterium Methanosarcina barkeri and can protect DNA against thermal denaturation. The conformation of protein MC1 has been investigated by means of predictive methods, infrared spectroscopy, circular dichroism and tryptophan fluorescence studies. Protein MC1 has a low amount of alpha-helix but contains antiparallel beta-sheet strands. The larger hydrophobic cluster which contains tryptophan at position 61 appears buried in the protein. Addition of salts induces the unfolding of the protein and makes the tryptophan indole ring more rigid. With respect to its primary structure and its conformation, protein MC1 appears radically different from the chromosomal DNA-binding protein II (also called HU-type protein) in eubacteria.

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins.

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.

Primary structure of the chromosomal proteins MC1a, MC1b, and MC1c from the archaebacterium Methanothrix soehngenii.

The chromosomal protein MC1 of Methanothrix soehngenii is a family of three variants a, b, and c. These are small basic polypeptides of 89, 87, and 90 residues, respectively. Their primary structures have been determined from automated sequence analyses of the intact proteins and from structural data provided by peptides derived from the variants by cleavage at aspartic acid, glutamic acid, arginine, and methionine residues. By comparison with variant b taken as reference, variants a and c present 18 and 24 differences, respectively. The extent of sequence homologies between protein MC1 from M. soehngenii and proteins MC1 from two other species of Methanosarcinaceae is only 60%. The sequences 17-35 and 45-58 of the protein MC1 appear well conserved. Deletions are observed in region 36-44. Many changes, most of them nonconservative, occur in the carboxyl-terminal third of the protein. However, proline residues at positions 68, 72, 76, and 82 remain strictly conserved. Predictive methods for secondary structures indicate a low content of alpha helix and beta sheet structures in proteins MC1.

Archaebacterial histone-like proteins. Purification and characterization of helix stabilizing DNA binding proteins from the acidothermophile Sulfolobus acidocaldarius.

Four DNA binding histone-like proteins have been purified from the nucleoid of the acidothermophilic archaebacterium Sulfolobus acidocaldarius to homogeneity employing DNA-cellulose chromatography and carboxymethylcellulose chromatography. The molecular weights of these proteins are in the range 8,000-12,500. Immunoblotting results suggest that a few antigenic determinants are common among these proteins which could not be detected by immunodiffusion. Spectroscopic properties of the proteins have been studied. The amino acid compositions of these proteins show both similarities and differences with histones and prokaryotic histone-like proteins. All of the four proteins bind native and denatured DNAs and single stranded RNA with differing affinities. Three of the proteins, denoted by HSNP (helix stabilizing nucleoid protein)-A, HSNP-C, and HSNP-C', show physiologically significant, strong, and synergistic effects in stabilizing duplex DNA against thermal denaturation with Tm increases in the range of 15-30 +/- degrees C.

Purification of glycerol dialkyl nonitol tetraether from Sulfolobus acidocaldarius.

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.

Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria.

The isoelectric points of most proteins from the extremely halophilic archaebacteria are between 4.0 and 4.65 which agrees with the generally high content of glutamic and aspartic acid in proteins from halobacteria. The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. We propose that these differences reflect the existence of two classes of halobacterial proteins, one resembling non-halophilic proteins, and the other possessing unique properties that may be related to salt dependence.

A simple chromatographic procedure for the detection of cyclized archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids.

Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.

Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp. CHTI 55 and its effect on the thermal stability of DNA.

In the deoxyribonucleoprotein complex of Methanosarcina sp. CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) (Mr 10,760) and MC2 (Mr 17,000). Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1. The effect of Methanosarcina sp. CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E. coli DNA-binding protein II. Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms. Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA.

Analysis and characterization of the folates in the nonmethanogenic archaebacteria.

A detailed analysis of the folate coenzymes in the nonmethanogenic archaebacteria has been performed. By using the Lactobacillus casei microbiological assay for folates, the levels of folates in Sulfolobus solfataricus and Sulfolobus acidocaldarius were found to be 3.7 and 8.3 ng/g (dry weight) of cells, respectively, compared with 88,000 and 28,000 ng/g (dry weight) of cells in Halobacterium halobium and Halobacterium strain GN-1, respectively. The levels of folates found in the Sulfolobus spp. were approximately 100 times less than those found in the typical eubacterium, whereas the levels in the halobacteria were approximately 10 times higher. The folate in Sulfolobus solfataricus was shown to consist of only 5-formyltetrahydropteroylglutamate, and the folate in Halobacterium strain GN-1 was shown to consist of only pteroyldiglutamate. The low folate levels in the Sulfolobus spp. are the same as those found in the methanogenic bacteria, suggesting that another C1 carrier may function in these cells.

Folic acid and pteroylpolyglutamate contents of archaebacteria.

Cell extracts of methanogens and the thermoacidophile Sulfolobus solfataricus contained little or no folic acid (pteroylglutamate) or pteroylpolyglutamate activity (less than 0.1 nmol/g [dry weight]). However, the halophile Halobacterium salinarum contained pteroylmono- or pteroyldiglutamates, and Halobacterium volcanii and Halobacterium halobium contained pteroyltriglutamates at levels equivalent to those in eubacteria (greater than 1 nmol/g [dry weight]).

Isolation, characterization and microsequence analysis of a small basic methylated DNA-binding protein from the Archaebacterium, Sulfolobus solfataricus.

DNA-binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus solfataricus strain P1, grown at 86 degrees C and pH 4.5. These proteins, which may have a histone-like function, were isolated and purified under standard, non-denaturing conditions, and can be grouped into three molecular mass classes of 7, 8 and 10 kDa. We have purified to homogenity the main 7 kDa protein and determined its DNA-binding affinity by filter binding assays and electron microscopy. The Stokes radius of gyration indicates that the protein occurs as a monomer. The complete amino-acid sequence of this protein contains 14 lysine residues out of 63 amino acids and the calculated Mr is 7149. Five of the lysine residues are partially monomethylated to varying extents and the methylated residues are located exclusively in the N-terminal (positions 4 and 6) and the C-terminal (positions 60, 62 and 63) regions only. The protein is strongly homologous to the 7 kDa proteins of Sulfolobus acidocaldarius with the highest homology to protein 7d. Accordingly, the name of this protein from S. solfataricus was assigned as DNA-binding protein Sso7d.

Tetraether lipid components from a thermoacidophilic archaebacterium. Chemical structure and physical polymorphism.

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.

Microsequence analysis of DNA-binding proteins 7a, 7b, and 7e from the archaebacterium Sulfolobus acidocaldarius.

DNA-binding proteins in eubacteria, such as Escherichia coli NS1 and NS2, are generally small basic molecules. In contrast, the archaebacterium Sulfolobus acidocaldarius contains three groups of DNA-binding proteins which have molecular masses of 7, 8, and 10 kDa. In the first group, five proteins (7a-7e) have been identified, while in the second and third group only two proteins each are present, denoted 8a and 8b and 10a and 10b, respectively. In this paper, we present the primary structures of proteins 7a, 7b, and 7e from the first group. All three proteins contain lysyl residues which are monomethylated to different extents. The modified lysines are found in the NH2-terminal regions of all 7-kDa proteins and in the COOH-terminal part of protein 7e. The sequences of the 7-kDa group are highly similar to each other. All of these macromolecules have been shown to interact specifically with DNA. Protein 7e of the 7-kDa group shows the tightest binding to DNA.