Polyamine distributions in thermophilic eubacteria belonging to Thermus and Acidothermus.

Triamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70 degrees C) Thermus species tested. Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine were detected in T. thermophilus HB8, T. filiformis Wai33 A1, T. flavus AT-62, and T. caldophilus GK24. The linear hexaamines and branched polyamines were absent in T. aquaticus YT-1, T. sp. X-1, T. sp. T2, and T. sp. T351, in which linear pentaamines were minor components. Moderately thermophilic Thermus ruber and Thermus sp. K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomospermidine. No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species. On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all the polyamines.

Crystals of protein L1 from the 50 S ribosomal subunit of Thermus thermophilus. Preliminary crystallographic data.

Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile. Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol. The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A. They diffract X-rays to 2.3 A resolution.

[Modification of a microdialysis method for crystallization of proteins]. / Modifikatsii mikrodializnogo metoda kristallizatsii belkov.

Several variants of microdialysis cells have been developed for growing protein crystals suitable for X-ray analysis. The cells have simple construction and are made of easily available materials. Using these cells, over ten proteins have been crystallized, six of them in the form suitable for 3D-structure determination.

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites on EF-Tu.

In a preceding paper [(1987) Nucleic Acids Res. 15, 5787-5801], we have used trans-diamminedichloroplatinum (II) to induce reversible RNA-protein crosslinks within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex and have identified two crosslinking sites on the tRNA. The aim of the present paper is to determine the crosslinking sites on EF-Tu. Two tryptic peptides located in domain I could be identified, a major one (residues 45-74) and a minor one (residues 117-154). The use of Staphylococcus aureus V8 protease led to the isolation of two major peptides (residues 56-68 and 64-68) and one minor peptide (118-124). These results are discussed in the light of the current knowledge of the topography of the EF-Tu/tRNA complex.

Physical properties of membrane lipids isolated from a thermophilic eubacterium (Thermus sp.).

Membranes from a thermophilic eubacterium, Thermus sp. strain SPS 11, isolated from thermal springs of São Pedro do Sul spa (Portugal), are characterized for having two main polar lipids, a glycolipid (GL) with four monosaccharide residues, which at 73 degrees C accounts for 95% of the carbohydrate in the total lipid extracts, and a glycophospholipid (PL) which at 73 degrees C accounts for about 90% of the lipid phosphorous. A complex mixture of carotenoids (CA) makes up 11% by weight of the total membrane lipids. The branched fatty acyl chains (iso C15 and iso C17) comprise about 90% of the alifatic moieties of the polar lipids of this bacterium. Moreover, when the growth temperature increases from 50 to 73 degrees C there is an increase of the iso C17/ iso C15 ratio and of the GL/PL ratio. We have studied the biophysical properties of bilayers (as multilamellar liposomes) prepared from GL, PL and the mixtures of PL, GL and CA in proportions found in the membranes of bacteria growing at their optimal growth temperature, using polarization of DPH fluorescence, low and wide-angle X-ray diffraction and differential scanning calorimetry. The three techniques agree in showing the presence of a broad phase transition from a gel (L beta) phase to a liquid-crystal (L alpha) phase between 8 and 30 degrees C, for all the lipid dispersions studied except for the GL. Although all the dispersions studied form a bilayer structure at all the temperatures studied, only the mixture of the three components (PL, GL + CA) avoids the phase separation present in the mixtures of PL + CA at temperatures lower than 30 degrees C and PL + GL at temperatures lower than 55 degrees C. Our results are compared with those of Pinheiro et al. (1978) obtained with the 31p-NMR technique and applied to the study of the same samples.

A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium.

The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in São Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.

A new naturally occurring polyamine containing a quaternary ammonium nitrogen.

A new polyamine, tetrakis(3-aminopropyl)ammonium, N+ (CH2CH2CH2NH2)4, was identified in cells of an extreme thermophile, Thermus thermophilus. This compound was chemically synthesized and its chemical properties were coincident with those of the amine isolated from the thermophile.

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus.

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

Characterization of a Yellowstone hot spring microbial community by 5S rRNA sequences.

The microorganisms inhabiting a 91 degrees C hot spring in Yellowstone National Park were characterized by sequencing 5S rRNAs isolated from the mixed, natural microflora without cultivation. By comparisons of these sequences with reference sequences, the phylogenetic relationships of the hot spring organisms to better characterized ones were established. Quantitation of the total 5S-sized rRNAs revealed a complex microbial community of three dominant members, a predominant archaebacterium affiliated with the sulfur-metabolizing (dependent) branch of the archaebacteria, and two eubacteria distantly related to Thermus spp. The archaebacterial and the eubacterial 5S rRNAs each constituted about half the examined population.

Evidence for N coordination to Fe in the [2Fe-2S] clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas.

Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties. Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical [2Fe-2S] clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J. A., Findling, K. L., Yoshida, T., Hille, R., Tarr, G. E., Hearshen, D. O., Dunham, W. R., Day, E. P., Kent, T. A., and Munck, E. (1984) J. Biol. Chem. 259, 124-133). We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins. The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz. The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz. The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings.

Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands.

We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.

Homocaldopentamine: a new naturally occurring pentaamine.

A new pentaamine was extracted from an extreme thermophile, Thermus thermophilus strain HB8, and its chemical structure was determined to be 1,16-diamino-4,8,12-triazahexadecane (see structure 1). A trivial name, homocaldopentamine, was proposed for the new naturally occurring polyamine.

A pentaamine is present in an extreme thermophile.

A pentaamine was found in cells of an extreme thermophile, Thermus thermophilus, grown at 80 degrees C. The chemical structure was determined to be 1,15-diamino-4,8,12-triazapentadecane, NH2(CH2)3NH(CH2)3NH(CH2)3NH(CH2)3NH2. A trivial name "caldopentamine" is proposed for the new naturally occurring polyamine.

Single crystals of 5S rRNA from Thermus thermophilus HB8 and preliminary X-ray diffraction data.

5S rRNA from extremely thermophilic bacteria, Thermus thermophilus HB8, was crystallized. Using a vapour diffusion method, single crystals were grown with sufficient sizes for x-ray diffraction study. The crystals belong to space group P3(1)21 (or enatiomorph P3(2)21) with a = 99 A, b = 99 A and c = 360 A. A unit cell contains probably 36 molecules corresponding to 6 molecules/asymmetric unit. A smaller pseudo-lattice seems to be constructed with the same lengths for a- and b-axis, and 120 A for c-axis. Reflections were observed up to 25 A on x-ray photographs.

Glycolipids from some extreme thermophilic bacteria belonging to the genus Thermus.

The lipids of Thermus aquaticus YT1, Thermus thermophilus HB8, Thermus sp. strains H and J (from Icelandic hot springs), and Thermus sp. strain NH (from domestic hot water) have been investigated. Each strain contained two major components, a glycolipid and a glycophospholipid, which have been isolated and analyzed. All of the strains contained as the principal component (41 to 57% of total lipid) a diacyldiglycosyl-(N-acyl)glycosaminylglucosylglycerol, but the five glycolipids differed in carbohydrate composition. The glycophospholipid appeared to be identical in each strain and contained an N-acylglucosamine residue. The principal fatty acids were C15 and C17 branched-chain compounds. This unique polar lipid composition should be of value in the classification of other thermophiles in the genus Thermus. The exceptionally high carbohydrate content of the lipids of these extreme thermophiles may be of significance in relation to the molecular basis of thermophily.

Properties of native and nicked elongation factor Tu from Thermus thermophilus HB 8.

Two alternative procedures for the isolation of the elongation factor Tu from Thermus thermophilus were compared and the properties of a specifically nicked EF-Tu . GDP were examined in detail. Although the native elongation factor possessed similar catalytic activities in all reactions investigated as the protein isolated by Arai et al. [Eur. J. Biochem. 92, 509-519 and 521-531 (1978)] it could not be crystallized. The nicked EF-Tu, consisting of two associated fragments with molecular weights of 41000 and 8000 respectively, was active in binding GDP, GTP and in the formation of Phe-tRNAPhe . EF-Tu . GTP ternary complex. However, it did not promote poly(U)-dependent synthesis of polyphenylalanine on Escherichia coli ribosomes. The isolated fragment of a molecular weight of about 41000 did not bind GDP. This activity could be reconstituted with the supplement of the small 8000-Mr fragment. It is demonstrated that, in contrast to the native EF-Tu, the nicked EF-Tu forms high-molecular-weight aggregates. Cleavage of the polypeptide chain of EF-Tu from T. thermophilus stimulates the crystallization of this protein.

Phylogenetic tree derived from bacterial, cytosol and organelle 5S rRNA sequences.

A phylogenetic tree was constructed by computer analysis of 47 completely determined 5S rRNA sequences. The wheat mitochondrial sequence is significantly more related to prokaryotic than to eukaryotic sequences, and its affinity to that of the thermophilic Gram-negative bacterium Thermus aquaticus is comparable to the affinity between Anacystis nidulans and chloroplastic sequences. This strongly supports the idea of an endosymbiotic origin of plant mitochondria. A comparison of the plant cytosol and chloroplast sub-trees suggests a similar rate of nucleotide substitution in nuclear genes and chloroplastic genes. Other features of the tree are a common precursor of protozoa and metazoa, which appears to be more related to the fungal than to the plant protosequence, and an early divergence of the archebacterial sequence (Halobacterium cutirubrum) from the prokaryotic branch.