J Biochem (Tokyo), 1990 Oct, 108(4), 572 - 8 Primary structure of the inorganic pyrophosphatase from thermophilic bacterium PS-3; Ichiba T et al.; The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase . The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792 . The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes. J Biochem (Tokyo), 1990 Oct, 108(4), 554 - 9 Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity; Konishi J et al.; Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses . The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases . Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1 . S . cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1 . Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase . These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases. Enzyme Microb Technol, 1990 Oct, 12(10), 736 - 42 Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems; Reiken SR et al.; Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications . Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters . This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data . Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis . The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase . Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate . These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors. Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 1258 - 63 The alpha 1 beta 1 heterodimer of ATP synthase; Ohta S et al.; The alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al . (1989) FEBS Lett . 249, 67) . The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg . On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity . The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate . The alpha and beta monomers were practically inactive . The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC. Eur J Biochem, 1990 Sep 24, 192(3), 609 - 20 Primary structure of the oligo-1,6-glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene; Watanabe K et al.; The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo-1,6-glucosidase was cloned within a 2.8-kb SalI-EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host . E . coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo-1,6-glucosidase in the cytoplasm . The cloned enzyme coincided absolutely with B . cereus oligo-1,6-glucosidase in its Mr (65,000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants . The nucleotide sequence of B . cereus oligo-1,6-glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides) . The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon . The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66,010 . The amino acid composition and Mr were comparable with those of B . cereus oligo-1,6-glucosidase . The predicted N-terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo-1,6-glucosidase . The deduced amino acid sequence of B . cereus oligo-1,6-glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo-1,6-glucosidase and from Saccharomyces carlsbergensis CB11 alpha-glucosidase, respectively . Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B . cereus oligo-1,6-glucosidase may take the similar (alpha/beta)8-barrel super-secondary structure, a barrel of eight parallel beta-strands surrounded by eight alpha-helices, in its N-terminal active site domain as S . carlsbergensis alpha-glucosidase and Aspergillus oryzae alpha-amylase. FEBS Lett, 1990 Sep 17, 270(1-2), 184 - 6 Effect of formate on Mössbauer parameters of the non-heme iron of PS II particles of cyanobacteria; Semin BK et al.; Mossbauer spectra were measured for PSII particles having an active water-splitting system . The particles were isolated from the thermophilic cyanobacterium Synechococcus elongatus enriched in 57Fe . The Mossbauer resonance absorption spectrum is a superposition of 3 doublets with the following quadrupole splitting and chemical shift: 1, delta = 0.40, delta = 0.85; II, delta = 1.35, delta = 2.35; III, delta = 0.25, delta = 1.65 . The delta and delta values of doublets I, II, III are characteristic of proteins with iron-sulphur center, non-heme iron of the reaction center of higher plants and of the oxidized cytochrome b-559 . Treatment with sodium formate to remove bicarbonate affects only the doublet of non-heme iron, causing its quadrupole splitting to reduce to 1.75 and the chemical shift to reduce to 0.90 . After washing out the formate, the Mossbauer spectrum of non-heme iron is restored . The data suggest that bicarbonate is a ligand for the non-heme iron of the reaction center of cyanobacteria. Nucleic Acids Res, 1990 Sep 11, 18(17), 5133 - 41 Nuclear pre-mRNA introns: analysis and comparison of intron sequences from Tetrahymena thermophila and other eukaryotes; Csank C et al.; We have sequenced 14 introns from the ciliate Tetrahymena thermophila and include these in an analysis of the 27 intron sequences available from seven T . thermophila protein-encoding genes . Consensus 5' and 3' splice junctions were determined and found to resemble the junctions of other nuclear pre-mRNA introns . Unique features are noted and discussed . Overall the introns have a mean A + T content of 85% (21% higher than neighbouring exons) with smaller introns tending towards a higher A + T content . Approximately half of the introns are less than 100 bp . Introns from other organisms (approximately 30 of each) were also examined . The introns of Dictyostelium discoideum, Caenorhabditis elegans and Drosophila melanogaster, like those of T . thermophila, have a much higher mean A + T content than their neighbouring exons (greater than 20%) . Introns from plants, Neurospora crassa and Schizosaccharomyces pombe also have a significantly higher A + T content (10%-20%) . Since a high A + T content is required for intron splicing in plants (58), the elevated A + T content in the introns of these other organisms may also be functionally significant . The introns of yeast (Saccharomyces cerevisiae) and mammals (humans) appear to lack this trait and thus in some aspects may be atypical . The polypyrimidine tract, so distinctive of vertebrate introns, is not a trait of the introns in the non-vertebrate organisms examined in this study. J Biochem (Tokyo), 1990 Sep, 108(3), 449 - 56 Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8; Yamada T et al.; Threo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli carrying a recombinant plasmid . The thermostable enzyme thus produced was extracted from the E . coli cells, purified, and crystallized . The enzyme was shown to be a dimer of identical subunits of molecular weight (4.0 +/- 0.5) x 10(4) . The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0 x 10(-5) M and that for NAD 6.3 x 10(-4) M . The optimum pH at 75 degrees C in the presence of 1.2 M KCl was around 7.2 . The presence of Mg2+ or Mn2+ was essential for the enzyme action . The enzyme was activated about 30-fold by the addition of 1 M KCl or RbCl . The high salt concentration decelerated the thermal unfolding of the enzyme, and accelerated the aggregation of the unfolded protein . Based on these effects, the molecular mechanism of the unusual stability of the enzyme is discussed. Appl Environ Microbiol, 1990 Sep, 56(9), 2677 - 83 Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product; Luthi E et al.; A xylanase encoded by the xynA gene of the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible lambda pR and pL promoters of the expression vector pJLA602 . Induction of up to 55 times was obtained by growing the cells at 42 degrees C, and the xylanase made up to 20% of the whole-cell protein content . The enzyme was located in the cytoplasmic fraction in E . coli . The temperature and pH optima were determined to be 70 degrees C and pH 5.5 to 6, respectively . The xylanase was stable for at least 72 h if incubated at 60 degrees C, with half-lives of 8 to 9 h at 70 degrees C and 2 to 3 min at 80 degrees C . The enzyme had high activity on xylan and ortho-nitrophenyl beta-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl beta-D-cellobioside . The gene was probably expressed from its own promoter in E . coli . Translation of the xylanase overproduced in E . coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined. FEMS Microbiol Rev, 1990 Sep, 7(1-2), 61 - 77 Molecular genetics of Streptococcus thermophilus; Mercenier A; The metabolism and genetics of Streptococcus thermophilus (presently Streptococcus salivarius ssp . thermophilus) have only been investigated recently despite its widespread use in milk fermentation processes . The development of recombinant DNA technology has allowed impressive progress to be made in the knowledge of thermophilic dairy streptococci . In particular, it has permitted a careful analysis of phenotypically altered variants which were derived from a mother strain by plasmid or chromosomal DNA reorganization . While natural phage defense mechanisms of S . thermophilus remain poorly documented, information on the bacteriophages responsible for fermentation failures has accumulated . The lysogenic state of two S . thermophilus strains has also been demonstrated for the first time . Gene transfer techniques for this species have been established and improved to the point that targeted manipulation of their chromosomal determinants is now feasible . Cloning and expression vectors have been constructed, and a few heterologous genes were successfully expressed in S . thermophilus . The first homologous genes, involved in carbohydrate utilization, have been cloned and sequenced, shedding some light on the molecular organization of key metabolic steps. Biomed Environ Sci, 1990 Sep, 3(3), 353 - 63 Microbiological analyses and inflammatory effects of settled dusts from rice and hay; Shen YE et al.; Fourteen samples of settled dust from two factories processing rice and wheat straw near Shanghai, China, were examined by dilution plating for total bacteria, gram-negative bacteria, thermophilic actinomycetes, and fungi . They were also examined for aflatoxin, endotoxin, and potential to stimulate production of human interleukin 1 beta (IL-1 beta) and to consume complement . The concentrations of total microorganisms were consistently greater than 10(7) CFU/g and ranged from 10(7) to 10(9) CFU/g . In general, the level of microbial contamination was greater in the hay dust samples than in the rice dust samples, with bacteria being the most numerous microorganisms observed followed by molds, thermophilic actinomycetes, and yeasts . The predominant fungi were species of Aspergillus, Cladosporium, Penicillium, Trichosporon, and Cryptococcus . No significant levels of aflatoxin were observed and the isolates of A . flavus examined lack significant aflatoxigenic potential . The levels of microorganisms in these samples, the types of organisms found, and the inflammatory mediators such as endotoxin suggest that workers exposed to these dusts may be at risk for respiratory illness. J Appl Bacteriol, 1990 Sep, 69(3), 384 - 9 Study of haemolytic activity of some Campylobacter spp . on blood agar plates; Arimi SM et al.; A total of 152 strains of Campylobacter jejuni, C . coli, C . laridis and C . fetus subsp . fetus were tested for haemolysis on blood agar plates . Distinct haemolysis was detected in 92.3% (96/104) of strains of C . jejuni and 21.7% (5/23) of strains of C . coli on sheep blood heart infusion agar after incubation for 4 d microaerobically at 42 degrees C . Haemolysis was also detected on horse blood heart infusion agar . Haemolysis was not detected at 37 degrees C except with one of 50 strains of C . jejuni tested at this temperature, which was weakly positive . Campylobacter laridis was not haemolytic; C . fetus subsp . fetus, which does not grow at 42 degrees C, showed no haemolysis at 37 degrees C . Blood agar (Oxoid, BA Base No . 2) was not suitable for testing for haemolysis by these organisms . A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis easier to detect . There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae . The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters. Biotechniques, 1990 Sep, 9(3), 276 - 81 Purification of a thermostable DNA polymerase from Thermus thermophilus HB8, useful in the polymerase chain reaction; Carballeira N et al.; A thermostable DNA polymerase, isolated from the thermophilic strain Thermus thermophilus HB 8 was purified by a five-step procedure which provides a high yield and a homogeneous preparation . The molecular weight was estimated to be 67,000 daltons and the extension rate was determined to be 1500 nucleotides per minute . The enzyme works in polymerase chain reaction conditions similar to those used for Taq polymerase from Thermus aquaticus. Bioorg Khim, 1990 Sep, 16(9), 1218 - 35 {Study of the structure of the photosynthetic reaction center of the green thermophilic bacteria Chloroflexus aurantiacus}; Kutuzov MA et al.; Analysis of the Chloroflexus aurantiacus reaction centre (RC) using both protein and recombinant DNA techniques resulted in determination of its polypeptide composition and the primary structures of its two subunits . A model of the polypeptide chains' folding in the membrane is suggested based on: i) homology between L- and M-subunits of Chloroflexus aurantiacus RC and their counterparts in purple bacteria; ii) comparison of their hydropathy plots, and iii) data on the tertiary structures of purple bacteria RCs . The role of a number of functionally important amino acid residues in the RC electron transport activity is discussed . Limited proteolysis of the RC under non-denaturing conditions was used to determine the contribution of the N-terminal regions to its thermal stability. Biokhimiia, 1990 Sep, 55(9), 1570 - 7 {A comparative study of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus by the tritium topography method}; Bobkova EV et al.; A comparative study of thermostability and amino acid composition of phenylalanyl-tRNA synthetases from E . coli and Thermus thermophilus HB8 has been carried out . In the thermophilic protein the proline, leucine, phenylalanine, arginine content was considerably increased, whereas that of asparagine, isoleucine, serine, threonine and lysine was decreased as compared to the mesophilic protein . Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of the both enzymes . In the E . coli enzyme the threonine residues were also easy to access, while on the surface of the thermophilic enzyme arginine residues were more abundant . A quantitative assay of the surface compositions revealed the increased exposure of (Leu + Ile) residues in the thermophilic protein as well as of the charged asparagine and arginine residues . A possible relationship of the observed effects to thermostability is discussed. Biokhimiia, 1990 Sep, 55(9), 1539 - 52 {Cloning and nucleotide sequence determination of the fus gene coding for the elongation factor G of Thermus thermophilus HB8}; Iakhnin AV et al.; A clone with the Thermus thermophilus HB8 fus gene coding for elongation factor G has been identified in a genomic library in plasmid pBR 322 by hybridization with labeled oligonucleotide 19 bases that are complementary in length to the 3'-end of the T . thermophilus fus gene . The fragment with the fus gene was recloned into the pTZ 18R plasmid . A restriction map of this fragment has been made . A set of short overlapping fragments of the fus gene has been obtained by nucleotide sequence unspecific linearization of the plasmid and by the restriction fragment subcloning into M13 mp18 and mp19 vectors . The nucleotide sequence of the fus gene was determined by the dideoxy chain termination method . Fus gene codes the elongation factor G 690 amino acids in length (Mr = 76756 Da) . The amino acid sequence of EF-G from T . thermophilus has a 59.7% homology with that of E . coli and a 29.0% homology with EF-2 of rat liver. FEMS Microbiol Rev, 1990 Sep, 7(1-2), 113 - 30 Exocellular polysaccharides produced by lactic acid bacteria; Cerning J; The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structure and their synthesis are outlined . Mutans streptococci, which include Streptococcus mutans and S . sobrinus produce soluble and insoluble alpha-glucans . The latter may contain as much as 90% alpha-1-3 linkages and possess a marked ability to promote adherence to the smooth tooth surface causing dental plaque . Dextrans produced by Leuconostoc mesenteroides are high molecular weight alpha-glucans having 1-6, 1-4 and 1-3 linkages, varying from slightly to highly branched; 1-6 linkages are predominant . Emphasis is put on exopolysaccharide producing thermophilic and mesophilic lactic acid bacteria, which are important in the dairy industry . The produced polymers play a key role in the rheological behaviour and the texture of fermented milks . One of the main problems in this field is the transitory nature of the thickening trait . This instability is not yet completely understood . Controversial results exist on the sugar composition of the slime produced, but galactose and glucose have always been identified with galactose predominating in most cases. Agric Biol Chem, 1990 Sep, 54(9), 2385 - 92 Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli; Motoshima H et al.; Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium . We cloned the AP-T gene from T . aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe . The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon . The molecular weight was calculated to be 44,820 . The AP-T was overproduced in E . coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter . The AP-T expressed in E . coli was heat stable and easily purified by heat treatment (80 degrees C, 30 min) . The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus. Eur J Biochem, 1990 Aug 28, 192(1), 25 - 31 Complete amino-acid sequence of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima; Schultes V et al.; 1 . The complete amino-acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophilic eubacterium Thermotoga maritima has been determined by classical automated sequence analysis of peptides derived by chemical fragmentation with cyanogen bromide and enzymatic cleavages with specific proteases . 2 . The protein contains 332 amino acids per subunit . Its sequence is as follows: (sequence; see text) 3 . Comparing the given sequence with those of the enzymes from the moderate and extreme thermophilic bacteria Bacillus stearothermophilus and Thermus aquaticus, 63% and 59% identity are observed . Alignment of the sequences of GAPDHs from a variety of sources yields one deletion (one amino acid) and one insertion (two amino acids) . 4 . Thermal stability is caused by minute adjustments of the local three-dimensional structure . Previous 'strategies of thermal adaptation' in terms of preferred amino-acid exchanges are not in accordance with the present sequence data. Eur J Biochem, 1990 Aug 28, 192(1), 17 - 24 Phylogenetic and biochemical evidence for a secondary structure model of a small cytoplasmic RNA from Bacilli; Struck JC et al.; 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 eukaryotic 7SL RNA, the RNA component of the signal recognition particle . To elucidate the higher-order structure of scRNA, we have combined a phylogenetic approach with a biochemical one . The sequence of scRNA from a thermophilic relative of B . subtilis, Bacillus stearothermophilus, was determined and compared with the B . subtilis scRNA . In addition, the solution structure of B . stearothermophilus scRNA was probed with single- and double-strand-specific nucleases . Both types of analysis support a secondary structure model for scRNA that strongly resembles 4.5S RNA and respective parts of 7SL RNA . The results provide further evidence for the suggestion of a functional relationship between these RNAs. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 1 - 7 Crystals of complexes mimicking protein biosynthesis are suitable for crystallographic studies; Hansen HA et al.; A complex of 70S ribosomes from Thermus thermophilus together with an average of 1.5-1.8 equivalents of PhetRNA(Phe) and a short mRNA chain, composed of 35 +/- 5 uridines, was crystallized under the conditions used for the growth of crystals of isolated ribosomes from the same source . Considering the reproducibility of their growth, their internal order and their shape, the crystals of the complex are superior to those of isolated ribosomes . In accord with previous three-dimensional reconstruction and modeling experiments, we conclude that the complex is less flexible and that an average population of complexes is more homogeneous than that of isolated 70S ribosomes . The crystals of the complex diffract to higher than 15 A resolution and can be irradiated with synchrotron X-ray beam at cryo-temperatures for days without noticeable decay . Since the crystals of the complex are apparently isomorphous with these of the isolated 70S ribosomes (P4(1)2(1)2; a = b = 526; c = 315 A), they should provide tool for phasing as well as for locating the mRNA and tRNA binding sites. Biochemistry, 1990 Aug 21, 29(33), 7584 - 92 Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima; Wrba A et al.; D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Thermotoga maritima, a hyperthermophilic eubacterium, has been isolated in pure crystalline form . The enzyme is a homotetramer with a subunit molecular mass of 37 kDa . The sedimentation coefficient of the native enzyme is 7.3 X 10(-13)s, the isoelectric point is 4.6, and the specific absorption coefficient A1%, 1cm 280nm = 8.4 . The enzyme shows extreme thermal stability: differential scanning calorimetry yields a transition temperature (Tm) of 109 degrees C for the NAD-saturated enzyme . Thermal deactivation occurs at T greater than 90 degrees C . The physicochemical characteristics of the enzyme suggest that its gross structure must be very similar to the structure of GAPDHs from mesophilic sources . The amino acid composition does not confirm the known "traffic rules" of thermal adaptation, apart from the Lys----Arg exchange . One reactive and at least two buried SH groups can be titrated with 5,5'-dithiobis(2-nitrobenzoate) . The highly reactive SH group is probably the active-site cysteine residue common to all known GAPDHs . The activation energy of the glyceraldehyde 3-phosphate oxidation reaction decreases with increasing temperature . This functional behavior can be correlated with the temperature-dependent changes of both the intrinsic fluorescence and the near-UV circular dichroism; both indicate a temperature-dependent structural reorganization of the enzyme . Hydrogen-deuterium exchange reveals significantly increased rigidity of the thermophilic enzyme if compared to mesophilic GAPDHs at 25 degrees C, thus indicating that the conformational flexibility is similar at the corresponding physiological temperatures.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1990 Aug 20, 214(4), 819 - 20 Crystals of threonyl-tRNA synthetase from Thermus thermophilus . Preliminary crystallographic data; Garber MB et al.; Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant . The crystals are very stable and diffract to at least 2.4 A . The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A. Eur J Biochem, 1990 Aug 17, 191(3), 715 - 20 Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462 . Purification, characterization and kinetic mechanism; Ohshima T et al.; Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis . The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa . The enzyme was much more thermostable than that from a mesophile, B . sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min . The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months . The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively . The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia . Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding . NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion . The products are sequentially released from the enzyme in the order L-alanine then NAD+ . A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction . A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis . The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM. J Biol Chem, 1990 Aug 15, 265(23), 13419 - 22 The 55-kDa polypeptide released from spinach thylakoid membranes with 1 M LiCl is not the beta subunit of chloroplast F1; Sato MH et al.; It was reported by Frasch et al . (Frasch, W . D., Green, J., Caguiat, J., and Mejia, A . (1989) J . Biol . Chem . 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity . We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract . However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase . Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex . Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide . Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively. Biochemistry, 1990 Aug 7, 29(31), 7237 - 44 Cloning, sequence determination, and expression of the genes encoding the subunits of the nickel-containing 8-hydroxy-5-deazaflavin reducing hydrogenase from Methanobacterium thermoautotrophicum delta H; Alex LA et al.; The genes frhA (1217 bp), frhB (845 bp), and frhG (710 bp) encoding the three known subunits, alpha, beta, and gamma, of the 8-hydroxy-5-deazaflavin (F420) reducing hydrogenase (FRH) from the thermophilic methanogen Methanobacterium thermoautotrophicum delta H have been cloned, sequenced, and shown to be tightly linked, indicative of a single transcriptional unit . The DNA sequence contains a fourth open reading frame, designated frhD (476 bp), encoding a polypeptide (delta) that does not copurify with the active enzyme . Expression of the frh gene cluster in Escherichia coli shows that four polypeptides are synthesized . When analyzed by SDS-PAGE, the proteins migrate with mobilities consistent with their calculated molecular weights . In order to understand the mechanism of H2 oxidation by this enzyme, localization of redox cofactors (Ni, Fe/S, FAD) to specific subunits and information on their structure is needed . This has been hindered due to the refractory nature of the enzyme to denaturation methods needed in order to obtain individual subunits with cofactors intact . In this paper we discuss the possible localization of the redox cofactors as implicated from the DNA-derived protein sequences of the subunits . The amino acid sequences of the subunits of the FRH are compared with those of other Ni-containing hydrogenases, including the methyl viologen reducing hydrogenase (MVH) of M . thermoautotrophicum delta H. J Biol Chem, 1990 Aug 5, 265(22), 12927 - 32 Characterization of ribonuclease P from the archaebacterium Sulfolobus solfataricus; Darr SC et al.; Ribonuclease P is the endonuclease that removes the leader fragments from the 5'-ends of precursor tRNAs . The enzyme isolated from eubacteria contains a catalytic RNA subunit . RNAs also copurify with eukaryotic RNase P, although catalysis by those RNAs has not been demonstrated . This paper reports the isolation and characterization of ribonuclease P from the thermoacidophilic archaebacterium Sulfolobus solfataricus . Archaebacteria are a primary evolutionary lineage, distinct from both eukaryotes and eubacteria . Ribonuclease P of S . solfataricus has reaction component requirements and a Km for substrate tRNA (2.5 X 10(-7) M) that are roughly similar to those reported for eubacterial and eukaryotic ribonuclease P . The temperature optimum for the reaction is 77 degrees C, reflecting the thermophilic character of the organism . The enzyme activity is not affected by treatment with micrococcal nuclease, suggesting that there is no RNA subunit or that it is protected from nuclease action . The density of the enzyme in cesium sulfate equilibrium density gradients is 1.27 g/ml, which is similar to that of protein . However, several RNAs between 200 and 400 nucleotides in size copurify with the enzyme activity on the density gradients, and one of them remains after micrococcal nuclease treatment . These properties of the S . solfataricus enzyme are compared with those of ribonuclease P from eukaryotes and eubacteria. Epidemiol Infect, 1990 Aug, 105(1), 73 - 8 Incidence of Campylobacter infection in infants in western Algeria and the possible protective role of breast feeding; Megraud F et al.; A case-control study aimed at comparing the incidence of campylobacter infection with that of other enteropathogens in infants was performed in Oran, Western Algeria . During a one-year period, infants consulting in a health centre were included if they had acute diarrhoea . The controls comprised infants going to the same centre for vaccination . Butzler medium Virion was used to look for thermophilic campylobacters . Campylobacters were isolated in 17.7% of the 411 patients and in 14.9% of the 247 controls . No statistically significant difference was found after stratification by age . In contrast, other enteropathogenic bacteria were rarely present . Among the potential factors of clinical expression of infection, breast feeding appeared to have a protective effect which was higher for campylobacter diarrhoea than that observed for other causes of diarrhoea . These data contrast with those previously published in Bangladesh and could be an incentive for promoting breast feeding in this country, where the tradition is decreasing far below the standard which is generally accepted. J Appl Bacteriol, 1990 Aug, 69(2), 235 - 40 Correlation between environmental monitoring of thermophilic campylobacters in sewage effluent and the incidence of Campylobacter infection in the community; Jones K et al.; Environmental monitoring of thermophilic campylobacters in liquid sewage effluent (primary settlement only) during 1988 and 1989 showed a prominent seasonality with distinct peaks in May and June (the average number of bacteria per 100 ml of effluent in months other than May and June was 2244 and the average for the peak months was 50,778) . Apart from September 1989, this seasonality coincided precisely with the seasonal variation of campylobacter enteritis in the community with similar distinct peaks in May and June (the incidence of infection in May and June was twice or three times that in the other months) . Sampling of sewers showed that the campylobacters in the sewage effluent came mainly from abbatoir and animal processing plants with only a minor input from the community . Therefore, the seasonal peaks in the sewage effluent and in the community may not be dependent on human infections but on zoonotic infections which may also peak in May and June. J Appl Bacteriol, 1990 Aug, 69(2), 185 - 9 Seasonal variation of thermophilic campylobacters in sewage sludge; Jones K et al.; The seasonal variation of thermophilic campylobacters in Lancaster's sewage sludge was studied over a 21 month period . The numbers in fresh sludge (from primary sedimentation) vary between approximately 200 and 5000/100 ml for most of the year but there was a large increase in May and June (in May 1988 there were 42,100 campylobacters/100 ml which is 17 times more than in the preceding April) . In 1989 there was a similar May/June peak but with lower numbers . This seasonal variation, measured by environmental monitoring, reflects the incidence of infections in the community . The same pattern was found in 2-d old sludge but the numbers were substantially lower (40% lower over the experimental period) . Thermophilic campylobacters were virtually absent from digested sludge and sludge prior to land distribution . Survival experiments confirm that campylobacters survive for only a few hours in both sterile and unsterile digested and undigested sludge . These results suggest that it is safe to dispose of Lancaster's digested sludge on land but there is still uncertainty about the ability of campylobacters to survive in sludge in the viable but non-culturable form. Biol Chem Hoppe Seyler, 1990 Aug, 371(8), 655 - 62 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; Zulli F et al.; 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 point-mutants of the two LDHs . The amino acids at positions 207, 209B, and 209C could be identified to confer the property of activation by Fru-P2 to the enzymes . This part of the enzyme is to a large extent also responsible for the different thermostabilities of these two proteins. Biochim Biophys Acta, 1990 Aug 1, 1040(1), 112 - 8 Purification and characterization of bisulfite reductase (desulfofuscidin) from Desulfovibrio thermophilus and its complexes with exogenous ligands; Fauque G et al.; A dissimilatory bisulfite reductase has been purified from a thermophilic sulfate-reducing bacterium Desulfovibrio thermophilus (DSM 1276) and studied by EPR and optical spectroscopic techniques . The visible spectrum of the purified bisulfite reductase exhibits absorption maxima at 578.5, 392.5 and 281 nm with a weak band around 700 nm . Photoreduction of the native enzyme causes a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm . When reduced, the enzyme reacts with cyanide, sulfite, sulfide and carbon monoxide to give stable complexes . The EPR spectrum of the native D . thermophilus bisulfite reductase shows the presence of a high-spin ferric signal with g values at 7.26, 4.78 and 1.92 . Upon photoreduction the high-spin ferric heme signal disappeared and a typical 'g = 1.94' signal of {4Fe-4S} type cluster appeared . Chemical analyses show that the enzyme contains four sirohemes and eight {4Fe-4S} centers per mol of protein . The molecular mass determined by gel filtration was found to be 175 kDa . On SDS-gel electrophoresis the enzyme presents a main band of 44 to 48 kDa . These results suggest that the bisulfite reductase contains probably one siroheme and two {4Fe-4S} centers per monomer . The dissimilatory bisulfite reductase from D . thermophilus presents some homologous properties with desulfofuscidin, the bisulfite reductase isolated from Thermodesulfobacterium commune (Hatchikian, E.C . and Zeikus, J.G . (1983) J . Bacteriol . 153, 1211-1220). J Bacteriol, 1990 Aug, 172(8), 4329 - 38 Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli; Zwickl P et al.; The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity . This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus . The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C . The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P . woesei was deduced from the nucleotide sequence of the coding gene . Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P . woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues . The coding gene of P . woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme. J Muscle Res Cell Motil, 1990 Aug, 11(4), 344 - 50 Self-interaction of dynein from Tetrahymena cilia; Wells C et al.; The molecular mass (Mr) and enzymic activity of the larger dynein species from Tetrahymena thermophila has been studied in the high (600 mM) to low (40 mM) ionic strength range . The apparent Mr is found to vary with both ionic strength (by sedimentation velocity and quasi elastic light scattering analysis) and with protein concentration at low ionic strength (by sedimentation equilibrium analysis) . These data indicate a strong self-interaction, resulting in dimer formation under low salt conditions . There is no evidence for the formation of species of higher than dimeric mass . A molecular mass for the dynein monomer of 1.64 x 10(6) daltons has been determined, a value rather lower than previous published estimates . The ATPase activity of dynein increases with increasing ionic strength . The possible relationship between this effect and the self-association phenomenon is discussed. Int J Pept Protein Res, 1990 Aug, 36(2), 201 - 7 Heat stable proteinase from Thermomonospora fusca . Characterization as a serine proteinase; Kristjansson MM et al.; An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone . This conclusion is further supported by the effect of various proteinase inhibitors on its activity . The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids . The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus. J Gen Microbiol, 1990 Aug, 136 ( Pt 8), 1537 - 41 Purification and properties of a thermostable fumarate hydratase from the archaeobacterium Sulfolobus solfataricus; Puchegger S et al.; Fumarate hydratase (EC 4.2.1.2) from the extremely thermophilic archaeobacterium Solfolobus solfataricus has been purified to homogeneity by a rapid purification procedure using affinity chromatography and high-performance size-exclusion chromatography, and the enzyme's physical and biochemical properties have been determined . The native enzyme has a molecular mass of 170 kDa and is composed of identical subunits with a molecular mass of 45 kDa, thus indicating a tetrameric structure similar to fumarases isolated from other organisms . The enzyme was active at temperatures ranging from 40 degrees C to 90 degrees C, with a maximum activity at 85 degrees C . The pH optimum for generation of fumarate was found to be pH 8.0 . The enzyme showed high stability to denaturation by heat and organic solvents. J Bacteriol, 1990 Aug, 172(8), 4672 - 81 Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1; Sayre MH et al.; 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, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage . We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis . Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed . These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus. Chem Phys Lipids, 1990 Aug, 55(2), 85 - 96 Monopolar-bipolar lipid interactions in model membrane systems; Mirghani Z et al.; 1H-NMR, dynamic light scattering and negative staining electron microscopy have been used to study the formation and physico-chemical properties of aqueous dispersions of mixtures of monopolar lipids extracted from Sulfolobus solfataricus . This microorganism is a thermophilic archaeobacterium growing optimally at about 85 degrees C and pH 3 . The two hydrolytic fractions of the membrane complex lipids that have been studied are: the symmetric lipid glycerol dialkyl glycerol tetraether (GDGT) and the asymmetric lipid glycerol dialkyl nonitol tetraether (GDNT) . Electron micrographs of pure and mixed GDNT and GDGT dispersions show the formation of complex structures . Only above a critical monopolar/bipolar lipid ratio, typical of the bipolar lipid, could closed structures be formed and good agreement was obtained in sizing with NMR, electron microscopy and dynamic light scattering . NMR spectra have been carried out at several temperatures from 25 degrees to 85 degrees C, to obtain information on the temperature-dependent structural, dynamic and permeability properties of the co-dispersed vesicles . The results are discussed in terms of the steric constraints and the chemico-physical interactions occurring among the different parts of the molecules and compared with previous studies performed with different physical techniques. Appl Microbiol Biotechnol, 1990 Aug, 33(5), 494 - 500 Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable alpha-amylases and pullulanases; Klingeberg M et al.; For the production of cell-free thermostable alpha-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads . The entrapment of bacteria was performed in full as well as in hollow spheres . An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60 degrees C using 0.4% starch as substrate . Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed . An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres . In the case of C . thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10(12) cells up to 700 U/10(12) cells . Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium . Proteins that had a molecular mass of less than 450,000 daltons could easily diffuse through the gel matrix . Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. Agric Biol Chem, 1990 Aug, 54(8), 2115 - 9 Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase as a source of angiotensin-converting enzyme inhibitors; Kohama Y et al.; 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 B . stearothermophilus ACE inhibitors BG-1, was the GAPDH peptide 68-77 (Gly-Lys-Glu-Ile-Ile-Val-Lys-Ala-Glu-Arg, IC50: 32 microM) . Another inhibitor, BG-2 (Gly-Lys-Met-Val-Lys-Val-Val-Ser-Trp-Tyr, IC50: 6 microM), correspond to GAPDH peptide 304-313 . These sequences were quite different from those of vertebrate GAPDH peptides and the venom peptide family with ACE inhibitory activity . BG-2 was found to be a non-competitive type inhibitor, differing from many natural peptide inhibitors . Thus, B . stearothermophilus GAPDH seemed to be a good source of new type ACE inhibitors, in addition to the advantages due to its thermophilic property. Agric Biol Chem, 1990 Aug, 54(8), 2069 - 76 Thermostable S-alkylcysteine alpha, beta-lyase from a thermophile: purification and properties; Kamitani H et al.; S-Alkylcysteine alpha, beta-lyase was found in a thermophile, Bacillus sp . 41A, which was newly isolated from soil, and purified to homogeneity from the cell extract . The enzyme has a molecular weight of about 76,000, and is composed of two subunits identical in molecular weight (39,000) . The enzyme requires pyridoxal 5'-phosphate as a coenzyme, and catalyzes alpha, beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, L-cystine, Se-methyl-L-selenocysteine, and O-methyl-DL-serine . However, S-methyl-D-cysteine, D-cystine, L-methionine, and L-norleucine were inert . The enzyme also catalyzes the beta-replacement reaction of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines . In addition to S-methyl-L-cysteine, Se-methyl-L-selenocysteine and O-methyl-DL-serine also serve as beta-substituent acceptors in the beta-replacement reaction . The enzyme is most active at 70 degrees C and stable at high temperatures . Automated Edman degradation provided the N-terminal sequence of the first 44 amino acids . The amino acid sequence in the vicinity of the lysyl residue to which pyridoxal 5'-phosphate is bound, was -Lys-His-Gln-Arg- by Edman degradation of the pyridoxyl peptide obtained by digestion with trypsin after reduction with sodium borohydride. Eur J Biochem, 1990 Jul 31, 191(2), 467 - 72 Isolation and physical properties of the DNA-directed RNA polymerase from Thermus thermophilus HB8; Wnendt S et al.; The DNA-directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method . The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for beta and beta', 40 kDa for alpha and 92 kDa for sigma . The RNA polymerase is active at elevated temperatures (65 degrees C) . Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity . The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T . thermophilus templates whose in vivo starts of transcription were characterized by nuclease S1 mapping. Biochem Biophys Res Commun, 1990 Jul 16, 170(1), 368 - 74 Xylan degradation by the thermophile Clostridium stercorarium: cloning and expression of xylanase, beta-D-xylosidase, and alpha-L-arabinofuranosidase genes in Escherichia coli; Schwarz WH et al.; Seven genes related to arabinoxylan degradation were isolated from a genomic library of the thermophilic bacterium Clostridium stercorarium . The cloned genes include a xylanase gene (xynA), two beta-D-xylosidase genes (bx1A and bx1B), two alpha-L-arabinofuranosidase genes (arfA and arfB), and two genes (celW and celX) encoding enzymes termed celloxylanases, which hydrolyze both xylans and beta-D-cellobiosides . The genes xynA, celX, and bxlB were found to encode the major xylanolytic enzyme activities induced by growth of C . stercorarium on xylan. Eur J Biochem, 1990 Jul 5, 190(3), 517 - 21 Purification and characterization of a DNA polymerase from the archaebacterium Thermoplasma acidophilum; Hamal A et al.; A thermophilic DNA polymerase has been purified to near homogeneity from the archaebacterium Thermoplasma acidophilum . Analysis of the purified enzyme by sodium dodecyl sulfate gel electrophoresis revealed a single polypeptide of 88 kDa which co-sediments with the DNA polymerase activity on sucrose gradients . Combination of sedimentation and gel filtration analyses indicates that this DNA polymerase is an 88-kDa monomeric enzyme in its native form . The DNA polymerase is resistant to aphidicolin, slightly sensitive to 2',3'-dideoxyribosylthymine triphosphate and inhibited by N-ethylmaleimide when preincubation with this reagent is performed at 65 degrees C . We find that a 3'----5' exonuclease activity is associated with the purified DNA polymerase; the two activities of the enzyme are optimal at 65 degrees C but the exonuclease activity is active in a broader range of lower temperatures and is more thermostable than the DNA polymerase activity. Gene, 1990 Jul 2, 91(1), 19 - 25 Cloning and sequencing the gene encoding 3-phosphoglycerate kinase from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus; Fabry S et al.; The nucleotide sequences of the gene (pgk) encoding 3-phosphoglycerate kinase (PGK) from the mesophilic archaebacterium, Methanobacterium bryantii, and from the closely related thermophile, Methanothermus fervidus, were determined . The deduced amino acid (aa) sequences show 61% identity with each other and 32-36% identity with the enzyme homologues from eubacteria and eukaryotes . As found for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-malate dehydrogenase, the relatedness between the archaebacterial aa sequences on the one hand and the eubacterial or eukaryotic sequences on the other is lower than that between the latter ones . Comparison of the aa sequence of PGK from mesophilic and thermophilic archaebacteria indicates an increase of the overall hydrophobicity and a decrease of the chain flexibility in the thermophilic enzyme, as already deduced from respective comparisons between GAPDH aa sequences of the same organisms . In addition, glycine residues are strikingly discriminated in the thermophilic PGK, which was also observed for GAPDH . Contrary to GAPDH, however, Lys and Arg residues are preferred in the thermophilic PGK . Lys to Arg substitutions are the most frequent cold-to-hot changes in PGK, whereas in GAPDH from the same organisms these changes do not occur. Plasmid, 1990 Jul, 24(1), 45 - 56 Plasmid-associated aggregation in Thermus thermophilus HB8; Mather MW et al.; Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth . Strain HB8 also contains two cryptic plasmids . We isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation . An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA . The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth . Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome . This is the first report of a phenotype associated with a plasmid from a Thermus strain. J Protozool, 1990 Jul-Aug, 37(4), 273 - 7 Regulation of ornithine decarboxylase activity in the growth cycle of Tetrahymena thermophila; Eichler W; Cells of the ciliated protozoan Tetrahymena thermophila, grown in proteose peptone medium up to late logarithmic phase, harvested by centrifugation, and resuspended in fresh medium to almost the same cell density, underwent one more division cycle within 5 h after inoculation, thereafter being definitely in full stationary phase . This growth cycle proved to be a useful tool to investigate the activation and deactivation of ornithine decarboxylase ODC1 in Tetrahymena: In late logarithmic phase the cells contained a very low specific activity of ODC of about 3 nmol CO2.h-1.mg-1 in the soluble protein fraction . After growth stimulation the activity was increased up to 100-fold within 1 h . This high activity was maintained for about 5 h-about as long as division activity-then rapidly declined with a half life time (t1/2) of about 15 min to the original low level . Inhibition assays with cycloheximide and actinomycin D revealed that: i . the rapid increase of ODC activity was biphasic with one component of translation of preexisting mRNA and one component of translation of newly transcribed mRNA; ii . the t1/2 of the mRNA of ODC was estimated to be about 2 h; iii . inhibition of protein biosynthesis before ODC inactivation at 5 h caused a decrease of ODC with a t1/2 of 55 min instead of 15 min . These findings suggest that ODC activity in Tetrahymena is regulated on both levels: transcription and translation and by an inactivating protein factor which is regulated at the level of biosynthesis. J Protozool, 1990 Jul-Aug, 37(4), 259 - 62 Conjugation involving dividing cells of Tetrahymena thermophila; Bliley MA et al.; Feulgen-stained preparations of mixtures of starved Tetrahymena thermophila cells of complementary mating types have revealed an atypical form of conjugation involving cells which have completed the nuclear events of cell division, but have not undergone cytokinesis . Both micronuclei in the dividing cells are induced to undergo meiosis, but in 21 of 23 cases, the anterior micronucleus was activated 1st, suggesting that the meiotic inducer is synthesized near the mating junction and diffuses posteriad . Despite the induction of two micronuclei, "triad" conjugants appear to regulate nuclear events so as to produce a normal outcome. Plasmid, 1990 Jul, 24(1), 1 - 11 Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8; Ashby MK et al.; The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented . IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication . There are at least 6 copies of IS1000 in the genome of T . thermophilus HB8 . A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica. Appl Environ Microbiol, 1990 Jul, 56(7), 2251 - 4 Cloning of alpha- and beta-galactosidase genes from an extreme thermophile, Thermus strain T2, and their expression in Thermus thermophilus HB27; Koyama Y et al.; The genes encoding thermostable alpha- and beta-galactosidases from an extremely thermophilic bacterium, Thermus strain T2, were cloned in Escherichia coli . The alpha-galactosidase gene was located just downstream from the beta-galactosidase gene . The genes were introduced into Thermus thermophilus HB27 with the aid of Thermus cryptic plasmid pTT8, and beta-galactosidases were expressed constitutively. Protein Seq Data Anal, 1990 Jul, 3(3), 257 - 62 N-terminal amino acid sequence analysis of small subunits of photosystem I reaction center complex from a thermophilic cyanobacterium, Synechococcus elongatus Nägeli; Enami I et al.; Four small subunits (14, 13, 10, and 8 kDa) of the photosystem I reaction center complex were isolated from a thermophilic cyanobacterium Synechococcus elongatus and their N-terminal amino acid sequences determined . Sequence analysis of the 10-kDa subunit revealed that the distribution of cysteine residues, Cys-X-X-Cys-X-X-Cys-X-X-X-Cys-Pro, is characteristic of bacterial-type ferredoxins, and that its partial sequence is highly homologous to that deduced from the chloroplast gene frx A of liverwort . This indicates that the 10-kDa polypeptide is an apoprotein carrying two iron-sulfur centers, FA and FB, assigned as {4Fe-4S} clusters, which mediated the light-activated transfer of electrons from P700 in photosystem I reaction center complex to soluble ferredoxin . The amino acid sequence of the 14-kDa polypeptide also showed similarity to that of the 20-kDa polypeptide from spinach chloroplast that can be chemically crosslinked with soluble ferredoxin . Thus, the 14-kDa polypeptide appears to be the ferredoxin 'docking' protein. Rev Argent Microbiol, 1990 Jul-Sep, 22(3), 137 - 41 Thermophilic Streptomyces found in sugarcane environments in Jujuy, Argentina; Carrilo L; The thermophilic Streptomyces strains isolated from: a) airborne dust of bagasse storing area, b) dry sugarcane residues and c) stored sugar manufacture wastes are described . The strains grown at maximal rates at 50 degrees C have a gray shade after 48 h . All the mature spores retain the malachite green stain . They were identified with Williams' clusters: S . chromofuscus, S . cyaneus, S . microflavus, S . antibioticus, S . halstedii and S . violaceusniger . The last three clusters coincide with the species found by other workers in bagasse. Appl Environ Microbiol, 1990 Jul, 56(7), 2200 - 5 Thermal ecology of Naegleria fowleri from a power plant cooling reservoir; Huizinga HW et al.; The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis . N . fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined . In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp . before and after thermal additions from a nuclear power plant . Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp . and pathogenic N . fowleri . Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis . N . fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir . The probability of isolating thermophilic Naegleria and pathogenic N . fowleri increased significantly with temperature . Repetitive DNA restriction fragment profiles of the N . fowleri Clinton Lake isolates and a known N . fowleri strain of human origin were homogeneous. J Bacteriol, 1990 Jul, 172(7), 4037 - 47 Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase; Poolman B et al.; The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined . Both genes are located immediately upstream of the S . thermophilus lac operon . To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning . The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans . The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity . In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively . Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E . coli cells containing plasmids encoding galM and galE . Expression of galE in E . coli was increased 300-fold when the gene was placed downstream of the tac promoter . The gene order for the gal-lac gene cluster of S . thermophilus is galE-galM-lacS-lacZ . The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis . Analysis of mRNA levels for the gal and lac genes in S . thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose . The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S . thermophilus cells matched the mRNA levels. Appl Microbiol Biotechnol, 1990 Jul, 33(4), 469 - 72 Effects of lipids on thermophilic anaerobic digestion and reduction of lipid inhibition upon addition of bentonite; Angelidaki I et al.; The effect of bentonite-bound oil on thermophilic anaerobic digestion of cattle manure was investigated . In digestor experiments, addition of oil was found to be inhibitory during start-up and the inhibitory effect was less pronounced when the oil was added in the form of bentonite-bound oil compared to when the oil was added alone . After adaptation of the digestors, very rapid degradation of oil was observed and more than 80% of the oil was degraded within a few hours after daily feeding . In batch experiments, glyceride trioleate was found to be inhibitory to thermophilic anaerobic digestion when the concentrations were higher than 2.0 g/l . However, addition of bentonite (a clay mineral) at concentrations of 0.15% and 0.45% was found to partly overcome this inhibition . Addition of calcium chloride in concentration of 3 mM (0.033% w/v) showed a similar positive effect on the utilization of oil, but the effect was lower than with bentonite. Appl Microbiol Biotechnol, 1990 Jul, 33(4), 367 - 71 Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis; Johri BN et al.; Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture . Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%-100% . The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium . The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads . Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium . A minimum of 8 h regeneration period was necessary for lipase synthesis . Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts . Tween 80 enhanced lipase activity of the immobilized protoplasts . Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion . Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity. Cell, 1990 Jun 29, 61(7), 1237 - 46 A programmed site-specific DNA rearrangement in Tetrahymena thermophila requires flanking polypurine tracts; Godiska R et al.; During macronuclear development in ciliates, precise deletion events eliminate thousands of specific DNA segments . Each segment is bounded by a unique pair of short direct repeats, but no other common feature has been reported . To determine the critical cis-acting sequences, we developed an in vivo system for analyzing this process in Tetrahymena . We show that sequences essential for recognition and excision of one such region are located within the 70 bp of DNA flanking either side of it . Three authentic splice sites and one cryptic site are each adjacent to an unusual polypurine tract (5'-A5G5) situated 40-50 bp distal to each terminal repeat . Removal of this tract or substitution of 3 bp within it abolishes splicing to the adjacent site . The normal chromosomal environment and the integrity of the eliminated sequence are not required for its removal . We believe the polypurine tract is a signal essential for excision of this sequence. J Mol Biol, 1990 Jun 20, 213(4), 631 - 2 Crystals of seryl-tRNA synthetase from Thermus thermophilus . Preliminary crystallographic data; Garber MB et al.; Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol . The crystals are very stable and diffract to at least 2 A . The crystals are monoclinic (space group P21) with cell parameters a = 87.1 A, b = 126.9 A, c = 63.5 A and beta = 109.7 degrees. FEMS Microbiol Lett, 1990 Jun 15, 58(1), 7 - 14 Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B; Nicholls DJ et al.; A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined . Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs . A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T . aquaticus B . Expression of the T . aquaticus B mdh gene in E . coli was found to be at a relatively low level . A simple method for purification of thermostable MDH from the E . coli clone containing the T . aquaticus B mdh gene is presented. Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 414 - 21 Cytochrome oxidase from thermophilic bacterium PS3 contains a fourth protein subunit; Gai WZ et al.; Monoclonal antibodies prepared against subunits II and IV of beef heart cytochrome oxidase were found to cross-react with thermophilic bacterial PS3 oxidase . Each individual antibody affects the enzymatic activity . "Western" blot analyses showed that subunit II antibodies of beef heart recognized subunit II of PS3 and subunit IV antibody likewise recognized a fourth protein subunit on slab gels . This fourth subunit previously thought to be a contaminant or a degradation product has a molecular weight of about 10,500 on SDS-gels, and appears to exist in stoichiometric amount . We have extracted this subunit from slab gels and compared its amino acid composition with that of subunit III. Appl Environ Microbiol, 1990 Jun, 56(6), 1981 - 3 Stability of antibiotics under growth conditions for thermophilic anaerobes; Peteranderl R et al.; It was shown that the inhibitory effect of kanamycin and streptomycin in a growing culture of Clostridium thermohydrosulfuricum JW 102 is of limited duration . To screen a large number of antibiotics, their stability during incubation under the growth conditions of thermophilic clostridia was determined at 72 and 50 degrees C by using a 0.2% yeast extract-amended prereduced mineral medium with a pH of 7.3 or 5.0 . Half-lives were determined in a modified MIC test with Escherichia coli, Staphylococcus aureus, and Bacillus megaterium as indicator strains . All compounds tested were similar at the two temperatures or more stable at 50 than at 72 degrees C . The half-life (t1/2) at pH 7.3 and 72 degrees C ranged from 3.3 h (k = 7.26 day-1, where k {degradation constant} = 1/t1/2) for ampicillin to no detectable loss of activity for kanamycin, neomycin, and other antibiotics . Apparently some compounds (e.g., lasalocid and neomycin) became more potent during incubation (k greater than 0) . A change to pH 5.0 caused some compounds to become more labile (e.g., kanamycin) and others (e.g., streptomycin) to become more stable than at pH 7.3. Appl Environ Microbiol, 1990 Jun, 56(6), 1631 - 5 Effect of growth rate and hydrophobicity on bacteria surviving protozoan grazing; Gurijala KR et al.; Measurements were made of the predation by Tetrahymena thermophila on several bacterial species in media containing heat-killed Escherichia coli cells to serve as an alternative prey . If grazing pressure was initially not intense on a mixture of bacterial species, the species that survived protozoan feeding at greater densities were those that grew quickly before the onset of active predation . If members of several species were incubated individually at similar initial densities with actively grazing T . thermophila, some species survived at ca . 10(4)/ml, some survived at ca . 10(2)/ml, and others were eliminated . Members of the first two groups but not the third group were able to multiply in the medium in the absence of the protozoan, but the growth rates in the protozoan-free medium did not correlate with the number of survivors . However, the species that persisted at the higher densities possessed highly hydrophobic cell surfaces . The size of the surviving population of four bacterial species whose growth was prevented by chloramphenicol correlated with the initial cell density that was incubated with T . thermophila . It is concluded that the individual species surviving predation on a mixture of species is related to the capacity of the bacterium to grow, the hydrophobicity of its cell surface, and the population density of the species before the onset of intense grazing. J Clin Microbiol, 1990 Jun, 28(6), 1284 - 7 DNA probe culture confirmation assay for identification of thermophilic Campylobacter species; Tenover FC et al.; We studied the ability of a new DNA probe-based assay system to correctly identify isolates of the thermophilic campylobacters Campylobacter jejuni, C . coli, and C . laridis grown in vitro . We examined 424 organisms, including 214 Campylobacter isolates and 210 other aerobic and anaerobic isolates . The probe assay, which uses a new homogeneous system in which all reactions take place within a single tube, demonstrated 100% accuracy, producing neither false-positive nor false-negative results . The assay does not, however, distinguish among C . jejuni, C . coli, and C . laridis. Mol Cell Biol, 1990 Jun, 10(6), 2960 - 5 Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron; Suh ER et al.; It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R . W . Davies, R . B . Waring, J . Ray, T . A . Brown, and C . Scazzocchio, Nature {London} 300:719-724, 1982) . We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila . Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site . Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site . These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron . These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against . Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site. J Appl Bacteriol, 1990 Jun, 68(6), 593 - 9 Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples; Korhonen LK et al.; Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics . Fifty-two strains of campylobacters were isolated from total of 1668 cultures . The various broth/medium combinations did not affect the dominance of C . jejuni over C . coli (total 49 C . jejuni and three C . coli) . The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium . Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants . Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate . Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants . Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants . However, these membranes retain campylobacters and must be cultured to avoid underestimation . From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium. J Biomol Struct Dyn, 1990 Jun, 7(6), 1269 - 77 Secondary structures of Tetrahymena thermophila rRNA IVS sequence involved in its self-splicing reactions: a new computer analysis; Benedetti G et al.; The secondary structures of Tetrahymena thermophila rRNA IVS sequence involved in the self-splicing reactions, are theoretically investigated with a refined computer method previously proposed, able to select a set of the deepest free energy RNA secondary structures under constraints of model hypotheses and experimental evidences . The secondary structures obtained are characterized by the close proximity of self-reactions sites and account for double mutations experiments, and differential digestion data. J Bacteriol, 1990 Jun, 172(6), 3490 - 5 Cloning and sequence analysis of tryptophan synthetase genes of an extreme thermophile, Thermus thermophilus HB27: plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T . thermophilus cells; Koyama Y et al.; Tryptophan synthetase genes (trpBA) of the extreme thermophile Thermus thermophilus HB27 were cloned by a novel method of direct plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T . thermophilus HB27 trpB cells . The nucleotide sequences of the trpBA genes were determined . The amino acid sequences deduced from the nucleotide sequences of Thermus trpB and trpA were found to have identities of 54.8 and 28.7%, respectively, with those of E . coli trpB and trpA genes . Low cysteine content (one in trpB; zero in trpA) is a striking feature of these proteins, which may contribute to their thermostability. J Mol Evol, 1990 Jun, 30(6), 514 - 21 Comparison of primary and secondary 26S rRNA structures in two Tetrahymena species: evidence for a strong evolutionary and structural constraint in expansion segments; Engberg J et al.; We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for two Tetrahymena species, T . thermophila and T . pyriformis . The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria . The majority of the nucleotide changes between the two Tetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments . These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes . The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality . However, our data show that a considerable selective constraint has operated to preserve the secondary structure of these segments . Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance . Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions. Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4576 - 9 Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya; Woese CR et al.; Molecular structures and sequences are generally more revealing of evolutionary relationships than are classical phenotypes (particularly so among microorganisms) . Consequently, the basis for the definition of taxa has progressively shifted from the organismal to the cellular to the molecular level . Molecular comparisons show that life on this planet divides into three primary groupings, commonly known as the eubacteria, the archaebacteria, and the eukaryotes . The three are very dissimilar, the differences that separate them being of a more profound nature than the differences that separate typical kingdoms, such as animals and plants . Unfortunately, neither of the conventionally accepted views of the natural relationships among living systems--i.e., the five-kingdom taxonomy or the eukaryote-prokaryote dichotomy--reflects this primary tripartite division of the living world . To remedy this situation we propose that a formal system of organisms be established in which above the level of kingdom there exists a new taxon called a "domain." Life on this planet would then be seen as comprising three domains, the Bacteria, the Archaea, and the Eucarya, each containing two or more kingdoms . (The Eucarya, for example, contain Animalia, Plantae, Fungi, and a number of others yet to be defined) . Although taxonomic structure within the Bacteria and Eucarya is not treated herein, Archaea is formally subdivided into the two kingdoms Euryarchaeota (encompassing the methanogens and their phenotypically diverse relatives) and Crenarchaeota (comprising the relatively tight clustering of extremely thermophilic archaebacteria, whose general phenotype appears to resemble most the ancestral phenotype of the Archaea. Enzyme Microb Technol, 1990 Jun, 12(6), 464 - 8 Production of cellulolytic enzymes by immobilized Sporotrichum thermophile; Singh A et al.; Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes . Immobilized mycelium was considerably less effective than free cells for cellulase productivity . Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium . Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency. Gene, 1990 May 31, 90(1), 51 - 9 Genes encoding the 7S RNA and tRNA(Ser) are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus; Haas ES et al.; Analysis of gene structure in the extremely thermophilic archaebacterium, Methanothermus fervidus, has revealed the presence of a cluster of stable RNA-encoding genes arranged 5'-7S RNA-tRNA(Ser)-16S rRNA-tRNA(Ala)-23S rRNA-5S rRNA . The genome of M . fervidus contains two rRNA operons but only one operon has the closely linked 7S RNA-encoding gene . The sequences upstream from the two rRNA operons are identical for 206 bp but diverge at the 3' base of the tRNA(Ser) gene . The secondary structures predicted for the M . fervidus 7S, 16S rRNA, tRNA(Ala) and tRNA(Ser) have been compared with those of functionally homologous molecules from moderately thermophilic and mesophilic archaebacteria . A consensus secondary structure for archaebacterial 7S RNAs has been developed which incorporates bases and structural features also conserved in eukaryotic signal-recognition-particle RNAs and eubacterial 4.5S RNAs. Nucleic Acids Res, 1990 May 25, 18(10), 2953 - 60 A conjugation-specific gene (cnjC) from Tetrahymena encodes a protein homologous to yeast RNA polymerase subunits (RPB3, RPC40) and similar to a portion of the prokaryotic RNA polymerase alpha subunit (rpoA); Martindale DW; The cnjC gene from the protozoan Tetrahymena thermophila was completely sequenced . The deduced gene product was found to have significant sequence similarity to the yeast and prokaryotic RNA polymerase subunits involved with subunit assembly . Since cnjC is active only during the sexual stage (conjugation) of Tetrahymena's life cycle, these results indicate it may be part of a novel type of transcriptional control . The yeast proteins to which the Tetrahymena cnjC is homologous are the 40 kd protein of RNA polymerases I and III (coded for by gene RPC40) and the third-largest subunit of RNA polymerase II (coded for by gene RPB3) . The degree of similarity of the cnjC protein to the two yeast subunits was found to be greater than the similarity of the two yeast subunits to each other . The alpha subunit of the core RNA polymerase from prokaryotes (coded for by gene rpoA) was found to have regions of similarity to the cnjC protein as well as to the subunits encoded by RPC40 and RPB3 . Regions of high conservation among the four proteins are noted . The significance of these results is discussed. Nucleic Acids Res, 1990 May 25, 18(10), 3035 - 44 Prediction of RNA secondary structure, including pseudoknotting, by computer simulation; Abrahams JP et al.; A computer program is presented which determines the secondary structure of linear RNA molecules by simulating a hypothetical process of folding . This process implies the concept of 'nucleation centres', regions in RNA which locally trigger the folding . During the simulation, the RNA is allowed to fold into pseudoknotted structures, unlike all other programs predicting RNA secondary structure . The simulation uses published, experimentally determined free energy values for nearest neighbour base pair stackings and loop regions, except for new extrapolated values for loops larger than seven nucleotides . The free energy value for a loop arising from pseudoknot formation is set to a single, estimated value of 4.2 kcal/mole . Especially in the case of long RNA sequences, our program appears superior to other secondary structure predicting programs described so far, as tests on tRNAs, the LSU intron of Tetrahymena thermophila and a number of plant viral RNAs show . In addition, pseudoknotted structures are often predicted successfully . The program is written in mainframe APL and is adapted to run on IBM compatible PCs, Atari ST and Macintosh personal computers . On an 8 MHz 8088 standard PC without coprocessor, using STSC APL, it folds a sequence of 700 nucleotides in one and a half hour. Biochim Biophys Acta, 1990 May 24, 1049(1), 27 - 32 cDNA cloning and expression of a Talaromyces emersonii beta-glucosidase determinant in Escherichia coli; Morrison J et al.; Intact mRNA has been isolated from the thermophilic fungus Talaromyces emersonii following growth on lactose-containing media and this has been used as template to produce cDNA . This cDNA has been cloned into the Escherichia coli expression system, pUC18 and this DNA was used to transform E . coli . A 2.1 kb fragment was isolated and shown to encode functional beta-glucosidase activity in E . coli . When the fragment was sub-cloned into a leaky strain of E . coli K-12, beta-glucosidase activity was detected in culture supernatants . The fragment was characterised further using restriction enzyme analysis and was also used as a probe in Northern and Southern blotting analyses of T . emersonii mRNA and genomic DNA, respectively . Results obtained from these analyses verified that the cloned insert DNA was of T . emersonii origin. J Mol Biol, 1990 May 20, 213(2), 289 - 302 Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits; Ito Y et al.; Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli . alpha and beta subunits deuterated to the level of 90% were obtained by culturing E . coli in 2H2O medium . Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering . The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation . The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively . In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex . The beta-beta distance is about 53 A . Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits . The centers of the alpha and beta subunits lie in the same plane, forming a hexagon . Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution. Nucleic Acids Res, 1990 May 11, 18(9), 2801 - 5 Archaebacterial reverse gyrase cleavage-site specificity is similar to that of eubacterial DNA topoisomerases I; Kovalsky OI et al.; ATP-dependent type I topoisomerases from extremely thermophilic archaebacteria--reverse gyrases--drive positive supercoiling of DNA . We showed that reverse gyrase from Desulfurococcus amylolyticus breaks the DNA at specific sites and covalently binds to the 5' end . In 30 out of 31 sites located in pBR322 DNA fragments, cleavage occurs at the sequence 5'---CNNN/---(N is any base) . The same rule was previously shown to hold for single-stranded DNA breakage by eubacterial topoisomerases I . The relative cleavage frequencies at different sites depend on Mg2+ and temperature . We discuss the possible physiological and mechanistic role of the above specificity of the bacterial topoisomerases I. Bioorg Khim, 1990 May, 16(5), 617 - 24 {Stepwise synthesis of oligonucleotides . XXXV . Native and immobilized polynucleotide phosphorylase from Thermus thermophilus in oligoribonucleotide synthesis}; Sedel'nikova EA et al.; A polynucleotide phosphorylase was isolated from the Thermus thermophilus protein fractions, obtained at different steps of purification of elongation factors, and immobilized on agarose activated with cyanogen bromide and macroporous glass modified with (3,3-diethoxypropyl)triethoxysilane . The preparations of the native and immobilized enzyme catalyzed rather efficiently the addition of adenylyl and guanylyl residues to oligonucleotide primers, in contrast to the E . coli and M . luteus polynucleotide phosphorylases . Tri-, tetra- and pentanucleotides with 3'-terminal guanosine and adenosine were obtained including structural analogues of the anticodon fragment 34-37 of yeast tRNA(Phe). FEMS Microbiol Lett, 1990 May, 57(1-2), 145 - 52 Production of novel pullulanases at high concentrations by two newly isolated thermophilic clostridia; Klingeberg M et al.; Two thermophilic bacteria, which are capable of growing on starch at 60-70 degrees C under anaerobic conditions, were isolated from a sugar refinery in Uelzen and from Solar lake in Israel . On the basis of their physiological characteristics they were identified as Clostridium thermohydrosulfuricum Uel 1 and C . thermohydrosulfuricum Sol 1, respectively . The product pattern of glucose polymer hydrolysis showed that both strains secreted enzymes that possess amylolytic and pullulytic activities . The major product formed was maltose . In addition, alpha-glucosidase activity could be detected in the supernatants of Uel 1 strain . Compared to most anaerobes investigated these isolates secreted extremely high concentrations of pullulanases in batch culture . Up to 85% of the total enzyme synthesized was detected in the culture fluid . Unlike the pullulanases of type I, which can only attack the alpha-1,6-glycosidic linkages, the pullulanases of both clostridial strains were also capable of hydrolyzing alpha-1,4-linkages . The enzyme system of both bacteria was found to be highly thermoactive; optimal activity was detected at pH 5.0 and 85 degrees C . Even at 95 degrees C and without the addition of metal ions still 15% to 25% of enzymatic activity was detectable. J Dairy Res, 1990 May, 57(2), 233 - 8 A survey of the microflora of raw and pasteurized milk and the sources of contamination in a milk processing plant in Addis Ababa, Ethiopia; Mahari T et al.; The microorganisms present in raw and pasteurized milk and the sources of contamination in the milk after it had arrived at the processing plant in Addis Ababa were studied . The lowest count registered for raw milk samples was 4 X 10(7) cfu/ml while the highest was 1 X 10(9) cfu/ml . Pasteurized milk had mesophilic aerobic counts of 7 X 10(5) cfu/ml as it left the pasteurizing unit, but the population increased 2- to 4-fold as a result of subsequent contamination . Of the total counts in raw milk, psychrophilic, thermoduric and thermophilic organisms made up 98.1, 1.4 and 0.5% respectively . In pasteurized milk, the amounts were 53.0, 39.5 and 7.5% respectively . Samples of milk pasteurized in the laboratory contained only 74.5% thermoduric and 25.5% thermophilic organisms . The isolates mostly belonged to the genera Bacillus, Streptococcus, Lactobacillus, Arthrobacter, Alcaligenes, Aeromonas and Pseudomonas . Cocci were more predominant than rod-shaped bacteria . Of the rod-shaped bacteria, 73% were Gram-negative . The utensils holding the raw and pasteurized milk and the plastic sheets used for bagging the pasteurized milk contributed unusually high numbers of bacteria which were either thermoduric or thermophilic . More isolates were obtained from the pasteurized than the raw milk . The keeping quality of the pasteurized milk was found to be much lower than that of the laboratory-pasteurized milk. Appl Environ Microbiol, 1990 May, 56(5), 1504 - 8 Occurrence of beta-glutamate, a novel osmolyte, in marine methanogenic bacteria; Robertson DE et al.; The unusual compound beta-aminoglutaric acid (beta-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria . We examined several methanogen species representing nine genera and found that beta-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9) . The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles . Studies using Methanococcus thermolithotrophicus showed that levels of beta-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65 degrees C), NH4+ (2 to 80 mM), Mg2+ (10 to 50 mM), or K+ (2 to 10 mM) in the medium . In contrast, soluble pools of beta-glutamate and L-alpha-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium . This dependence of beta-glutamate and L-alpha-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells. Mol Cell Biol, 1990 May, 10(5), 2070 - 80 Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila; Yu GL et al.; The macronuclear rRNA genes (rDNA) in the ciliate Tetrahymena thermophila are normally palindromic linear replicons, containing two copies of the replication origin region in inverted orientation . A circular plasmid containing a single Tetrahymena rRNA gene (one half palindrome) joined to a tandem repeat of a 1.9-kilobase (kb) rDNA segment encompassing the rDNA replication origin and known replication control elements was used to transform Tetrahymena macronuclei by microinjection . This plasmid was shown previously to have a replication advantage over the rDNA allele of the recipient cell strain (G.-L . Yu and E . H . Blackburn, Proc . Natl . Acad . Sci . USA 86:8487-8491, 1990) . During vegetative cell divisions, the circular and palindromic rDNAs were rapidly replaced by novel, successively longer linear rDNAs that eventually contained up to 30 tandem 1.9-kb repeats, resulting from homologous but unequal crossovers between the 1.9-kb repeats . We present evidence to show that increasing the number of copies of the replication control regions increases the replicative advantage of the rDNA, the first such situation for a cellular nuclear replicon in a eucaryote. Mol Biol (Mosk), 1990 May-Jun, 24(3), 788 - 94 {Express method of isolation of mammalian phenylalanine-tRNA-synthetase and preparation of monoclonal antibodies against this enzyme}; Vartanian OA et al.; A rapid and efficient procedure for isolating homogeneous beef liver phenylalanyl-tRNA synthetase (EC.6.1.1) was developed that enables to purify the enzyme 5000 fold and to achieve the activity of 8 e.a.u . per mg of protein . The molecular mass of the native enzyme was estimated to be 260 kDa, for alpha subunit - 59 kDa, and for beta - 72 kDa . Two cellular clones were derived by means of hybridization of immunised splenocytes with myeloma cells . They secrete monoclonal antibodies, designated P6 and P1 2, that bind to human placental and bovine liver phenylalanyl-tRNA synthetases but not to the same enzymes from E . coli and T . thermophilus . P6 and P1 2 antibodies do not affect the aminoacylation capacity of human or bovine phenylalanyl-tRNA synthetases . By immunoblotting, it was shown that P6 antibodies recognize the alpha subunit of the enzyme. Mol Biol (Mosk), 1990 May-Jun, 24(3), 736 - 43 {Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli}; Kozlov DG et al.; Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C . thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E . coli cells has been identified . Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined . The pul enzyme was shown to be a protein with molecular weight of approximately 60,000 . It was found that both pullulanase and glucoamylase activities resides in pullulanase . The intracellular distribution of pullulanase was studied . An E . coli strain that produces large amounts of thermostable pullulanase has been constructed. Mol Biol (Mosk), 1990 May-Jun, 24(3), 781 - 7 {Amplification of DNA sequences in Epstein-Barr virus and human immunodeficiency virus using DNA polymerase from Thermus thermophilus}; Glukhov AI et al.; Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV) . DNA-polymerase from Thermus thermophilus (the molecular mass of 80 to 86 kDa) differs in its physico-chemical properties from DNA-polymerase from the Thermus acquaticus (the molecular mass of 62 to 68 kDa) . To amplify the specific EBV DNA sequence oligonucleotide primers for the virus replicon region (oriP-region) were used . As a result of amplification, a specific 405 b.p . DNA fragment was produced . Primers for the virus Gag region were used for amplification of HIV DNA . The possibility to conduct amplification cycles under two temperature conditions was demonstrated. J Biochem (Tokyo), 1990 May, 107(5), 661 - 5 Effect of unusual polyamines on cleavage of DNA by restriction enzymes; Kirino H et al.; The effect of unusual polyamines, such as thermine, caldopentamine, caldohexamine, tris(3-aminopropyl)amine, or tetrakis(3-aminopropyl)ammonium, on the activities of various restriction endonucleases was investigated by using an Escherichia coli plasmid as a substrate, which contains a high GC content fragment from an extreme thermophile . Restriction enzymes used were SmaI, BanII, NaeI, RsaI, and TaqI . Most of the polyamines tested were inhibitory to the enzyme activities . The larger and more branched a polyamine was, the more the activities of nucleases were inhibited . The inhibition was positively correlated with the polyamine concentration . The sites protected by a polyamine were identical to those protected by other polyamines, and also identical to those which were less sensitive to the restriction enzyme in the absence of polyamines . No sequence specificity was seen among these sites. Carbohydr Res, 1990 May 1, 198(2), 313 - 21 Structure of an exocellular polysaccharide produced by Streptococcus thermophilus; Doco T et al.; Streptococcus thermophilus strains grown on skimmed milk produced a viscosifying, exocellular, and water-soluble polysaccharide which contains D-glucose, D-galactose, and N-acetyl-D-galactosamine in the ratio of 1:2:1 . Methylation analysis identified the glycosidic linkages in the tetrasaccharidic repeating-unit, and Smith degradation and nitrous deamination after N-deacetylation gave the sequence of monosaccharides in the repeating-unit . The anomeric configurations of the sugar residues were determined by oxidation of the peracetylated polysaccharide with chromium trioxide and by 1H- and 13C-n.m.r . spectroscopy . The following structure was assigned to the repeating unit of the polysaccharide,----3)-beta-D-Galp-(1----3)-{alpha-D-Galp-(1----6)}-beta- D- Glcp-(1----3)-alpha-D-GalpNAc-(1----. Appl Microbiol Biotechnol, 1990 May, 33(2), 239 - 44 Kinetic studies of acetate fermentation by Methanosarcina sp . MSTA-1; Clarens M et al.; The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp . MSTA-1 . The optimum temperature for growth ranged around 55 degrees C, and optimum pH was 6.5-7.5, giving a minimum generation time of 12.6-13.9 h (mu max = 0.050-0.055 h-1) and a maximum value of the specific acetate consumption rate (qmaxs) of 14-20 mmol/g cells per hour . Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature . Methanosarcina sp . MSTA-1 showed a low affinity for acetate substrate . Growth at 55 degrees C and at constant pH 7 resulted in a Km value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. J Biol Chem, 1990 Apr 25, 265(12), 6576 - 81 Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli; Terada I et al.; Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium . The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues) . When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted . However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction . On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction . When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction . The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor . These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E . coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order . These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus. J Biol Chem, 1990 Apr 25, 265(12), 6874 - 8 Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease; Takagi H et al.; Sites for Cys substitutions to form a disulfide bond were chosen in subtilisin E from Bacillus subtilis, a cysteine-free bacterial serine protease, based on the structure of aqualysin I of Thermus aquaticus YT-1 (a thermophilic subtilisin-type protease containing two disulfide bonds) . Cys residues were introduced at positions 61 (wild-type, Gly) and 98 (Ser) in subtilisin E by site-directed mutagenesis . The Cys-61/Cys-98 mutant subtilisin appeared to form a disulfide bond spontaneously in the expression system used and showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate . The thermodynamic characteristics of these enzymes were examined in terms of enzyme autolysis (t1/2) and thermal stability (Tm) . The half-life of the Cys-61/Cys-98 mutant was found to be 2-3 times longer than that of the wild-type enzyme . Similar results were obtained by differential scanning calorimetry . The disulfide mutant showed a Tm of 63.0 degrees C, which was 4.5 degrees C higher than that observed for the wild-type enzyme . Under reducing conditions, however, the characteristics of the mutant enzyme were found to revert to those of the wild-type enzyme . These results strongly suggest that the introduction of a disulfide bond by site-directed mutagenesis enhanced the thermostability of subtilisin E without changing the catalytic efficiency of the enzyme. FEBS Lett, 1990 Apr 24, 263(2), 361 - 4 Conformational changes of aminoacyl-tRNA and uncharged tRNA upon complex formation with polypeptide chain elongation factor Tu; Haruki M et al.; The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T . thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA) . Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region . Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized . On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA . These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP. Biochim Biophys Acta, 1990 Apr 19, 1038(2), 260 - 7 Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing; Harauz G et al.; Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of ribosomes from the thermophilic fungus Thermomyces lanuginosus into their characteristic views . Three predominant views were elucidated, called here overlap, non-overlap and top, showing reproducible detail approaching 1.8 nm resolution . The overlap and non-overlap forms of the fungal ribosomes appeared to be similar to those from the eubacterium Escherichia coli, despite differences in rRNA composition . The non-overlap projection predominated for the fungal complexes, suggesting different adsorption properties for ribosomes from the two species . Additionally, the top view has not been previously described for eubacteria . No major morphological differences could be detected between the fungal and eubacterial ribosomes at the resolution achieved in this study, suggesting a strong conservation of tertiary structure of this macromolecular complex despite the evolutionary gap between these two organisms. Biochem Biophys Res Commun, 1990 Apr 16, 168(1), 372 - 8 Aromatic rings of tyrosine residues at adenine nucleotide binding sites of the beta subunits of F1-ATPase