Crystallization and preliminary X-ray diffraction analysis of the secreted protein Athe_0614 from Caldicellulosiruptor bescii.

The Athe_0614 protein is a component of the extracellular proteins secreted by the anaerobic, extremely thermophilic and cellulolytic bacterium Caldicellulosiruptor bescii. The recombinant protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized using polyethylene glycol 2000 monomethyl ether as a precipitant. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 48.4, b = 42.2, c = 97.8 Å, ß = 96.1°, and diffracted to 2.7 Å resolution using synchrotron radiation.

Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium.

A thermophilic, anaerobic, spore-forming, dissimilatory Fe(III)-reducing bacterium, designated strain SR4T, was isolated from sediment of newly formed hydrothermal vents in the area of the eruption of Karymsky volcano on the Kamchatka peninsula. Cells of strain SR4T were straight-to-curved, peritrichous rods, 0.4-0.6 micron in diameter and 3.5-9.0 microns in length, and exhibited a slight tumbling motility. Strain SR4T formed round, refractile, heat-resistant endospores in terminally swollen sporangia. The temperature range for growth was 39-78 degrees C, with an optimum at 69-71 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.3-6.5. Strain SR4T grew anaerobically with peptone as carbon source. Amorphous iron(III) oxide present in the medium stimulated the growth of strain SR4T; cell numbers increased with the concomitant accumulation of Fe(II). In the presence of Fe(III), strain SR4T grew on H2/CO2 and utilized molecular hydrogen. Strain SR4T reduced 9,10-anthraquinone-2,6-disulfonic acid, sulfite, thiosulfate, elemental sulfur and MnO2. Strain SR4T did not reduce nitrate or sulfate and was not capable of growth with O2. The fermentation products from glucose were ethanol, lactate, H2 and CO2. The G + C content of DNA was 32 mol%. 16S rDNA sequence analysis placed the organism in the genus Thermoanaerobacter. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain SR4T (= DSM 12299T) should be assigned to a new species, Thermoanaerobacter siderophilus sp. nov.

The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.

Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encode D-xylose isomerase and D-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli D-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent D-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [14C]palmitate. The induced E. coli avidly bound D-[14C]xylose, yielding additional evidence that T. ethanolicus XylF is the D-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)3IX3(DN)(SGP)X3(GS)X(LIVA) 2X2A. Transcription of the monocistronic 1.3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xylF transcription initiates from two +1 sites, both situated within the xylAB operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity D-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.

Two-dimensional paracrystalline glycoprotein S-layers as a novel matrix for the immobilization of human IgG and their use as microparticles in immunoassays.

In the present study, cup-shaped 1-3 microns large cell wall fragments from Thermoanaerobacter thermohydrosulfuricus L111-69 covered with a hexagonal S-layer lattice composed of glycoprotein subunits were shown to act as a matrix for the immobilization of human IgG. After cross-linking the S-layer glycoprotein lattice with glutaraldehyde (S-layer microparticles), IgG was either bound to carbodiimide activated carboxyl groups from acidic amino acids from the protein moiety or to the carbohydrate chains activated with cyanogen bromide or oxidized with periodate. After determining the binding capacity of the S-layer lattice for human IgG, the orientation of the immobilized antibody molecules was investigated using anti-human IgG peroxidase conjugates with different specificity. Attachment of S-layer microparticles with covalently bound human IgG to microplates precoated with anti-human IgG of different specificity led to clear correlations between the amount of applied human IgG and the absorption values in the immunoassays. The steepest absorption curves were obtained when human IgG was bound to the carbohydrate chains exposed on the surface of the S-layer lattice. This confirmed that the location and the accessibility of the immobilized antibodies on S-layer microparticles is of major importance for the response in immunoassays. In addition to the high reproducibility of the amount of IgG which could be bound to the S-layer lattice and the high reproducibility of the absorption curves in the immunoassays, one major advantage of using cup-shaped S-layer microparticles can be seen in the considerable increase of the actual surface available for binding processes and immunological reactions.

The single-ring Thermoanaerobacter brockii chaperonin 60 (Tbr-EL7) dimerizes to Tbr-EL14.Tbr-ES7 under protein folding conditions.

Chaperone proteins assist in the folding of some newly synthesized proteins and inhibit protein aggregation. The Thermoanaerobacter brockii chaperonin proteins (Tbr-EL and Tbr-ES) have recently been purified and characterized [Truscott, W.N., Høj, P. B., & Scopes, R. K. (1994) Eur. J. Biochem. 222, 277-284]; Tbr-EL was a single seven-membered toroid, unlike most GroELs which exist as double toroids. Using high-resolution gel filtration chromatography, we have resolved the purified Tbr-EL into single ringed (Tbr-EL7) and double ringed (Tbr-EL14) species. The latter contained tightly bound Tbr-ES co-chaperonin (Tbr-EL14.Tbr-ES7). In the presence of Mg.ATP and either Escherichia coli GroES (Eco-ES) or Tbr-ES (i.e., under protein folding conditions), the isolated Tbr-EL7 rapidly dimerized to the Tbr-EL14.Eco-ES7 or Tbr-EL14.Tbr-ES7 complexes. The doubly toroidal species thus formed contained > or = 6 molecules tightly bound ADP and one GroES7 and are similar to the asymmetric chaperonin complex isolated from Thermus thermophilus [Taguch, H., Konishi, J., Ishii, N., & Yoshida, M. (1991) J. Biol. Chem. 266, 22411-22418]. The isolated Tbr-EL7 and Tbr-EL14.Tbr-ES7 hydrolyzed ATP at approximate to 2 and 1 min-1, respectively. Addition of a molar excess of Eco-ES7 to the isolated Tbr-EL7 reduced the ATPase activity to 1 min-1, consistent with the formation of Tbr-EL14.Eco-ES7. Eco-ES7 failed to inhibit the Tbr-El14.Tbr-ES7 complex. The isolated Tbr-EL14.Tbr-ES7 complex did not support the folding of Rubisco under nonpermissive conditions. Only when the complex was supplemental with additional GroES was folding of Rubisco observed; i.e., one molar equivalent of GroES was not sufficient for folding. Both Tbr-EL7 and Tbr-EL14.Tbr-ES7 bound on unfolded [35S] Rhodospirillum rubrum Rubisco per mole particle. In contrast, Eco-EL14 bound 2 mol of protein per mole particle, consistent with each toroid having a peptide binding site. Eco-EL14.Eco-ES7 complex only bound one unfolded protein, thus GroES binding blocks one GroEL peptide binding site. Addition of Eco-ES7 to a Eco-EL14.Rubisco2 complex did not result in the displacement of one molecule of Rubisco but in the formation of a ternary Eco-EL14.Rubisco2.Eco-ES7 complex.

A new family of very long chain alpha,omega-dicarboxylic acids is a major structural fatty acyl component of the membrane lipids of Thermoanaerobacter ethanolicus 39E.

A new family of alpha,omega-dicarboxylic, very long chain fatty acids was isolated and characterized from the lipids of thermophilic anaerobic eubacterium, Thermoanaerobacter ethanolicus 39E. After the isolation of the membrane, the fatty acyl components were converted to methyl esters by acid-catalyzed methanolysis. The esterified fatty acyl components were purified by a variety of chromatographic techniques and analyzed by gas chromatography (GC) and GC-mass spectrometry (MS). One of the isolated, esterified alpha,omega-dicarboxylic, very long chain fatty acids was characterized by mass spectrometry, 1H and 13C NMR spectroscopy and Fourier transform infrared spectroscopy. NMR experiments used included double quantum filtered correlated spectroscopy (DQF-COSY) to establish spin connectivities and polarization transfer (DEPT) to measure the multiplicity of carbon signals split by protons. Based on these results, the structures of the other components could be deduced from their mass spectra. The new family of very long chain fatty acid methyl esters are alpha,omega-13,16-dimethylheptacosanedioate dimethyl ester (C29), alpha,omega-13,16-dimethyloctacosanedioate dimethyl ester (C30), alpha,omega-13,16-dimethylnonacosanedioate dimethyl ester (C31), and alpha,omega-13,16-dimethyltriacotanedioate dimethyl ester (C32). This family of fatty acids make up about 40% of fatty acyl components of the membrane of Thermoanaerobacter ethanolicus 39E. Almost all (> 90%) of the very long chain, alpha,omega-dicarboxylic fatty acid was alpha,omega-13,16-dimethyloctacosanedioic acid. A careful analysis of the structures of the alpha,omega-dicarboxylic acid strongly implies that the synthetic mechanism for formation is by tail-to-tail (omega)coupling of regular iso-branched fatty acids across opposite sides of the membrane.