One-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography.

Bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. Lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. Coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-Sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. The procedure is applicable to macroamphiphiles with various lipid structures as was demonstrated using the diacylglycerol-containing lipoglycan of Micrococcus luteus, the lipid A-containing lipopolysaccharide of Salmonella typhimurium, and the diglyceryl tetraether lipoglycans of Thermoplasma acidophilum and Thermoplasma volcanicum. On elution from octyl-Sepharose, separation into molecular species of different compositions was observed with the lipopolysaccharide of S. typhimurium and the lipoglycan of T. volcanicum. It was also shown that, after phenol/water extraction, membrane lipids are completely recoverable from the phenol layer, which makes it possible to isolate lipids along with macroamphiphiles from the same sample of bacteria.

Occurrence of coenzyme F420 and its gamma-monoglutamyl derivative in nonmethanogenic archaebacteria.

Analysis of the fluorescent compounds extracted from six different species of halobacteria and one species each of Sulfolobus and Thermoplasma revealed the universal occurrence of coenzyme F420, (N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy -4-pentoxyhydroxyphosphinyl]-L-lactyl]-L-gamma-glutamyl]-L -glutamic acid), or its gamma-monoglutamyl derivative or both. The total amount (approximately 100 pmol/mg [dry weight]) of these compounds found in the halobacteria studied was approximately 5% of the amount previously reported for methanogenic bacteria. The amount of F420 found in the Sulfolobus and Thermoplasma strains was approximately 1% of that found in the halobacteria. The major compound in all but one of the examined strains was the gamma-monoglutamyl derivative of F420; one strain of halobacteria contained only F420. For the halobacterium-derived samples, the additional glutamic acid was shown to be linked by a gamma-glutamyl peptide bond to the terminal glutamic acid of the F420 core structure by enzymatic hydrolysis of the samples with three different gamma-glutamyltranspeptidases. The product of this enzymatic hydrolysis was F420 with one less glutamic acid in the side chain.

Preliminary X-ray diffraction studies on a ferredoxin from the thermophilic archaebacterium, Thermoplasma acidophilum.

A ferredoxin from the thermophilic archaebacterium, Thermoplasma acidophilum, is supposed to contain two (4Fe-4S) active centers; one center could be linked by four cysteine residues to the protein and the other bonded with three cysteines and an unknown group. This ferredoxin has been crystallized by salting-out against 2.3 M-ammonium sulfate solution. The space group is P21212 with cell dimensions of a = 59.20 A, b = 52.77 A and c = 41.28 A. Four molecules pack in the unit cell with Vm = 2.03 A3/dalton.

Primary structure of elongation factor 2 around the site of ADP-ribosylation is highly conserved from archaebacteria to eukaryotes.

Elongation factor 2 (EF-2) from eukaryotes and archaebacteria can be ADP-ribosylated by diphtheria toxin (DT) [(1977) Annu. Rev. Biochem. 46, 69-94; (1980) Nature 287, 250-251]. The primary structure of the ADP-ribose accepting region in EFs from the archaebacteria Thermoplasma acidophilum Halobacterium cutirubrum and Methanococcus vannielli was determined in order to elucidate the degree of conservation compared with 4 previously established eukaryotic sequences [(1971) FEBS Lett. 103, 253-255]. Within a 9-residue sequence including the site of ADP-ribosylation 5 positions were found to be occupied by the same amino acid in all the archaebacterial and eukaryotic factors studied. There were more differences among the 3 archaebacterial sequences than among the 4 eukaryotic ones.

Lipoglycans from mycoplasmas.

Lipoglycans , distinguishable from bacterial lipopolysaccharides, are associated with the cytoplasmic membranes of several genera of Mollicutes, namely Acholeplasma, Mycoplasma neurolyticum , Anaeroplasma , and Thermoplasma. Structurally, the lipoglycans are long heteropolysaccharides covalently linked to a lipid. The exact structures of three have been determined. Thermoplasma oligosaccharide is attached to a diglycerol tetraether ; A. granularum to a diacyl glycerol. The lipoglycan from A. axanthum is unique by its possession of glycerol phosphate and galactose phosphate side chains and the occurrence of fatty acids in N-acyl linkages. Only one molecular species of lipoglycan occurs in a given species. These lipoglycans possess a variety of biological activities. The terminal three sugar residues define their antigenic specificity; they attach to specific receptors on mammalian cells; they exhibit pyrogenicity in rabbits and clot Limulus lysate; they stimulate the production of IgM antibody both in vivo and in vitro; they modulate the immune response to T-cell dependent antigens; they exhibit immunosuppressive and immunostimulatory activities.

The nucleotide sequence of the tRNAMMet from the archaebacterium Thermoplasma acidophilum.

Using in vitro labelling techniques, a tRNAMMet from Thermoplasma acidophilum, a member of the Archaebacteriae, has been shown to have the sequence: pGCCGGG Gs4UGGCUCANCUGGAGGAGC m2(2)GCCGGACmUCAUt6AAUCCGGAGGUCUCGGG psi psi CmGAUCCCCGAUCCCGGCACCAOH. Despite the small genome size of this non-parasitic organism, eight modified nucleosides are present, one of which is typically eubacterial, one of which is typically eukaryotic and some of which appear to be unique to the archaebacteria. There is no close sequence homology between this tRNA and that of any other methionine tRNA so far sequenced (less than 70%) but it has almost 90% homology with the nucleotide sequence proposed by Eigen and Oswatitsch for the ancestral quasi-species.

A histone-like protein (HTa) from Thermoplasma acidophilum. I. Purification and properties.

A histone-like protein (HTa) has been isolated from cell extracts of Thermoplasma acidophilum by column chromatography on DNA-cellulose, hydroxylapatite, and Sephadex G-75, HTa elutes from DNA-cellulose in two fractions, one of which contains an 80-residue form of the protein with an NH2-terminal sequence of Val-Gly. The other fraction apparently contains the 89-residue species, in addition to a 90-residue form of the protein with the NH2-terminal sequence Met-Val. The sequence of 47 residues from the NH2 terminus of the 89-residue protein was established by automated Edman degradation. HTa is characterized by the following properties: 22% of its residues are lysine and arginine; the lysine:arginine ratio is 2.33; the absorption spectrum of the protein is distinctive due to the lack of tryptophan and the presence of 1 tyrosine and 5 phenylalanine residues; and the protein stabilizes DNA against thermal denaturation (Stein, D. B., and Searcy, D. G. (1978) Science 202, 219-221) and condenses DNA into spherical particles. All of these characteristics indicate that HTa resembles eukaryotic histones, but there are distinctive differences.

A histone-like protein (HTa) from Thermoplasma acidophilum. II. Complete amino acid sequence.

The complete amino acid sequence of the DNA-binding histone-like protein (Hta) from Thermoplasma acidophilum has been established by sequence studies directly on the protein and on tryptic, chymotryptic, and thermolysin peptides derived from the protein. The sequence of the 89-residue form of HTa is: H2N-Val-Gly-Ile-Ser-Glu-Leu-Ser-Lys-Glu-Val-Ala-Lys-Lys-Ala-Asn-Thr-Thr-Gln-Lys -Val-Ala-Arg-Thr-Val-Ile-Lys-Ser-Phe-Leu-Asp-Glu-Ile-Val-Ser-Glu-Ala-Asn-Gly-Gl y-Gln-Lys-Ile-Asn-Leu-Ala-Gly-Phe-Gly-Ile-Phe-Glu-Arg-Arg-Thr-Gln-Gly-Pro-Arg-L ys-Ala-Arg-Asn-Pro-Gln-Thr-Lys-Lys-Val-Ile-Glu-Val-Pro-Ser-Lys-Lys-Lys-Phe-Val- Phe-Arg-Ala-Ser-Ser-Lys-Ile-Lys-Tyr-Gln-Gln-COOH The molecular weight calculated from the sequence is 9,934. Another form of HTa probably differs only by the presence of an additional residue (methionine) at the NH2 terminus (the calculated molecular weight of this form is 10,065). HTa resembles eukaryotic histones in several ways, including some sequence homology, HTa also shows sequence homology with the Escherichia coli DNA-binding proteins NS1 (or HU-1) and NS2 (or HU-2).

Nucleoprotein subunit structure in an unusual prokaryotic organism: Thermoplasma acidophilum.

The freeliving thermophilic mycoplasma, Thermoplasma acidophilum, has a small acid-soluble protein tightly bound to its DNA. This protein is similar to eukaryotic histones in both size and amino acid composition. Here we report that the protein condenses DNA into globular particles that are about half the size of eukaryotic nucleosomes. Our conclusions are based primarily upon the following observations: (1) Nuclease digestion produced DNA fragments of 40 base-pairs. (2) The ratio of protein to DNA was such that 4--5 molecules of protein were associated with each 40 base-pair segment of DNA. (3) Protein crosslinking reagents produced tetramers of the histone-like protein. (4) Electron microscopy revealed globular particles 5--6 nm in diameter. (5) Each globular particle reduced the apparent contour length of the DNA by 40 base-pairs. Thus, each nucleoprotein particle is apparently composed of 40 base-pairs of DNA coiled around four molecules of proteins.

Thermoplasma acidophilum histone-like protein. Partial amino acid sequence suggestive of homology to eukaryotic histones.

Thermoplasma acidophilum is a freeliving mycoplasma-like organism that has a small basic protein tightly bound to its DNA. The N-terminal sequence of this protein has been determined. It has a distanct but statistically significant homology to eukaryotic histones H2A, H3, and to Escherichia coli protein HU.

Sequence and glycosidic bond arrangement of sugars in lipopolysaccharide from Thermoplasma acidophilum.

The lipopolysaccharide from Thermoplasma acidophilum is a linear polymer with the structure [Manp-(alpha 1 leads to 2)-Manp-(alpha 1 leads to 4)-Manp-(alpha 1 leads to 3)]8-Glcp-(alpha 1 leads to 1)-diglycerol tetraether as established by Smith degradation, methylation, acetolysis and CrO3 oxidation.

Purification and partial characterization of a procaryotic glycoprotein from the plasma membrane of Thermoplasma acidophilum.

The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56 degrees C and pH 2.0. Its plasma membrane possessed 21--22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched alpha 1 leads to 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunciton with water turgor, may contribute to the rigidity of the membrane and the cation binding.

Phylogenetic affinities between eukaryotic cells and a thermophilic mycoplasma.

Thermoplasma acidophilum, a thermophilic mycoplasma, has several unusual features suggesting a possible relationship to eukaryotic cells. One feature is a histone-like protein that is associated with the DNA, condensing it into subunits similar to those in eukaryotic chromatin. A second feature is an association of cytoplasmic proteins that resembles eukaryotic actin and myosin. These two components are widely distributed in different groups of eukaryotic cells, but are typically lacking in prokaryotic cells. Furthermore, T. acidophilum lacks cytochromes and respires by enzymes that apparently are not coupled to oxidative phosphorylation. This primitive type of respiration resembles that of microbodies, another feature which is represented in the cytoplasm of all groups of eukaryotic cells. Furthermore, since T. acidophilum lacks a cell wall and appears to have a primitive correlate of endocytosis, it would appear to be mechanically capable of acquiring a symbiotic mitochondrion. Thus, our observations are consistent with the symbiotic hypothesis for the origin of eukaryotic cells. We suggest that an organism similar to T. acidophilum was the host cell for the original symbiosis, becoming the nucleus and cytoplasm of modern eukaryotic cells.

Ultrastructural localization of Thermoplasma acidophilum surface carbohydrate by using concanavalin A.

Surface carbohydrate, presumably the lipopolysaccharide, of Thermoplasma acidophilum was visualized by means of the concanavalin A, horseradish peroxidase, and diaminobenzidine cytochemical staining procedure.

Long-chain diglycerol tetraethers from Thermoplasma acidophilum.

The C40 isopranol-containing glycerol ether residues which characterize the complex lipids of the extreme thermoacidophile Thermoplasma acidophilum were isolated and purified from the glycolipid and phospholipid fractions. The glycerol ether, as well as the acetate and methoxy derivatives were characterized by thin-layer, gel-permeation and gas-liquid chromatography, infrared, nuclear magnetic resonance and mass spectrometry and by vapor phase osmometry. The glycerol ethers are proposed to be unique fully saturated diglycerol tetraethers, primarily C86H172O6, Mr 1300, which contain two sn-2,3-glycerol residues bridged through ether linkages by two C40 isopranoid branched diols.

Distribution and composition of lipopolysaccharides from mycoplasmas.

Polymeric carbohydrates containing glycerol and fatty acids were isolated from whole cells and membranes of mycoplasmas by hot aqueous phenol extraction and gel filtration. Lipopolysaccharides were found to occur in four species of Acholeplasma, two of Anaeroplasma, and in Mycoplasma neurolyticum. None were detected in Spiroplasma citri or in five species of Mycoplasma. All lipopolysaccharides contained both neutral and N-acylated amino sugars in ratios varying from 1:1 to 3:1. The neutral sugars found in varying distribution were glucose, galactose, and mannose. The amino sugars included fucosamine, an unidentified deoxyhexosamine, galactosamine, and glucosamine. Fucosamine and glucose were the only sugars common to all lipopolysaccharides. The fatty acids were similar to those found in the lipids of each organism.