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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 ...19892502433
subchronic toxicity studies in dogs and in utero rats fed diets containing bacillus stearothermophilus alpha-amylase from a natural or recombinant dna host.beagle dogs and fischer 344 rats were fed diets containing 0, 36 or 72 units bacillus stearothermophilus alpha-amylase (bsa)/g food or of bacillus subtilis alpha-amylase (cbsa)/g food. the dogs (four/sex/group) received treated diets for 13 wk. for the rat studies, the parental (f0) generation (12 males and 24 females/group for the bsa study, and 26 rats/sex/group for the cbsa study) received treated diets for 13 or 4 wk, respectively, before breeding and through weaning of the f1 pups; 20 f1 ra ...19892807104
novel procedures for derivatization of ribosomes for crystallographic studies.undecagold and tetrairidium clusters have been used for the preparation of heavy-metal derivatives of ribosomal particles, necessary for the evaluation of phases in the x-ray structure determination of these large particles. to obtain specific binding, monofunctional reagents of the clusters were prepared and were covalently bound to free sulfhydryl groups on the surface of the ribosome. in addition, a mutant of bacillus stearothermophilus which lacks one ribosomal protein (bl11) was grown. the ...19892808418
characterization and application of a thermostable primary transport system: cytochrome-c oxidase from bacillus stearothermophilus.cytochrome-c oxidase from bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by deae-cellulose, hydroxyapatite- and gel-filtration chromatography. the enzyme is a typical cytochrome-aa3-type oxidase which binds carbon monoxide and is sensitive to classical oxidase inhibitors like cyanide and azide. the purified enzyme is composed of three different subunits (57, 37 and 22 kda). the subunit with intermediate molecular mass contains a covalently attached ...19892536327
permeability studies on protective gloves used in dental practice.the recent guidelines issued by the dhss and the bds recommend that to prevent cross-infection gloves should be worn by dentists and their assistants whilst treating all patients. little information is available on the permeability of the various types of glove available; this pilot study was therefore conducted, to test five commonly used brands of gloves for permeability to air, electrolytes, dye and bacteria. tests were carried out before and after using the gloves for varying periods. from t ...19892912478
addition of a methyl group changes both the catalytic velocity and thermostability of the neutral protease from bacillus stearothermophilus.specific activity was compared between wild-type (wt) neutral protease from bacillus stearothermophilus and mutant protease (m1; gly144 replaced by ala144) with enhanced thermostability. when casein was used as a substrate, m1 showed 1.5-times higher specific activity than that of wt. in contrast, the specific activities of m1 for soluble reduced lysozyme and insulin b chain were lower than those of wt by 17.2 and 13.2%, respectively. after digestion of the insulin a chain by these enzymes, the ...19892673840
nucleotide sequences of genes encoding heat-stable and heat-labile glyceraldehyde-3-phosphate dehydrogenases; amino acid sequence and protein thermostability.the structural gene (gapst) encoding glyceraldehyde-3-phosphate dehydrogenase (gpdh; ec 1.2.1.12) from bacillus stearothermophilus has been cloned in escherichia coli using plasmid pbr322 as a vector; the homologous gene (gapco) from bacillus coagulans was cloned from a phage lambda library. expression of the cloned gap genes revealed that, as in the wild-type (wt) organisms, the gpdh from b. stearothermophilus (gpdh-st) was intrinsically heat stable (hs) and that from b. coagulans (gpdh-co) hea ...19892684782
isolation and identification of restriction endonuclease bsisi. 19901691485
direct phase determination for the molecular envelope of tryptophanyl-trna synthetase from bacillus stearothermophilus by x-ray contrast variation.monoclinic crystals of bacillus stearothermophilus tryptophanyl-trna synthetase grown in the presence of substrate tryptophan (space group p2(1)) display evidence of a low-resolution trigonal space group (p321). the origin and averaging transformations for the local 32 point group of this unusually clear sixfold non-crystallographic symmetry may be inferred without prior estimation of the electron density. this local symmetry was exploited in conjunction with solvent density contrast variation t ...19902310535
the rabbit muscle phosphofructokinase gene: cdna cloning and sequencing.a partial cdna for rabbit muscle phosphofructokinase (rm-pfk) has been cloned and sequenced. the nucleotide sequence of the cdna agrees with the previously determined rm-pfk genomic sequence. in addition, the amino acid sequence deduced from the cdna is nearly identical to the rm-pfk sequence previously determined by peptide analysis. a significant degree of homology exists when the amino acid sequence of rm-pfk is compared with the sequences of bacillus stearothermophilus pfk or escherichia col ...19902147292
characterization of the bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in saccharomyces cerevisiae.recombinant clones containing the manganese superoxide dismutase (mnsod) gene of bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of escherichia coli, allowed us to define the region of dna which encodes the mnsod structural gene and to identify a promoter region immediately upstream from the ...19902407726
[defective function of bioindicators for steam sterilization].it can be shown, that under certain conditions commercially available indicators with bacillus stearothermophilus and packages of native spores from soil prepared according to din 58 946/4 react differently to treatment in a lab-type steam sterilizer. the differences were most evident when incomplete evacuation of air had to be supposed. these results lead to the conclusion that some bioindicators are not able to show the inefficient function of steam sterilizers caused by local residuals of air ...19902393488
designs for a broad substrate specificity keto acid dehydrogenase.variations have been made to the structure of the nicotinamide adenine dinucleotide (nad) dependent l-lactate dehydrogenase from bacillus stearothermophilus at regions of the enzyme that we believe determine specificity toward different alpha-hydroxy acids (rchohcoo-, r = ch3, c2h5, etc.). two regions of ldh that border the active site (but are not involved in the catalytic reaction) were altered in order to accommodate substrates with hydrophobic side chains larger than that of the naturally pr ...19902271542
the nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis.directed mutagenesis has been used to study the nicotinamide subsite of the glycolytic nad-dependent glyceraldehyde-3-phosphate dehydrogenase (gapdh). residue asn313 is involved together with the carboxyamide moiety of the nicotinamide ring in a complex network of hydrogen bonding interactions which fix the position of the pyridinium ring of nad to which hydride transfer occurs at the c-4 position in the catalytic reaction. the asparagine side-chain has been replaced by that of the thr and ala r ...19902126460
composition of polar lipid acyl chains of bacillus stearothermophilus as affected by temperature and calcium.bacillus stearothermophilus was grown within the temperature range of 48 to 68 degrees c in a complex medium and in the range of 45 to 72 degrees c in the presence of 2.5 mm ca2+. the main fatty acids of lipid extracts contain 15 to 17 carbon atoms, mostly branched-chain species. the most prevalent saturated straight-chain fatty acid is n-c16. the total amount of branched-chain species decreases with increasing temperature of growth from 48 to 68 degrees c, whereas the straight-chain species inc ...19902369582
colorimetric glucose assay using thermostable glucokinase.a method for assaying glucose in serum or plasma samples using a thermostable glucokinase was developed. glucokinase from bacillus stearothermophilus was coupled with glucose-6-phosphate dehydrogenase to produce nadph, which reduced the tetrazolium dye mtt to its formazan. detection of the product at 660 nm allowed samples containing up to 30 mmol/l glucose to be assayed with an endpoint method. use of the optimal wavelength for formazan detection, 570 nm, increased sensitivity for nadph detecti ...19902310155
electron transferase activity of diaphorase (nadh: acceptor oxidoreductase) from bacillus stearothermophilus.electrochemical kinetic measurements were carried out for electron-transfer between nadh and the oxidized forms of mediators (ferrocenylmethanol (fma), ferrocenyl-1-ethanol (fea), n,n,n',n'-tetramethylphenylenediamine (tmpd), co(phen)2+(3) and fe(cn)4-(6)) catalyzed by diaphorase (nadh: acceptor oxidoreductase, ec 1.6.99.-) purified from bacillus stearothermophilus. cyclic voltammograms for the mediators with excess nadh in the presence of diaphorase gave steady-state currents. the quantitative ...19902317516
alanine dehydrogenases from two bacillus species with distinct thermostabilities: molecular cloning, dna and protein sequence determination, and structural comparison with other nad(p)(+)-dependent dehydrogenases.the gene encoding alanine dehydrogenase (ec 1.4.1.1) from a mesophile, bacillus sphaericus, was cloned, and its complete dna sequence was determined. in addition, the same gene from a moderate thermophile, b. stearothermophilus, was analyzed in a similar manner. large parts of the two translated amino acid sequences were confirmed by automated edman degradation of tryptic peptide fragments. each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residu ...19902340274
identification of a reversible structural transition in the metal-depleted glycerol dehydrogenase from bacillus stearothermophilus.evidence is presented to demonstrate that the zn2+ -depleted, inactive form of the glycerol dehydrogenase from bacillus stearothermophilus exists in one of two possible conformations in equilibrium, the position of which is temperature sensitive. the conformation of the metal-depleted enzyme favoured by higher temperatures (20-40 degrees c) is able to bind zn2+ and regain catalytic activity, whereas that favoured at lower temperatures (0-10 degrees c) is unable to bind metal ions and is thus ina ...19902294019
cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of bacillus stearothermophilus.a 2641-bp ecori fragment of dna that encodes the c-terminal part of the dihydrolipoyl acetyltransferase (e2) component and the dihydrolipoamide dehydrogenase (e3) component of the pyruvate dehydrogenase complex of bacillus stearothermophilus has been cloned in escherichia coli. its nucleotide sequence was determined. a 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another ecori fragment of ...19902253629
structure determination and refinement of bacillus stearothermophilus lactate dehydrogenase.structures have been determined of bacillus stearothermophilus "apo" and holo lactate dehydrogenase. the holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. the "apo" lactate dehydrogenase structure was solved by use of the known apo-m4 dogfish lactate dehydrogenase molecule as a starting model. phases were refined and extended from 4 a to 3 a resolution by means of the noncrystallographic molecular 222 symmetry. the r-factor was reduced to 28.7%, using 2.8 a resol ...19902330370
cloning and nucleotide sequences of the bacillus stearothermophilus neutral protease gene and its transcriptional activator gene.both the neutral protease gene (nprs) and its transcriptional activator gene (npra) from bacillus stearothermophilus telne were cloned in bacillus subtilis by using ptb53 as a vector plasmid. the presence of the npra gene enhanced protease synthesis by about fivefold. the nucleotide sequences of nprs and its flanking regions were determined. nprs was composed of 1,653 base pairs and 551 amino acid residues. a shine-dalgarno (sd) sequence was found 9 bases upstream from the translation start site ...19902203733
nucleotide sequences of bacillus stearothermophilus ribosomal protein genes: part of the ribosomal s10 operon.restriction fragments from bacillus stearothermophilus chromosomal dna were cross-hybridized with the escherichia coli ribosomal protein l2 gene rplb. a 2-kb ecori fragment which showed cross-hybridization was cloned into the m13 phage and sequenced by the dideoxy chain-terminating method. comparison of the deduced amino-acid sequences with the corresponding sequences of e. coli ribosomal proteins showed that this fragment contains the region encoding the c-terminus of l2, the genes encoding s19 ...19902222862
cloning and sequence analysis of the genes encoding the alpha and beta subunits of the e1 component of the pyruvate dehydrogenase multienzyme complex of bacillus stearothermophilus.a 4175-bp ecori fragment of dna that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (e1) of the pyruvate dehydrogenase multienzyme complex of bacillus stearothermophilus has been cloned in escherichia coli. its nucleotide sequence was determined. open reading frames (pdha, pdhb) corresponding to the e1 alpha subunit (368 amino acids, mr 41,312, without the initiating methionine residue) and e1 beta subunit (324 amino acids, mr 35,306, without the initiating ...19902200674
probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis.by combining our knowledge of the crystal structure of the glycolytic nad-dependent glyceraldehyde-3-phosphate dehydrogenase (gapdh) and the sequence of the photosynthetic nadp-dependent gapdh of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. by use of site-directed mutagenesis, the amino acids leu 187 and pro 188 of gapdh from bacillus stearothermophilus have been replaced with ala 187 and ser 188, which occur ...19902223764
dynamic ultraviolet sterilization of different implant types.this paper investigates the use of the dynamic ultraviolet sterilization process with various dental implants, stainless steel orthopedic cortical bone screws, and polysulfone polymer healing caps. these biomaterials were inoculated with the spores of bacillus subtilis and bacillus stearothermophilus. they were then exposed to dynamic ultraviolet radiation in the chamber of a bud ultraviolet device. samples were incubated in trypticase soy broth at 37 degrees c and 56 degrees c, and they were su ...19902133336
[molecular cloning and structural-functional analysis of the arginine biosynthesis genes of the thermophilic bacterium bacillus stearothermophilus].genes encoding arginine biosynthesis of bacillus stearothermophilus strain 718 were cloned in the mutant arga strain of the escherichia coli k-12. the arg genes were shown to be located on the 3.7 kb dna fragment in the following order: arga--arge--argb. the expression of the arga gene of b. stearothermophilus on the multicopy vehicle is twofold higher in argr- strain of e. coli k-12 than is isogenic argr+ strain. according to hybridization analysis arga genes of b. stearothermophilus and b. sub ...19902074006
recognition of trna(tyr) by tyrosyl-trna synthetase.in this review, i have brought together and compared the available data on the interaction between trna(tyr) and tyrosyl-trna synthetases (tyrts) of prokaryotic origins. the amino acid sequences of the heterologous tyrts that can charge escherichia coli trna(tyr), show that the residues involved in the binding and recognition of tyrosine are strictly conserved whereas those involved in the interaction with trna(tyr) are only weakly similar. the results of in vivo genetic complementation experime ...19902126463
does single-amino-acid replacement work in favor of or against improvement of the thermostability of immobilized enzyme?thermostabilities of kanamycin nucleotidyltransferase and of its mutants that became thermostable, in the free state, because of single-amino-acid replacements were studied after immobilization of the enzymes on cyanogen bromide-activated sephadex g-200 particles. lys in place of gln at position 102 decreased the thermostability of the immobilized enzyme, whereas replacement with other amino acids enhanced it.19902176451
[expression of xyle gene in bacillus stearothermophilus].using the gene expression vector pfdc11 for bacillus stearothermophilus cu21, a recombinant plasmid pfdx1 was constructed, which carries the pseudomonas-derived xyle gene coding for catechol 2,3-dioxygenase (cato2ase). cato2ase activity can be detected from cu21 (pfdx1) cells grown at 48 degrees c. this result indicates that the promoterless xyle gene can be expressed in a thermophilic host under the direction of a promoter from a thermophilic bacterium. by selecting kmr mutants at elevated grow ...19902372405
studies on the interactions of glycerol dehydrogenase from bacillus stearothermophilus with zn2+ ions and nadh.the interactions of the essential divalent cation, zn2+, with the binary complex formed between glycerol dehydrogenase (glycerol:nad+ 2-oxidoreductase, ec 1.1.1.6) and its coenzyme nadh have been examined by fluorescence spectroscopy. both the metallo and non-metallo form of the enzyme bind the coenzyme nadh. the addition of zn2+ ions to a solution of the binary complex formed between metal-depleted enzyme and nadh results in a rapid increase in fluorescence emission at 430 nm. this has been use ...19902378897
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-broadenin ...19902156700
purification and characterization of thermostable beta-mannanase and alpha-galactosidase from bacillus stearothermophilus.bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. the hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange ...19902176449
presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant escherichia coli strain.a recombinant plasmid (pmk57) was constructed by cloning the bacillus stearothermophilus alpha-amylase gene into puc8; plasmid pmk79 was then derived from pmk57 by inserting the bacterial (vitreoscilla) hemoglobin gene into the latter plasmid. both pmk57 and pmk79 were transformed into escherichia coli strain jm 103 to make strains mk57 and mk79, respectively. both mk57 and mk79 produced alpha-amylase and mk79 produced hemoglobin. mk79 outgrew mk57 in shake flasks in lb medium, the advantage of ...19902136531
structural basis of the allosteric behaviour of phosphofructokinase.comparison between the crystal structures of low- and high-affinity forms of phosphofructokinase shows a close coupling between the change of quaternary structure and local changes triggered by binding of the allosteric effectors. these concerted changes link all the substrate and effector sites in the tetramer, and explain the change of affinity for the cooperative substrate.19902136935
evidence that chemical modification of a positively charged residue at position 189 causes the loss of catalytic activity of iron-containing and manganese-containing superoxide dismutases.the escherichia coli, bacillus stearothermophilus, and human manganese-containing superoxide dismutases (mnsods) and the e. coli iron-containing superoxide dismutase (fesod) are extensively inactivated by treatment with phenylglyoxal, an arginine-specific reagent. arg-189, the only conserved arginine in the primary sequences of these four enzymes, is also conserved in the three additional fesods and five of the six additional mnsods sequenced to date. the only exception is saccharomyces cerevisi ...19902186704
thermostable alanine racemase. its structural stability.the gene encoding thermostable alanine recemase from bacillus stearothermophilus was cloned and expressed in e. coli. the enzyme was purified to homogeneity from cell extracts of e. coli carrying a plasmid designated picr4. the alanine racemase gene sequenced was found to contain an open reading frame of 1158 nucleotides. the molecular weight of the enzyme subunit was estimated to be 43,341. the alpha-helical and beta-structure contents were calculated to be about 34 and 26%, respectively, from ...19902192620
expression in escherichia coli of a sub-gene encoding the lipoyl domain of the pyruvate dehydrogenase complex of bacillus stearothermophilus.a sub-gene encoding the lipoyl domain (residues 1-85) of the lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of bacillus stearothermophilus was over-expressed in escherichia coli. approx. 80% of the domain was unlipoylated but most of the remainder was correctly lipoylated on lys-42 and could be reductively acetylated by the b stearothermophilus enzyme complex. a small proportion (approx. 4%) of the domain carried an aberrant substituent, possibly an octanoyl group, on lys- ...19902192914
amino acid sequences of the ribosomal proteins hl30 and hmal5 from the archaebacterium halobacterium marismortui.the complete amino acid sequences of the ribosomal proteins hl30 and hmal5 from the archaebacterium halobacterium marismortui were determined. protein hl30 was found to be acetylated at its n-terminal amino acid and shows homology to the eukaryotic ribosomal proteins yl34 from yeast and rl31 from rat. protein hmal5 was homologous to the protein l5 from escherichia coli and bacillus stearothermophilus as well as to yl16 from yeast. hmal5 shows more similarities to its eukaryotic counterpart than ...19902198942
expression of the copy dna for human a4 and b4 l-lactate dehydrogenases in escherichia coli.the human ldh-a and ldh-b cdnas, containing the coding regions for the l-lactate dehydrogenase a4 (m) and b4 (h) polypeptides respectively have been cloned into escherichia coli to place the cdnas under the control of hybrid e. coli/bacillus stearothermophilus transcriptional and translational signals. human a4- and b4-isoenzymes are produced in e. coli cells harbouring the expression plasmids phldha22 and phldhb10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. the t ...19902205297
structure and function of l-lactate dehydrogenases from thermophilic and mesophilic bacteria, x. analysis of structural elements responsible for the differences in thermostability and activation by fructose 1,6-bisphosphate in the lactate dehydrogenases from b. stearothermophilus and b. caldolyticus by protein engineering.the amino-acid sequences of the lactate dehydrogenases (ldh) from b. stearothermophilus and b. caldolyticus differ at only 10 positions. the properties of these enzymes however show substantial differences. the ldh from b. stearothermophilus is activated by fru-p2 and has a higher thermostability (10 degrees c) than the enzyme from b. caldolyticus which cannot be activated by fru-p2. to correlate these functional differences to the structural properties, we have constructed a set of hybrid- and ...19902206453
a comparative study of ribosomal and dna binding protein ii from two thermophilic bacteria, bacillus caldolyticus strain ep 00275 and bacillus stearothermophilus.ribosomal and dna binding proteins (dna bp ii) from an extreme thermophilic bacterium, b. caldolyticus strain ep 00275, were investigated for stability and crystallization and compared to the homologous proteins from b. stearothermophilus. two-dimensional gel electrophoresis of both types of proteins, the amino acid composition and the sequences of some of the peptides of dna bp ii revealed a close relationship between each other. the physico-chemical characteristics of dna bp ii were similar bu ...19902226856
use of a hapten specific anti-dansyl antibody for the localization of ribosomal proteins by immuno electron microscopy.the fluorescent reagent dansyl chloride has been used as an immunological marker for the electron microscopic localization of ribosomal proteins on the surface of 50s ribosomal subunits. the proteins bstl1 from bacillus stearothermophilus and ecol1 from escherichia coli were dansylated to various degrees and reconstituted into the l1-deficient e. coli 50s subunits from mutant mv17-10. using antibodies specific to dansyl chloride, both proteins were mapped at the lateral protuberance near the pep ...19902242000
[construction of promoter probe vector pfdc4 and gene expression vector pfdc11 with high transformation efficiency in bacillus stearothermophilus].a 0.5kb fragment from sau3a-digested total dna of bacillus stearothermophilus cu21 was cloned with the vector ppgv5, a derivative of ppl703. the insertion of this fragment can activate the expression of the promoteless cat-86 gene on the cloning vector in both b. stearothermophilus and bacillus subtilis hosts. when the 0.54 kb fragment is present in ppgv5 in either orientation, the transformation efficiency of the plasmid is increased about 10(3) to 10(4) fold in cu21 protoplasts. southern hybri ...19902242283
[a novel type of phase variation regarding integrated and free states of plasmid pfdx163 in bacillus stearothermophilus cu21].pfdx1 is a recombinant plasmid which carries a foreign gene xyle. by selecting for kanamycin-resistant mutants of bacillus stearothermophilus cu21(pfdx1) at higher temperature, a variant strain cu21-163 was obtained. this strain harbors a mutant plasmid pfdx163, which was formed by insertion of a 2.0kb h-fragment from the cu21 genome onto the plasmid pfdx1. pfdx163 was supposed to be integrated into the cu21 chromosome via homologous recombination of h-fragments. the cu21-163 strain consists of ...19902252599
isolation and characterization of bacillus stearothermophilus 30s and 50s ribosomal protein mutations.bacillus stearothermophilus mutations which confer resistance to or dependence on a variety of ribosome-targeted antibiotics have been isolated. many of these mutations produce ribosomal proteins with altered mobilities in a two-dimensional gel electrophoresis system. this collection of altered thermophilic ribosomal proteins will be useful in examining ribosomal structure and function.19902254291
proteolysis of bacillus stearothermophilus if2 and specific protection by gtp.translation initiation factor if2 from bacillus stearothermophilus (741 amino acids, mr = 82,043) was subjected to trypsinolysis alone or in the presence of gtp. following electroblotting and automated amino acid sequencing of the resulting peptides, the location and the sequential order of the main cleavage sites were identified. trypsinolysis of if2 ultimately generates two compact domains: a 24.5 kda c-terminal fragment and a 40 kda g-fragment which is obtained only in the presence of gtp whi ...19902265694
genetic map of the bacillus stearothermophilus nub36 chromosome.a circular genetic map of bacillus stearothermophilus nub36 was constructed by transduction with bacteriophage tp-42c and protoplast fusion. sixty-four genes were tentatively assigned a cognate bacillus subtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding, accumulation of intermediates, or their relative order in a linkage group. although the relative position of many genes on the bacillus stearothermophilus and bacillus subtilis genetic map appear ...19902298700
conserved residues of liquefying alpha-amylases are concentrated in the vicinity of active site.three dimensional structure of three liquefying type bacillus alpha-amylases were modeled based on sequence analyses and refined structure of aspergillus oryzae enzyme. the models suggest that the overall folding motif of alpha-amylases is conserved. the active site, substrate binding and stabilizing calcium binding residues are conserved and concentrated in a cleft between two domains. they constitute the core of alpha-amylases to which other, less conserved regions are attached. the bacterial ...19902302216
a single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme.using site-directed mutagenesis, the nadh-linked lactate dehydrogenase from bacillus stearothermophilus has been specifically altered at a single residue to shift the coenzyme specificity towards nadph. the single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine. this substitution was made to reduce steric hindrance on binding of the extra phosphate group of nadph and to remove the negative charge of the aspartate group. the resultant ...19902302233
a new type ii restriction endonuclease, bsma i, from bacillus stearothermophilus. 19902308865
simultaneous comparison of several sequences. 19902314287
isolation and identification of restriction endonuclease bsiki. 19902326209
random mutagenesis used to probe the structure and function of bacillus stearothermophilus alpha-amylase.mutations that cover the sequence of bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in escherichia coli, which also secreted the product. ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. a molecular model of the enzyme was constructed using the coordinates of takaamylase a and a consensus a ...19902330367
metal ion dependence of phosphorothioate atp analogues in the bacillus stearothermophilus tyrosyl-trna synthetase reaction.pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [atp alpha s and atp beta s; eckstein, f., & goody, r. (1976) biochemistry 15, 1685-1691; yee, d., armstrong, v. w., & eckstein, f. (1979) biochemistry 18, 4116-4123] were performed in the presence of mg2+, co2+, and cd2+ as the divalent metal ion cofactor. a modest preference of 5.5-fold in kappa 3/ka' (where kappa 3 ...19902334722
identification of a new type ii restriction endonuclease, bsaai. 19902339077
maltogenic amylases of bacillus stearothermophilus. 19901696223
phylogenetic and biochemical evidence for a secondary structure model of a small cytoplasmic rna from bacilli.small cytoplasmic rna (scrna; 271 nucleotides) is an abundant, stable rna identified in the gram-positive eubacterium bacillus subtilis. several findings suggest an important role of scrna in protein biosynthesis: it shares structural and biochemical features with the escherichia coli 4.5s rna (114 nucleotides), a molecule known to be involved in this process, and it can complement the essential function of 4.5s rna in vivo. the common apical hairpin motif of scrna and 4.5s rna also exists in eu ...19901698156
enhancement of thermostability of neutral proteases. 19902075976
thermostable glucokinase from bacillus stearothermophilus and its analytical application. 19902075989
atp binding plays a role in the selection of amino acid substrate by aminoacyl-trna synthetases. 19902075999
nucleotide sequence and cloning in bacillus subtilis of the bacillus stearothermophilus pleiotropic regulatory gene degt.the regulatory gene (degt) from bacillus stearothermophilus nca1503 which enhanced production of extracellular alkaline protease (apr) was cloned in bacillus subtilis with ptb53 as a vector. when b. subtilis mt-2 (npr- [deficiency of neutral protease] apr+) was transformed with the recombinant plasmid, pdt145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. in contrast, when b. subtilis db104 (npr- apr-) was used as a host, the transformant wi ...19902104607
vapor-phase hydrogen peroxide as a surface decontaminant and sterilant.the feasibility of utilizing vapor-phase hydrogen peroxide (vphp) as a surface decontaminant and sterilant was evaluated in a centrifuge application. the prototype vphp decontamination system, retrofitted into a beckman l8-m ultracentrifuge, was designed to vaporize a 30% (wt/wt) solution of aqueous hydrogen peroxide continuously injecting and withdrawing vphp in a deep-vacuum flow-through system. vphp cycles of 4, 8, 16, and 32 min were examined for cidal activity against spores of bacillus sub ...19902106287
construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage spo1 dna-binding protein tf1.the bacillus subtilis bacteriophage spo1 encodes the dna-binding protein tf1, a homolog of the ubiquitous type ii dna-binding proteins that are components of bacterial chromatin. the known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the tf1 gene. at the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, ...19902115873
characterization of a thermostable bacillus stearothermophilus alpha-amylase.liquefying-type bacillus stearothermophilus alpha-amylase was characterized. the coding gene was cloned in bacillus subtilis and the enzyme was produced in three different host organisms: b. stearothermophilus, b. subtilis, and escherichia coli. properties of the purified enzyme were similar irrespective of the host. temperature optimum was at 70-80 degrees c and ph optimum at 5.0-6.0. the enzyme was stable for 1 h in the ph range 6.0-7.5 at 80 degrees c. the enzyme was stabilized by ca2+, na+, ...19902119192
glutamyl-trna synthetases of bacillus subtilis 168t and of bacillus stearothermophilus. cloning and sequencing of the gltx genes and comparison with other aminoacyl-trna synthetases.the glutamyl-trna synthetase (glurs) of bacillus subtilis 168t aminoacylates with glutamate its homologous trna(glu) and trna(gln) in vivo and escherichia coli trna(1gln) in vitro (lapointe, j., duplain, l., and proulx, m. (1986) j. bacteriol. 165, 88-93). the gltx gene encoding this enzyme was cloned and sequenced. it encodes a protein of 483 amino acids with a mr of 55,671. alignment of the amino acid sequences of four bacterial glurss (from b. subtilis, bacillus stearothermophilus, e. coli, a ...19902120226
thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.differential scanning calorimetry (dsc) was used to analyze thermal transitions in two strains of the thermophile bacillus stearothermophilus (atcc 12016 and wat), the mesophile bacillus megaterium and the psychrotroph bacillus psychrophilus. the observed transitions, representing lipid melting and dna and protein unfolding, are compared to the maximum growth temperature (tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of gro ...19902121283
growth of bacillus stearothermophilus on glycerol in chemostat culture: expression of an unusual phenotype.bacillus stearothermophilus grew readily on glycerol in carbon-limited chemostat culture and expressed a high carbon conversion efficiency. however, the strain of organism used (probably b. stearothermophilus var. nondiastaticus) proved particularly sensitive to glycerol, both respiration and growth being severely impeded by any surfeit of this compound. sensitivity was found to correlate with an exceptionally high level of expression of glycerol kinase [activities of more than 80 mumol min-1 (m ...19902121901
purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated bacillus stearothermophilus strain.we isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. based on taxonomical research, this bacterium was identified as bacillus stearothermophilus. each extracellular enzyme was separated by hydrophobic chromatography by using a toyopearl hw-65 column, followed by gel filtration with a sephacryl s-200 column. each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatog ...19902123854
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 ...19901690648
site-directed mutagenesis of a thermostable alpha-amylase from bacillus stearothermophilus: putative role of three conserved residues.the relationship between structure, activity, and stability of the thermostable bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. mutation of his-238 to asp involved in ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the ph and temperature optima. replacement of asp-331 by glu in the active site caused almost total inactivation. interestingly, in prolonged incubation this mut ...19901694530
heat sterilization of bioindicators in propylene glycol and propylene glycol-water mixtures: arrhenius equation, thermodynamic data, and z values.our interest in calculating the thermodynamic data by means of the arrhenius equation was based on two observation: (a) the thermal death time increases considerably when the bioindicators bacillus subtilis var. niger and bacillus stearothermophilus are sterilized in nonaqueous hydrophilic solutions as found in propylene glycol (pg) with low water concentrations; and (b) the inactivation kinetics of bac. stearothermophilus does not follow a first-order reaction. the frequency factor a and the en ...19902128895
evaluation of sterilization monitoring for dental offices in ohio. 19902129135
physiology and performance of thermophilic microorganisms in sewage sludge treatment processes. 19901368146
bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase as a source of angiotensin-converting enzyme inhibitors.the more potent inhibitory activity against angiotensin-converting enzyme (ace) was excised from a glyceraldehyde-3-phosphate dehydrogenase (gapdh) preparation of bacillus stearothermophilus by heating at 120 degrees c in 1 m acoh-20 mm hcl, as compared with gapdh preparations of yeast and pig. sufficient excision of b. stearothermophilus ace inhibitors required a longer proteolysis time of 60 min. two inhibitors were then purified by gel-permeation and reverse-phase chromatographies. one of the ...19901368612
random point mutation analysis of the signal peptide cleavage area of bacillus stearothermophilus alpha-amylase. 19911368751
production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from bacillus stearothermophilus jts 859.5-methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from bacillus stearothermophilus jts 859. the process was carried out in a column reactor at 60 degrees c for 17 d without any bacterial contamination under non-aseptical conditions. half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees c and 70 deg ...19911367490
molecular cloning and nucleotide sequence determination of the bacillus stearothermophilus nca 1503 superoxide dismutase gene and its overexpression in escherichia coli.the gene (sod) encoding bacillus stearothermophilus mn-superoxide dismutase (mnsod) has been cloned in escherichia coli and its entire nucleotide sequence determined. with the exception of the post-translationally cleaved n-terminal methionine residue, the predicted amino acid sequence exhibits complete identity to the previously determined amino acid sequence. the recombinant mnsod was shown to be functionally active in e. coli both in vitro and in vivo, and was expressed to 49% of the soluble ...19911367808
binding of a chaperonin to the folding intermediates of lactate dehydrogenase.when bacillus stearothermophilus ldh dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [smith, c. j., et al. (1991) biochemistry 30, 1028-1036]. the kinetics of ldh refolding are consistent with an unbranched progression through these states. the escherichia coli chaperonin, groel, binds with high affinity to the completely denatured form and more weakly to the earliest folding inte ...19911680001
effectiveness of dental office instrument sterilization procedures.to evaluate instrument sterilization procedures in minnesota, biological indicators were used to monitor 406 sterilizers in 381 dental offices. findings suggest a general improvement in instrument performance over that of a decade ago, but sterilization failure rates are still too high. sterilizer operator errors are a major cause of sterilization failures. bis are useful in monitoring sterilization performance only when sterilization procedures are performed consistently and competently by well ...19911660501
plasmids in bacillus stearothermophilus coding for bacteriocinogeny and temperature resistance.obligately thermophilic strains of bacillus stearothermophilus were screened for the presence of plasmids by agarose gel electrophoresis. all strains in our collection contained large plasmids (20 x 10(6)-80 x 10(6)) and were divided into four groups with respect to their plasmid pattern and production of bacteriocins. the major plasmid species were designated pse407 (38.7 x 10(6)), pse409 (29.0 x 10(6)), pse411 (21.5 x 10(6)), and pse410 (23.5 x 10(6)). their physical endonuclease maps were con ...19911661014
effects of aeration during growth of bacillus stearothermophilus on proton pumping activity and change of terminal oxidases.the effects of aeration during bacterial growth on the proton translocating activity of the respiratory chain of b. stearothermophilus atcc 8005, which is stable enough for measurement of the h+/o ratio by an oxygen pulse method, were examined. for endogenous and ascorbate-n,n,n',n'-tetramethyl p-phenylene diamine (tmpd) respiration, h+/o ratios of around 6 and 2 were obtained using resting cells grown under highly aerated conditions. the values were about 4 and 0 when cells were grown under lim ...19911665485
a synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.acylphosphonic acids, r-co-po(oh)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. acetylphosphonic acid inhibited nad+ reduction by pyruvate with the pyruvate dehydrogenases from escherichia coli and bacillus stearothermophilus. the inhibition was competitive with pyruvate, with ki of 6 microm for the e. coli en ...19911669440
a chaperonin from a thermophilic bacterium, thermus thermophilus, that controls refoldings of several thermophilic enzymes.a chaperonin has been purified from a thermophilic bacterium, thermus thermophilus. it consists of two kinds of proteins with approximate mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. its weak atpase activity is inhibited by sulfite and activated by bicarbonate. atp causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. the t. thermophilus chaperonin c ...19911682319
determination of peptide regions on the surface of the eubacterial and archaebacterial ribosome by limited proteolytic digestion.limited proteolysis was used in combination with two-dimensional gel electrophoresis, blotting, and amino acid sequence analysis to investigate the surface of intact ribosomal subunits at the peptide and amino acid level. surface sites of 14 ribosomal proteins from escherichia coli 50s subunits were determined using proteases with different specificities. to assess the evolutionary conservation of ribosomal topography among eubacteria, large subunits from bacillus stearothermophilus were also su ...19911751495
effect of glycosylation of bacterial amylase on stability and active site conformation.with a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. several group specific inhibitors exhibited dose-dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. the ph optima of native and glycosylated enzyme remained the same indicating t ...19911715313
single-stranded dna plasmid, vector construction and cloning of bacillus stearothermophilus alpha-amylase in lactobacillus.vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to taqi-digested dna of a cryptic plasmid from lactobacillus plantarum. the minimal region of lactobacillus plasmid dna that was required for dna replication was defined and a single-stranded dna intermediate replication system was observed. homologies with other origins of replication of plasmids from gram-positive bacteria, replicating via rolling circle mechanism, were found. it was shown that the c ...19911961976
the effect of kirromycin on the metal ion coordination in complexes of elongation factor tu from bacillus stearothermophilus as inferred from the 17o-55mn superhyperfine interaction.the binding of the antibiotic kirromycin to elongation factor tu (ef-tu) leads to a severe line broadening of the q-band manganese epr lines of ef-tu.mn, ef-tu.mn.gdp, as well as ef-tu.mn.gdp.pi indicating that the coordination sphere of the metal ion is changed by this interaction. the number of coordinated water molecules was determined from the 17o-55mn superhyperfine coupling observable in h2(17)o enriched water; it is ph-dependent in the ef-tu.mn.gdp complex, at ph 6.8 probably four water m ...19911648403
sodium ion-dependent amino acid transport in membrane vesicles of bacillus stearothermophilus.amino acid transport in membrane vesicles of bacillus stearothermophilus was studied. a relatively high concentration of sodium ions is needed for uptake of l-alanine (kt = 1.0 mm) and l-leucine (kt = 0.4 mm). in contrast, the na(+)-h(+)-l-glutamate transport system has a high affinity for sodium ions (kt less than 5.5 microm). lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. the stimulatory effect of monensin on the steady-state accumulation le ...19911670936
cloning of a chromosomal alpha-amylase gene from bacillus stearothermophilus.we have cloned and sequenced a gene for a heat-stable alpha-amylase from a natural isolate of bacillus stearothermophilus. previously, it had been shown that b. stearothermophilus amylase genes may be harboured on indigenous plasmids. we have found that our isolate harbours the amylase gene only on the chromosome and not on its indigenous plasmid.19911903751
thermostable alanine racemase of bacillus stearothermophilus. construction and expression of active fragmentary enzyme.limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (galakatos, n. g., and walsh, c. t. (1987) biochemistry 26, 8475-8480). we have constructed a mutant gene that tandemly encodes the two polypeptides of the bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. the mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologica ...19911906880
two lipoyl domains in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of streptococcus faecalis.a fragment of dna incorporating the gene, pdhc, that encodes the dihydrolipoamide acetyltransferase (e2) chain of the pyruvate dehydrogenase multienzyme complex of streptococcus faecalis was cloned and a dna sequence of 2360 bp was determined. the pdhc gene (1620 bp) corresponds to an e2 chain of 539 amino acid residues, mr 56,466, comprising two lipoyl domains, a peripheral subunit-binding domain and an acetyltransferase domain, linked together by regions of polypeptide chain rich in alanine, p ...19911908789
bst dna polymerase permits rapid sequence analysis from nanogram amounts of template.a simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded dna. this approach employs the thermostable dna polymerase from bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. the procedure requires few pipetting steps, no preannealing step and very short reaction time. this method can significantly reduce the cost associated with d ...19911954022
bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily.bacterial catalase-peroxidases are enzymes containing 0.5-1.0 heme per subunit. the identical subunits are generally 80 kda in size, and the sequenced subunits of e. coli, s. typhimurium and b. stearothermophilus contain 726-731 amino acid residues per subunit. the heme-containing peroxidases of plants, fungi and yeast are monomeric, homologous and 290-350 residues in size. analyses of the amino acid sequences indicate that the double length of the bacterial peroxidases can be ascribed to gene d ...19911954228
effect of simultaneous application of heat and pressure on the survival of bacterial spores.the effect of simultaneous application of moderate hydrostatic pressure (10-300 atm) and heat on the survival of the bacillus stearothermophilus spores in a flow-through system was investigated. a high heterogeneity of the sensitization of spores to heat by pressure was found. a higher degree of reduction of heat resistance was observed at the low than at the high temperatures tested. the simultaneous application of moderate pressure and heat can not be applied for the preservation of liquid foo ...19911955421
kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-trna synthetase analyzed by pyrophosphate-exchange kinetics.the first step of the reaction catalyzed by the aminoacyl-trna synthetases is the formation of enzyme-bound aminoacyl adenylate. the steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and atp. a simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-trna synthetase from bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identi ...19911645192
the identification of a structurally important cysteine residue in the glycerol dehydrogenase from bacillus stearothermophilus.evidence is presented to demonstrate that the zn2+ metallo-enzyme glycerol dehydrogenase from the thermophile bacillus stearothermophilus has one cysteine residue per subunit which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme. modification of the metal-depleted enzyme by methyl methanethiosulphonate prevents the reactivation of the enzyme by zn2+ ions and induces dissociation of the oligomer into subunits. the rate of reaction of the cysteine residu ...19912009285
fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1.3-di(2-pyrenyl)propane.intramolecular excimer formation of 1,3-di(2-pyrenyl)propane was used to study the fluidity of liposomes prepared from membrane polar lipids of bacillus stearothermophilus. on the basis of spectral data, local polarity and polarizability parameters were established suggesting that the probe molecules are located well inside the membranes, but displaced towards the polar head groups of the phospholipid molecules. the excimerization rate is very sensitive to lipid phase transitions and pretransiti ...19912018528
isolation and identification of restriction endonuclease bsebi. 19911653952
improving the thermostability of the neutral protease of bacillus stearothermophilus by replacing a buried asparagine by leucine.amino acids buried in the hydrophobic interior of a protein with polar side chain atoms, which are not involved in hydrogen bonding or electrostatic interactions, have an adverse effect on protein stability. replacing such residues by hydrophobic ones may render a protein more stable. asparagine 241, which is buried in the neutral protease of bacillus stearothermophilus, was replaced by leucine by site-directed mutagenesis. this mutation increased the stability of the protein by 0.7 +/- 0.1 degr ...19912026247
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