Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF-Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)-(Rp)-[beta-17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line-broadening of the EPR spectrum of Mn2+ at the active site of EF-Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF-Tu.GDP.Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2'(3')-O-(N-methylanthraniloyl)-GDP is 6.2 x 10(-3) s-1 at 25 degrees C and pH 6.8], we conclude that the broadened signal arises from the EF-Tu.Mn.GDP.Pi complex, the predominant steady-state species. During the hydrolysis of GTP the Mn2+ remains bound to the beta-phosphate oxygen of GDP which arises from the beta pro-S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF-Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)-[beta-17O]GTP in the corresponding complex in the presence of EF-Ts.

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.

Modification of Bacillus subtilis elongation factor Tu by N-tosyl-L-phenylalanyl chloromethane abolishes its ability to interact with the 3'-terminal polynucleotide structure but not with the acyl bond in aminoacyl-tRNA.

Modification of B. subtilis EF-Tu by N-tosyl-L-phenylalanyl chloromethane destroyed its ability to promote protein synthesis and resulted in selective dissociation of the two binding activities of the protein for aminoacyl-tRNA. The modified EF-Tu was completely ineffective in the protection of the 3'-terminal CCA structure of tRNA against pancreatic ribonuclease, while remaining almost fully active in the protection of the ester bond between the 3'-terminal adenosine and the amino acid residue in aminoacyl-tRNA.

The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus.

The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy-terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an Mr of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.

[1H-NMR study of protein L7/L12 from bacterial ribosomes]. / Issledovanie belka L7/L12 iz bakterial'nykh ribosom metodom 1H-IaMR.

The 500 MHz 1H-NMR spectra of dimeric protein L12 from ribosomes shows a limited number of unusually sharp signals at room temperature. This is interpreted as evidence for substantial segmental flexibility of the region in the protein molecule. We have analysed the extent of the flexible region and also the size of the organized structures of the molecule. Thus residues 37-50 were found to be highly mobile whereas the N-terminal and C-terminal region are organized into folded domains.

Three-dimensional location of ribosomal protein BL2 from Bacillus stearothermophilus, a key component of the peptidyl transferase center.

Protein BL2 from Bacillus stearothermophilus has been localized by immunoelectron microscopy on the interface side of the 50 S subunit, beneath the angle formed between the central protuberance and the L1 protuberance. The immuno-electron microscopic data suggest that the interface region of the 50 S particle is not as flat as most of the proposed three-dimensional models suggest, but instead there is a significant concavity. Since several studies demonstrated that BL2 is implicated in peptidyl transferase activity or at least located close to the peptidyl transferase center, the location of protein BL2 also provides information as to the location of this important functional domain.

Asparaginyl-rhamnose: a novel type of protein-carbohydrate linkage in a eubacterial surface-layer glycoprotein.

The subunits of the crystalline surface layer of Bacillus stearothermophilus, strain NRS 2004/3a contain carbohydrates covalently linked to protein. Hydrolysis of a glycopeptide obtained by pronase digestion of the glycoprotein and analysis of the fragments revealed that rhamnose is N-glycosidically linked to the amide nitrogen of an asparaginyl residue.

Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30.

The structures of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and L30 from Bacilus stearothermophilus display a remarkably similar fold in which alpha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet. A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 A. The principal difference is an extra alpha-helix in L12CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002.

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55 degrees C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. No variation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50 degrees C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.

Isolation and structure determination of a diacetamidodideoxyuronic acid-containing glycan chain from the S-layer glycoprotein of Bacillus stearothermophilus NRS 2004/3a.

A glycan isolated from the surface-layer glycoprotein of Bacillus stearothermophilus strain NRS 2004/3a was shown by 1H- and 13C-n.m.r. spectroscopy to have the tetrasaccharide repeating-unit ----4)-beta-ManpA2,3(NAc)2-(1----3)-alpha-GlcpNAc-(1----4)-beta- ManpA2,3(NAc)-(1----6)-alpha-Glcp(1----.

Molecular sieving through S layers of Bacillus stearothermophilus strains.

The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.

Relation of the heat resistance of bacterial spores to chemical composition and structure. II. Relation to cortex and structure.

The relation between the amount of cortex, measured as total hexosamine, as diaminopimelic acid and as muramic lactam, and the heat resistance of spores of five different strains of Bacillus stearothermophilus was studied. Electron micrographs of thin sections of the spores were made to relate the structure of the spores to chemical and thermal characteristics. It was found that the amount of the cortex was significantly related to heat resistance of the spores. Strains with more electron-dense and better organized cortices were found to express higher heat resistance.

The complete amino acid sequence of the ribosomal 'A' protein (L12) from Bacillus stearothermophilus.

The complete amino acid sequence of the ribosomal 'A' protein (Bst L12) has been determined from Bacillus stearothermophilus. The protein contains 122 amino acids and has a composition of Asp4, Ans3, Thr6, Glu20, Gln2, Pro3, Gly9, Ala23, Val13, Met2, Ile11, Leu8, Phe2, Lys15, Arg1 and a molecular mass of 12737 Da.

Characterization of single crystals of the large ribosomal particles from a mutant of Bacillus stearothermophilus.

Single, three-dimensional crystals of 50 S ribosomal subunits, from a mutant of Bacillus stearothermophilus that lacks the protein L11, have been characterized using a synchrotron X-ray source. The crystals of the mutated particles grow under the same conditions and are isomorphous to those of the wild type of the same bacteria. They are orthorhombic, contain at least one 2-fold screw axis, and have unit cell dimensions of a = 350(+/- 10) A, b = 670(+/- 10) A, and c = 910(+/- 10) A. They diffract to 15 to 18 A resolution at 4 degrees C and are stable in the synchrotron beam for several hours.

Structural and chemical characterization of S-layers of selected strains of Bacillus stearothermophilus and Desulfotomaculum nigrificans.

The structures, amino acid- and neutral sugar compositions of the crystalline surface layers (S-layers) of four selected strains each of Bacillus stearothermophilus and Desulfotomaculum nigrificans were compared. Among the four strains of each species a remarkable diversity in the molecular weights of the S-layer subunits and in the geometry and constants of the S-layer lattices was apparent. The crystalline arrays included hexagonal (p6), square (p4) and oblique (p2) lattices. In vitro self-assembly of isolated S-layer subunits (or S-layer fragments) led to the formation of flat sheets or open-ended cylindrical assembly products. The amino acid composition of the S-layers exhibited great similarities and was predominantly acidic. With the exception of the S-layers of two strains of B. stearothermophilus (where only traces of neutral sugars could be detected), all other S-layer proteins seemed to be glycosylated. Among these strains significant differences in the amount and composition of the glycan portions were found. Based on this diversity interesting questions may be asked about the biological significance of the carbohydrate units of glycoproteins in prokaryotic organisms.

Structure of a rhamnan from the surface-layer glycoprotein of Bacillus stearothermophilus strain NRS 2004/3a.

The structure of a glycan from the surface-layer glycoprotein of Bacillus stearothermophilus strain NRS 2004/3a has been studied by 1H- and 13C-n.m.r. spectroscopy. The results indicate the glycan to be a polymer of the trisaccharide repeating-unit ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-beta-L-++ +Rhap-(1----.

Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus".

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP. Genes coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.

Differential scanning calorimetry of bacteria.

Thermograms obtained by differential scanning calorimetry of a range of bacteria of different heat resistances were compared. Equations were derived to calculate the rate at which the numbers of viable organisms in a calorimeter decline as the temperature is raised at a constant rate. Vegetative bacteria scanned at 10 degrees C min-1 showed multi-peaked thermograms with four major peaks (denoted m, n, p and q) occurring in the regions 68-73, 77-84, 89-99 and 105-110 degrees C respectively. Exceptions were that peak m (the largest peak) occurred at 79-82 degrees C in Bacillus stearothermophilus and an additional peak, r, was detected in Escherichia coli at 119 degrees C. At temperatures below the main peak m there were major differences in thermograms between species. There was a direct relationship between the onset of thermal denaturation and the thermoresistance of different organisms. Heat-sensitive organisms displayed thermogram features which were absent in the more heat-resistant types. When samples were cooled to 5 degrees C and re-heated, a small endothermic peak, pr, was observed at the same temperature as p. Peaks p and pr were identified as the melting endotherms of DNA. In all vegetative organisms examined, maximum death rates, computed from published D and z values, occurred at temperatures above the onset of thermal denaturation, i.e. cell death and irreversible denaturation of cell components occurred within the same temperature range.

The primary structure of ribosomal protein L2 from Bacillus stearothermophilus.

The complete amino acid sequence of ribosomal protein L2 from the moderate thermophile Bacillus stearothermophilus has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with Staphylococcus aureus protease, trypsin and chymotrypsin, as well as by chemical cleavage with o-iodosobenzoic acid. The protein contains 275 amino acid residues and has a calculated molecular mass of 30201 Da. Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli and from spinach and tobacco chloroplasts reveals that 60% of the residues of protein L2 from B. stearothermophilus are identical to those of the protein from E. coli and 45% are identical to those found in the two chloroplast proteins. There are extended regions of totally conserved sequence at positions 54-58 (GGGHK), 81-86 (EYDPNR), and 224-230 (MNPVDHP) in all four proteins.

Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus.

Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus. Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [14C]tryptophan per A260 unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNAtrp were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNAtrp has been crystallized under several different conditions.