Thermophile

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 are not necessary for ATPase activity; Odaka M et al.; Using site-directed mutagenesis, Tyr-307, Tyr-341, or Tyr-364, supposedly located at the adenine nucleotide binding site(s) of the beta subunits of F1-ATPase from the thermophilic bacterium PS3, was replaced with Phe or Cys . The alpha 3 beta 3 complexes reconstituted from the alpha subunits and individual mutant beta subunits hydrolyzed ATP . Thus, neither the hydroxyl groups nor the aromatic rings in these positions are required for ATPase activity of F1-ATPase.

Biotechniques, 1990 Apr, 8(4), 430 - 7
A simplified biochemistry for DNA sequencing; Vizard D et al.; A simplified method of DNA sequencing by dideoxy chain termination is developed that approaches a single-step protocol . Utilizing the sequencing advantages contributed by a thermophilic polymerase and a guanine analog, stable sequencing reaction concentrates have been obtained that readily perform the entire sequencing reaction simply by adding prepared DNA to each of the four reaction concentrates required by this method . The mechanics and dynamics of these reactions have been investigated and the capacity of these reactions to withstand normal user variation is demonstrated . This study focuses on one form of this simplified method embodied in the FASTaq DNA sequencing kit.

Appl Environ Microbiol, 1990 Apr, 56(4), 971 - 6
Microbiological aspects of aerobic thermophilic treatment of swine waste; Beaudet R et al.; A thermophilic strain (D2) identified as a Bacillus sp . was isolated from an aerobic digestor of swine waste after several months of operation at 55 degrees C . Aerobic thermophilic batch treatment of swine waste inoculated with strain D2 was studied in a 4-liter fixed-bed reactor . Stabilization of the waste was achieved in less than 30 h when the original chemical oxygen demand (COD) was between 15 and 20 g/liter or in less than 48 h when the COD was around 35 g/liter . When the COD was higher than 30 g/liter, the pH of the waste reached 9.2 to 9.5 during the treatment, and periodic adjustment of the pH to 8.5 was necessary to maintain the activity of the biofilm . In this reactor, ammoniacal nitrogen was completely eliminated by desorption in less than 72 h of incubation . The different packing materials used resulted in similar rates of degradation of organic matter . The thermophilic treatment was also efficient in the 75-liter digestor, and stabilization was achieved in approximately 50 h . A bank of 22 thermophilic bacterial strains originating from different environments and adapted to the thermophilic treatment of swine waste was established . This thermophilic treatment allows, in one step, rapid stabilization of the waste, elimination of the bad smell, and complete elimination of ammonia nitrogen by stripping.

J Biochem (Tokyo), 1990 Apr, 107(4), 597 - 602
A cytochrome o-type oxidase of the thermophilic bacterium PS3 grown under air-limited conditions; Sone N et al.; A cytochrome o-type oxidase from the thermophilic bacterium PS3 grown under air-limited conditions was purified by ion-exchange chromatography in the presence of a non-ionic detergent . The enzyme was composed of three subunits (60, 30, and 16 kDa) and seemed to contain two molecules of heme b as prosthetic groups . It contained no detectable copper . The reduced enzyme showed absorption bands at 426 and 558.5 nm, and a characteristic spectral change upon binding CO . It oxidized several cytochromes c and artificial dyes such as N,N,N',N'-tetramethyl-p-phenylenediamine and phenazonium methosulfate at appreciable rates . Its Km for O2 was low (0.09 microM) . It was capable of transmembrane electron transfer, because when reconstituted into liposomes, it generated a membrane potential upon oxidation without pumping protons.

Mol Gen Mikrobiol Virusol, 1990 Apr, (4), 10 - 4
{Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells}; Molotov SV et al.; The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322 . The corresponding gene has been found to be located on the Pst1 DNA fragment . The restriction map of this 6 kb fragment has been constructed . The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis . The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria . The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length . Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.

Gen Physiol Biophys, 1990 Apr, 9(2), 189 - 202
Thermal stability of electron transport in PS II membranes and particles from the thermophilic cyanobacteria; Kaurov YuN et al.; Thermal stability of the ferricyanide (FC) and dichlorophenolindophenol (DCIP) reducing reactions was investigated in isolated membrane preparations and PS II particles with active water splitting system from the cyanobacterium Synechococcus elongatus . In a hypotonic medium, the thermostability was seen to be much higher for the DCIP than for the FC reduction reaction . After the addition of high concentrations of polyethylene glycol (Mwt = 4000) or sodium citrate to the medium, the FC reduction reaction appeared to be more temperature resistant . Data on the effects of temperature, DCMU and detergents on the electron transfer rate in PS II provide evidence suggesting that the different thermal stabilities of the two reactions are due to different physico-chemical properties of the electron donor sites to FC and DCIP . The data suggest that regions of contact between individual macromolecular complexes of the electron transport chain are the most labile sites of the photosynthetic apparatus . The role of the composition and properties of the intracellular medium on thermostability are emphasized.

Appl Environ Microbiol, 1990 Apr, 56(4), 1017 - 24
Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile "Caldocellum saccharolyticum"; Luthi E et al.; A lambda recombinant bacteriophage coding for xylanase and beta-xylosidase activity has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." Partial Sau3AI fragments of the lambda recombinant DNA were ligated into pBR322 . A recombinant plasmid with an insertion of ca . 7 kilobases of thermophilic DNA expressing both enzymatic activities was isolated . The location of the genes has been established by analyzing deletion derivatives, and the DNA sequence of 6.067 kilobases of the insert has been determined . Five open reading frames (ORFs) were found, one of which (ORF1; Mr 40,455) appears to code for a xylanase (XynA) which also acts on o-nitrophenyl-beta-D-xylopyranoside . Another, ORF5 (Mr 56,365), codes for a beta-xylosidase (XynB) . The xynA gene product shows significant homology with the xylanases from the alkalophilic Bacillus sp . strain C125 and Clostridium thermocellum.

Mol Gen Genet, 1990 Apr, 221(2), 187 - 94
Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: phylogenetic implications; Sanangelantoni AM et al.; The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli . The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes . The homology between the archaebacterial and the eubacterial enzymes is higher (42%-49%) than that found with the eukaryotic counterpart (less than 20%) . This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.

Vet Rec, 1990 Mar 31, 126(13), 305 - 6
Factors responsible for the introduction and spread of Campylobacter jejuni infection in commercial poultry production; Kazwala RR et al.; Campylobacter jejuni and related thermophilic campylobacters were not found in a hatchery or in chicks aged less than seven days . However, an increasing proportion of chicks aged two weeks and older shed campylobacters in their droppings . It was shown that a likely source of C jejuni for young chicks was the environment in the immediate vicinity of the rearing houses, and that infection could readily be introduced on the footwear and clothing of farm staff . Thermophilic campylobacters were found in the air, litter and drinking water containers in the rearing and finishing houses.

Biochem Biophys Res Commun, 1990 Mar 30, 167(3), 1341 - 7
The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: a thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction; Salhi S et al.; A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70 degrees C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.) . The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S . acidocaldarius DNA polymerase, for the same DNA target, was equivalent . The ability of S . acidocaldarius DNA polymerase to perform P.C.R . under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligible advantage over the Taq DNA polymerase.

Eur J Biochem, 1990 Mar 30, 188(3), 623 - 32
Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus; Honka E et al.; L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized . The primary structure of the protein was deducted from the coding gene . The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate . The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism . With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes . The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes . The phylogenetic meaning of this relationship is discussed.

Nature, 1990 Mar 29, 344(6265), 467 - 8
Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA; Robertson DL et al.; The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties . We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro . Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel . This allows evolution experiments to be carried out in response to artificially imposed selection constraints . We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates . It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity . We selected for variant forms of the enzyme that could best react with a DNA substrate . This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme . The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.

Nature, 1990 Mar 29, 344(6265), 405 - 9
DNA cleavage catalysed by the ribozyme from Tetrahymena; Herschlag D et al.; An RNA enzyme derived from the self-splicing intervening sequence of Tetrahymena thermophila catalyses sequence-specific cleavage of an oligodeoxyribonucleotide substrate . Compared with RNA, the DNA substrate is bound very weakly and is cleaved very slowly, revealing the importance of the RNA 2'-hydroxyl group in both the binding and chemical steps . The finding that catalysis by RNA can extend to DNA substrates indicates new possibilities for the transposition of intervening sequences and for the design of DNA cleavage agents with novel sequence specificities.

FEBS Lett, 1990 Mar 26, 262(2), 249 - 52
A fourth subunit is present in cytochrome c oxidase from the thermophilic bacterium PS3; Sone N et al.; A new putative subunit was found in cytochrome c oxidase (aa3-type) of the thermophilic bacterium PS3 . The N-terminal amino acid sequence of this approximately 12 kDa protein coincides with the deduced sequence of an open reading frame found downstream from the gene encoding subunit I of the PS3 cytochrome oxidase {(1988) J . Biochem . 103, 606-610} . This small hydrophobic protein, composed of 109 amino acid residues after the initial methionine residue has been processed, shows homology with the subunit IV (cyoD product) of cytochrome bo-type quinol oxidase of Escherichia coli.

Biochemistry, 1990 Mar 20, 29(11), 2876 - 84
Mapping the effector region in Thermus thermophilus elongation factor Tu; Peter ME et al.; Native elongation factor Tu from Thermus thermophilus is initially attacked by various endoproteases in a region spanning amino acid residues 40-70 . By comparing the hydrolysis rates of nucleotide-free and GDP-bound EF-Tu, only a small difference was observed for the tryptic cleavage at Arg-59 . Protease V-8 attacks Glu-55 only in a GDP/GTP form, whereas this enzyme exclusively hydrolyze Asn-64 in nucleotide-free EF-Tu, even when the protein had been previously cleaved at Arg-59 . Binding of GDP leads to a 42-fold decreased rate of hydrolysis by the Lys-C protease at Lys-52 . It also reduces the accessibility of Lys-275 to trypsin, reflecting a "long-range" effect from nucleotide binding domain I to domain II . Only slight differences were observed in the rate of hydrolysis at all positions in the GDP- versus the GTP-bound form . The intrinsic GTPase activity was slightly reduced in trypsin-treated EF-Tu, significantly impaired in EF-Tu cleaved at Lys-52, and completely abolished in EF-Tu cleaved at Asn-64 . No ribosome-induced GTPase activity was observed for protease-cleaved EF-Tu's . Treatment of these proteins with periodate-oxidized GDP or GTP followed by cyanoborohydride led to covalent modification of the new N-terminus located exclusively within region 52-60 . The highest reactivity was shown by the N-terminus of Glu-56 . Additionally, lysine residues in the native protein sensitive to affinity labeling {Peter, M.E., Wittmann-Liebold, B., & Sprinzl, M . (1988) Biochemistry 27, 9132-9139} lost their reactivity upon cleavage of EF-Tu in region 52-60, suggesting an altered structure of the cleaved protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Ukr Biokhim Zh, 1990 Mar-Apr, 62(2), 97 - 9
{Isolation of tyrosyl-tRNA-synthetase from Thermus thermophilus HB-27}; Iaremchuk AD et al.; A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65 . The yield of the purified enzyme was 1.6 mg per 1 kg of T . thermophilus cells . The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.

Z Naturforsch {C}, 1990 Mar-Apr, 45(3-4), 179 - 86
Antifungal antibiotics from Calcarisporium thermophilum: a new source of 15-azahomosterol derivatives; Chrisp P et al.; Two antifungal metabolites isolated from Calcarisporium thermophilum were identified as 15-azahomosterols related to the compounds previously isolated from Geotrichum flavo-brunneum . By full spectral comparison with authentic 15-aza-24-methylene-D-homocholesta-8,14-dien-3 beta-ol (A 25822 B) from G . flavo-brunneum, the Calcarisporium metabolites were characterized as the 4 alpha-methyl- and 4,4-dimethyl-analogues of A 25822 B . Several minor members of the series were also detected, and tentatively identified by MS analysis . The 15-azahomosterols exhibited good antifungal activity towards Candida parapsiliosis, though the activities were somewhat lower than that of the 4-demethyl derivative A25822 B . Calcarisporium thermophilum is the second microorganism known to synthesize these unusual 15-azahomosterol derivatives.

Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 346 - 57
{Correlation of enzyme thermostability and its surface hydrophobicity (using a modified alpha-chymotrypsin as an example)}; Melik-Nubarov NS et al.; Basing on the hypothesis that contact of hydrophobic surface clusters of proteins with water is thermodynamically disadvantageous, it is suggested to carry out the hydrophilization of protein surface by covalent modification in order to increase its thermostability . Hydrophilic fragments were introduced into the surface of alpha-chymotrypsin using acylation by anhydrides of aromatic carboxylic acids and reductive alkylation by aliphatic aldehydes . As a result of the hydrophilization the stability of the enzyme against irreversible thermoinactivation increased thousand-fold . The correlation is observed between the degree of hydrophilization of the protein surface and the increase in thermostability of modified alpha-chymotrypsin . The level of thermostability achieved by covalent modification of alpha-chymotrypsin is practically equal to thermostability of proteinases from extreme thermophiles, the most stable proteolytic enzymes currently known.

Antibiot Khimioter, 1990 Mar, 35(3), 34 - 6
{Antibiotic resistance of Campylobacter and its epidemiologic significance}; Aleksandrova NZ et al.; Sensitivity of 179 strains of thermophilic Campylobacter to 21 chemotherapeutic drugs was studied . Activity of the antibacterial agents against the pathogens was estimated by the MICs . The MIC50 and MIC90 were also determined . All the Campylobacter strains were sensitive to gentamicin, chloramphenicol, neomycin, furazolidone and furagin and resistant to cefazolin, polymyxin E, rifampicin, vancomycin and bacitracin . Differences in the attitude of the Campylobacter isolates from various sources and patients of various age groups to the chemotherapeutic drugs were detected . Possible consideration of the results of comparison of R spectra of Campylobacter strains and the levels of their resistance to antimicrobial drugs as epidemiological markers is discussed.

Biol Chem Hoppe Seyler, 1990 Mar, 371(3), 273 - 82
Regulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila . Effects of L-arginine metabolites and polyamines; Eichler W; Growth of Tetrahymena thermophila in a synthetic nutrient medium with or without the essential amino acid L-arginine was studied in the presence or absence of the arginine metabolites L-citrulline and L-ornithine and the polyamines putrescine, spermidine, and spermine . The effects of the growth conditions on the stimulations of the enzymes of the arginine metabolic and polyamine biosynthetic pathway, arginine deiminase (ADI), citrulline hydrolase (CH), ornithine decarboxylase (ODC), and ornithine-oxo-acid aminotransferase were determined . Tetrahymena cells were unable to grow in the absence of L-arginine and the amino-acid utilization was greatly impaired . None of the metabolites or polyamines was able to substitute for arginine . In the presence of arginine, Tetrahymena cultures grew well and citrulline and ornithine did not alter the growth behaviour in any way . In the presence of putrescine, the lag period was decreased from 3 h to 2 h . Spermidine and spermine acted similar to putrescine but less pronounced . The stimulation of the activity of ADI, the key enzyme of arginine degradation, was absolutely dependent upon the presence of arginine in the medium: in the absence of arginine, the low ADI activity which was present in the cells before inoculation was decreased to zero levels within 30 min . In the presence of arginine, the stimulation of ADI was not altered by citrulline and ornithine but putrescine, spermidine, and spermine decreased ADI-stimulation to half of the control values . The stimulation of CH activity in the presence of arginine was not altered by any added metabolite or polyamine . In the media without arginine, stimulation of CH was greatly reduced, in the presence of ornithine more than in its absence, and even more in the presence of putrescine and spermidine . Stimulation of ODC activity in the presence of arginine was not affected by citrulline and ornithine but in the presence of polyamines it was rapidly decreased to unstimulated levels after an initial ca . 10-fold increase . The "hyperstimulation" of ODC in the absence of free arginine was reduced to normal in the presence of citrulline, the stimulation was decreased even below normal levels in the presence of ornithine and polyamines decreased ODC activity to zero levels . O delta T activity was stimulated more in the presence of arginine than in its absence . In both cases the stimulation was enhanced in the presence of polyamines and only in the absence of arginine--by ornithine.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1990 Mar, 172(3), 1271 - 5
Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila; Jablonski PE et al.; The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis . More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells . Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells . Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila . Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum . All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.

J Diarrhoeal Dis Res, 1990 Mar-Jun, 8(1-2), 34 - 6
Prevalence of Campylobacter infections in animals and children in Haryana, India; Kakkar M et al.; To study the prevalence of infections with Campylobacter spp in Haryana, India, a stool sample was collected using a rectal swab from 30 buffaloes, 62 cattle, 95 pigs and 94 children and was bacteriologically cultured . The subjects were either apparently healthy or had diarrhoea . The organisms were isolated in a medium containing reducing agents and antibiotics, and culture plates were incubated in a candle jar at 42 degrees C . 63% of all thermophilic Campylobacter were cultured from rectal swabs taken from young livestock and children with diarrhoea . Of 32 isolates fully identified, 23 were C . jejuni, 8 were C . coli and 1 was C . laridis . The C . jejuni isolates belonged to the Lior's biotype II.

Biokhimiia, 1990 Mar, 55(3), 525 - 33
{Separation and comparative characteristics of subunits of phenylalanyl-tRNA synthetase from Escherichia coli MRE-600 and Thermus thermophilus HB8}; Bobkova EV et al.; Separation of alpha- and beta-subunits of phenylalanyl-tRNA-synthetases from E . coli MRE-600 and Thermus thermophilus HB8 using FPLC has been carried out for the first time . The separated subunits of both enzymes do not possess any detectable tRNA-amino-acylation activity . It was found that in the case of the E . coli enzyme beta-subunits exist in solution mainly in the monomeric form with negligible formation of beta 2-dimers, while alpha-subunits predominantly form alpha 2-dimers over the same concentration range . In the case of Thermus thermophilus phenylalanyl-tRNA-synthetase, both alpha- and beta-subunits mainly exist in the dimeric form . The putative mechanisms of alpha-subunit aggregation and of subunit association for alpha 2 beta 2-type enzymes are discussed.

J Biochem (Tokyo), 1990 Mar, 107(3), 331 - 8
Recognition sites of tRNA by a thermostable tRNA(guanosine-2'-)-methyltransferase from Thermus thermophilus HB27; Matsumoto T et al.; Recognition sites of tRNA by tRNA(guanosine-2'-)-methyltransferase (Gm-methylase) {EC 2.1.1.34} from an extreme thermophile, Thermus thermophilus HB27, were studied by two independent methods--fragment reactions and footprinting analyses, using yeast tRNA(Phe) and Escherichia coli tRNA(fMet) as substrates . None of the tRNA-derived oligonucleotides which have the G-G sequence but are not long enough to form the "stem-loop" structure could be methylated by Gm-methylase . The 5'-half fragments having the intact D-"stem-loop" structure served as substrates for Gm-methylase, with a similar Vmax but 6-8 times larger Km, as compared with the intact tRNAs . The results of footprinting analyses were consistent with the foregoing findings . Gm-methylase protected only the D-loop region of tRNA from RNase T1 attack, but other parts of tRNA extending from the amino acid stem to the T arm became more sensitive to RNase T1, suggesting a considerable change of tRNA tertiary structure due to complex formation with Gm-methylase . These results indicate that a D-"stem-loop" structure is a prerequisite for recognition by Gm-methylase.

Allergy Proc, 1990 Mar-Apr, 11(2), 101 - 2, discussion 97-9
Farmer's lung: thermophilic actinomycetes as a source of "farmer's lung hay" antigen . 1963; Pepys J et al.; Mouldy hay was produced in the laboratory by sterilising good hay, inoculating with aqueous suspensions of microorganisms, and incubating at 40 degrees or 60 degrees C . Extracts were tested for presence of farmer's lung hay (F.L.H.) antigen by agar-gel double-diffusion and immunoelectrophoresis tests against sixteen to twenty sera from patients with farmer's lung . F.L.H . antigen developed in hay after: (1) inoculating with mixed microbial suspensions from antigenically active hay; (2) inoculation with mixed suspensions of pure cultures of thermophilic actinomycetes, after raising the pH of the hay to 70 either by prior inoculation with fungi or by infiltration with ammonia vapour; and (3) inoculation at pH 70 with pure cultures of Thermopolyspora polyspora or with Micromonospora vulgaris . F.L.H . antigen did not develop in hay after inoculation with fungi only, or with six other actinomycetes tested, or after prior heating (though some sera reacted to fungal antigens in all these extracts) . T . polyspora is the richest source yet found of F.L.H . antigen, and inhalation of an extract by affected subjects produces some of the features of farmer's lung . Pure cultures can produce F.L.H . antigen on artificial media without hay . Spores and mycelium are rich in F.L.H . antigen, and inhalation of the spores may play a part in farmer's lung disease . Other antigens relevant to farmer's lung may be found in other actinomycetes, not yet cultured.

Med Clin North Am, 1990 Mar, 74(2), 291 - 306
Environmentally mediated disorders of the respiratory tract; Utell MJ et al.; Although much of the evidence in environmental lung disease remains equivocal, some environmental exposures are known to be clinically relevant . Ambient air pollution remains of concern as a source of morbidity, particularly for susceptible populations such as persons with asthma, chronic obstructive pulmonary disease, or cardiac disease and the elderly . The adverse effects of several components of indoor air pollution have been established . Environmental tobacco smoke contributes to lower-respiratory illness in infants; office workers exposed to thermophilic actinomycetes contaminating ventilation systems have developed hypersensitivity pneumonitis; and in the home, components of house dust and fungus spores may provoke asthma via immediate hypersensitivity . The evidence is less compelling for a link between other exposures and disorders of the respiratory tract . For example, formaldehyde may be responsible for provoking vague respiratory symptoms and even nasal cancers; however, the associations are unproved . Likewise, the relation between low-level exposure to asbestos and the development of lung cancer, although a concern, is not conclusively established . The clinician should be aware of practical measures for patients who inquire about air cleaning . Often, relatively simple solutions are effective . A knowledge of sources and exposures as well as an understanding of the principles of inhalation lung injury should prove useful in directing patient care.

J Infect, 1990 Mar, 20(2), 123 - 7
Biotypes, serogroups and antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli in Chile; Figueroa G et al.; Phenotypic markers were studied in 105 strains of thermophilic campylobacters isolated from human beings, animals and drinking water in Santiago, Chile . The strains were identified as Campylobacter jejuni (n = 49) and Campylobacter coli (n = 56) . Biotypes I and II (Lior schema) accounted for 96% C . jejuni isolates, the other 4% being biotype IV but the two biotypes of C . coli were about equally represented . A total of 28 serogroups (Lior's heat-labile antigens) were identified . Lior 13, 9, 79, 2 and 4 were prevalent among the C . jejuni, while Lior 8, 21 and 29/75 were prevalent among the C . coli isolates . These serogroups accounted for 73% all isolates . The distribution of biotypes and serogroups in patients and asymptomatic persons were similar . Human campylobacters were often resistant to ampicillin (31%) but sensitive to erythromycin and furazolidone . Swine C . coli isolates proved resistant to streptomycin (46%), tetracycline (38%) and erythromycin (15%) . Determination of phenotypic and serological characters provides valuable epidemiological markers in the study of campylobacter infections.

J Bacteriol, 1990 Mar, 172(3), 1345 - 51
Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species; Sung MH et al.; Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp . strain YM-2 . The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing . The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra . The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent . The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate . The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli . The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E . coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.

Ital J Biochem, 1990 Mar-Apr, 39(2), 83 - 99
Purification and properties of a thermophilic and thermostable DNA polymerase from the archaebacterium Sulfolobus solfataricus; Rella R et al.; A DNA-dependent DNA polymerase was obtained in homogenous form from the thermoacidophilic archaebacterium Sulfolobus solfataricus . The enzyme, purified 706-fold, has a molecular mass of about 110000 daltons as determined by gel filtration and by glycerol gradient centrifugation . It requires Mg++ for its activity and has a pH optimum of 7.7 . The activity is sharply dependent on the ionic strength . The enzyme is thermostable; its properties and activity requirements were characterized . The features of this enzyme are compared to those of other DNA polymerases isolated either from prokaryotes or eukaryotes.

Appl Environ Microbiol, 1990 Mar, 56(3), 697 - 701
Pyrite oxidation by thermophilic archaebacteria; Larsson L et al.; Three species of thermophilic archaebacteria of the genera Sulfolobus (Sulfolobus acidocaldarius and S . solfataricus) and Acidianus (Acidianus brierleyi) were tested for their ability to oxidize pyrite and to grow autotrophically on pyrite, to explore their potential for use in coal desulfurization . Only A . brierleyi was able to oxidize and grow autotrophically on pyrite . Jarosite was formed during the pyrite oxidation, resulting in the precipitation of sulfate and iron . The medium composition affected the extent of jarosite formation.

J Bacteriol, 1990 Mar, 172(3), 1478 - 84
Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp . strain B12; Trent JD et al.; The extreme thermophile Sulfolobus sp . strain B12 exhibits an acquired thermotolerance response . Thus, survival of cells from a 70 degrees C culture at the lethal temperature of 92 degrees C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88 degrees C for 60 min or longer before being exposed to the lethal temperature . In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known as heat shock proteins . In this Sulfolobus species, it correlates with the preferential synthesis of primarily one major protein (55 kilodaltons) and, to a much lesser extent, two minor proteins (28 and 35 kilodaltons) . Since the synthesis of all other proteins was radically reduced and these proteins were apparently not degraded or exported, their relative abundance within the cell increased during the time the cells were becoming thermotolerant . They could not yet be related to known heat shock proteins . In immunoassays, they were not cross-reactive with antibodies against heat shock proteins from Escherichia coli (DnaK and GroE), which are highly conserved between eubacteria and eucaryotes . However, it appears that if acquired thermotolerance depends on the synthesis of protective proteins, then in this extremely thermophilic archaebacterium it depends primarily on one protein.

Biochim Biophys Acta, 1990 Feb 26, 1033(2), 148 - 53
Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Hanner M et al.; An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus . The molecular weight of the native enzyme was about 320,000, as calculated by gel-filtration studies, and a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis . The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5 . The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA . The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+ . Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect . Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.

Biochim Biophys Acta, 1990 Feb 23, 1042(3), 338 - 43
2-(7,13-Dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis; Kaya K et al.; A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila . Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid . By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid . When cells of T . thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from {2(n)-3H}taurine and {1-14C}stearic acid, both the radioactivities were detected in taurolipid A, B and C . Furthermore, the ratio of the radioactivities of 3H and 14C in the lysotaurolipids were the same as that of the lipotaurine . From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.

Eur J Biochem, 1990 Feb 22, 188(1), 195 - 201
Lactate dehydrogenase from the extreme thermophile Thermotoga maritima; Wrba A et al.; Lactate dehydrogenase was isolated from the extreme thermophilic eubacterium Thermotoga maritima . The enzyme is stereospecific for L(+)-lactate . It represents a homotetramer of 144 kDa molecular mass, with a sedimentation coefficient of s20,w approximately 7 S . Under physiological temperature conditions, the enzyme shows high catalytic efficiency with a broad pH optimum at pH 7.0 +/- 1.0, and long-term stability up to 80 degrees C . The coenzyme, NAD+, and the effector fructose 1,6-bisphosphate {Fru(1,6)P2} increase the thermal stability: at 90 degrees C (pH 6.0), the liganded enzyme exhibits a half-life of thermal inactivation of 150 min . The enhanced rigidity of the enzyme at ambient temperature is reflected by an anomalously high stability toward guanidine denaturation: the midpoint of the equilibrium transition being 1.6 M guanidine hydrochloride . Under optimum conditions of the enzyme assay, the Michaelis constants (Km) for NADH, NAD+, pyruvate and L(+)-lactate at 55 degrees C, and in the absence of Fru(1,6)P2, are 0.03 mM, 0.09 mM, 3.7 mM and 410 mM, respectively; Fru(1,6)P2 as a positive effector shifts the Km values for pyruvate and L(+)-lactate to 0.06 mM and 25 mM, respectively . The Km values for the coenzyme are not affected . Neither Mn2+ nor other divalent cations have any activating effect . In contrast to lactate dehydrogenases from eukaryotes, the N-terminus of the enzyme from Th . maritima is not acetylated . Comparison of the 30 N-terminal amino acid residues with lactate dehydrogenase from Thermus aquaticus shows a high degree of similarity . This also holds if the two lactate dehydrogenases are compared with the glyceraldehyde-3-phosphate dehydrogenases from the same organisms.

Biochemistry, 1990 Feb 20, 29(7), 1823 - 8
13C and 15N NMR studies on the interaction between 6,7-dimethyl-8-ribityllumazine and lumazine protein; Vervoort J et al.; The interaction between the prosthetic group 6,7-dimethyl-8-(1'-D-ribityl)lumazine and the lumazine apoproteins from two marine bioluminescent bacteria, one from a relatively thermophilic species, Photobacterium leiognathi, and the other from a psychrophilic species, Photobacterium phosphoreum, was studied by 13C and 15N NMR using various selectively enriched derivatives . It is shown that the electron distribution in the protein-bound 6,7-dimethyl-8-ribityllumazine differs from that of free 6,7-dimethyl-8-ribityllumazine in buffer . The 13C and 15N chemical shifts indicate that the protein-bound 6,7-dimethyl-8-ribityllumazine is embedded in a polar environment and that the ring system is strongly polarized . It is concluded that the two carbonyl groups play an important role in the polarization of the molecule . The N(3)-H group is not accessible to bulk solvent . The N(8) atom is sp2 hybridized and has delta+ character . Nuclear Overhauser effect studies indicate that the 6,7-dimethyl-8-ribityllumazine ring is rigidly bound with no internal mobility . The NMR results indicate that the interaction between the ring system and the two apoproteins is almost the same.

J Biol Chem, 1990 Feb 15, 265(5), 2483 - 7
The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan; Yoshida M et al.; The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions . Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C . The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM {14C}Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined . Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex . Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex . This labeled peptide was shown to contain Tyr-beta 307 derivatized with {14C}Nbf-Cl by automatic amino acid sequence analyses . From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits . It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with {14C}Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.

Eur J Biochem, 1990 Feb 14, 187(3), 573 - 80
Purification and properties of an endo-1,4-xylanase excreted by a hydrolytic thermophilic anaerobe, Clostridium thermolacticum . A proposal for its action mechanism on larchwood 4-O-methylglucuronoxylan; Debeire P et al.; An extracellular xylanase from a thermophilic anaerobe, Clostridium thermolacticum, was purified 400-fold by ion-exchange chromatography and gel filtration . The purified enzyme had a specific activity of 31,670 nkat/mg of protein at 60 degrees C, a molecular mass of 39 kDa and a pI of 4.9 . The enzyme exhibited maximal activity at 80 degrees C (1 h assay) and at pH 6.0-6.5 . There was little loss of activity after 4 days at 60 degrees C and the enzyme was stable in the wide pH range 3-11 . Examination of the hydrolysis products of larchwood xylan indicated that it was an endoxylanase; at the early stage of the reaction, xylose (Xyl)-containing oligosaccharides of 3-12 residues were released and after a prolonged incubation time, the neutral end-products were Xyl2 and Xyl3 . Kinetic studies of the hydrolysis of xylose-containing oligosaccharides of 4-7 residues showed that the tetrasaccharide was hydrolysed more slowly than the pentasaccharide, while the calculated Km and V values for pentasaccharide and hexasaccharide were similar . The primary structures of the XylnGlcA produced by long-term hydrolysis of larchwood glucuronoxylan were determined on the basis of their carbohydrate composition, by methylation analysis and by 1H-NMR and 13C-NMR spectroscopies . These data allowed us to propose a model for the mode of action of this endoxylanase on larchwood 4-O-methylglucuronoxylan.

FEBS Lett, 1990 Feb 12, 261(1), 8 - 10
Sulfobacillus thermosulfidooxidans: a new lineage of bacterial evolution?
Karavajko GI, Bulygina ES, Tsaplina IA, Bogdanova TI, Chumakov KM.
The nucleotide sequence of 5 S ribosomal RNA (rRNA) of type strain Sulfobacillus thermosulfidooxidans VKM B-1269 was determined . This organism represents a group of moderately thermophilic acidophilic chemolithotrophic bacteria, able to use ferrous and sulfur compounds as the sole energy source . 5 S rRNA of this bacterium is drastically different from all other known bacterial 5 S rRNA sequences . It is suggested that S . thermosulfidooxidans represents a new lineage of bacterial evolution, that diverged from other bacteria at an early step of their evolution.

Biochim Biophys Acta, 1990 Feb 6, 1042(2), 198 - 203
Identification of pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from Tetrahymena thermophila; Kaya K et al.; A pentahydroxystearic acid-containing taurolipid (taurolipid C) was found in cells of Tetrahymena thermophila . The lipid accounted for about 1.2% of the total taurolipids of the cells . The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolyses products were identified by mass and nuclear magnetic resonance spectrometries, as taurine, non-hydroxy fatty acid and 2,3,7,12,13-pentahydroxystearic acd, a novel fatty acid . The NMR spectra of the intact and acetylated lipid showed that the carboxyl group of the pentahydrxyostearic acid was combined with the amino group of taurine, and the hydroxy group at C-3 was esterified with non-hydroxy fatty acids . From these results, the pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from T . thermophila was identified as 2-(3-acyloxy-2,7,12,13-tetrahydroxyoctadecanoylamino)ethanesulf oni c acid.

Biochemistry, 1990 Feb 6, 29(5), 1216 - 25
Molecular weight determination of an active photosystem I preparation from a thermophilic cyanobacterium, Synechococcus elongatus; Schafheutle ME et al.; An active photosystem I (PSI) complex was isolated from the thermophilic cyanobacterium Synechococcus elongatus by a procedure consisting of three steps: First, extraction of photosystem II from the thylakoids by a sulfobetaine detergent yields PSI-enriched membranes . Second, the latter are treated with Triton X-100 to extract PSI particles, which are further purified by preparative isoelectric focusing . Third, anion-exchange chromatography is used to remove contaminating phycobilisome polypeptides . The purified particles show three major bands in sodium dodecyl sulfate gel electrophoresis of apparent molecular mass of 110, 15, and 10 kDa . Charge separation was monitored by the kinetics of flash-induced absorption changes at 820 nm . A chlorophyll/P700 ratio of 60 was found . When the particles are stored at 4 degrees C, charge separation was stable for weeks . The molecular mass of the PSI particles, determined by measurement of zero-angle neutron scattering intensity, was 217,000 Da . The PSI particles thus consist of one heterodimer of the 60-80-kDa polypeptides and presumably one copy of the 15- and 10-kDa polypeptides, respectively.

J Mol Biol, 1990 Feb 5, 211(3), 633 - 44
Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 A; Duerring M et al.; The structure of the phycobiliprotein phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 2.7 A resolution by X-ray diffraction methods on the basis of the molecular model of C-phycocyanin from the same organism . Hexagonal phycoerythrocyanin crystals of space group P6(3) with cell constants a = b = 156.86 A, c = 40.39 A, alpha = beta = 90 degrees, gamma = 120 degrees are almost isomorphous to C-phycocyanin crystals . The crystal structure has been refined by energy-restrained crystallographic refinement and model building . The conventional crystallographic R-factor of the final model was 19.2% with data to 2.7 A resolution . In phycoerythrocyanin, the three (alpha beta)-subunits are arranged around a 3-fold symmetry axis, as in C-phycocyanin . The two structures are very similar . After superposition, the 162 C alpha atoms of the alpha-subunit have a mean difference of 0.71 A and the 171 C alpha atoms of the beta-subunit differ by 0.51 A . The stereochemistry of the chiral atoms in the phycobiliviolin chromophore A84 is C(31)-R, C(4)-S . The configuration of the chromophore is C(10)-Z, C(15)-Z and the conformation C(5)-anti, C(9)-syn and C(14)-anti like the phycocyanobilin chromophores in phycoerythrocyanin and C-phycocyanin.

Mol Gen Genet, 1990 Feb, 220(3), 475 - 7
Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene; Haspel G et al.; The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced . It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55,323 . Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli . The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance . The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.

Biol Chem Hoppe Seyler, 1990 Feb, 371(2), 103 - 10
Structure and function of L-lactate dehydrogenases from thermophilic, mesophilic and psychrophilic bacteria, IX . Identification, isolation and nucleotide sequence of two L-lactate dehydrogenase genes of the psychrophilic bacterium Bacillus psychrosaccharolyticus; Vckovski V et al.; Two genes encoding for L-lactate dehydrogenase (LDH) from the psychrophilic bacterium Bacillus psychrosaccharolyticus (DSM 6) were cloned and their nucleotide sequence determined using a pEMBL vector and gene hybridization probes . The deduced amino-acid sequence of the gene from clone pLDH(X), which is located on a 5.87-kb HindIII-fragment, shows an identity of 86% as compared with the sequence of the wildtype LDH(P) from B . psychrosaccharolyticus and consists of 319 amino acids . Clone pLDH(P) contained a gene on a 4-kb HindIII-EcoRI fragment, of which the amino-acid sequence is identical with the enzyme isolated from B . psychrosaccharolyticus . The nucleotide sequences of LDH(P) and LDH(X) show 77% identity . Both genes are expressed in E . coli and the proteins could be isolated as shown by enzyme activity tests and determination of the N-terminal amino-acid sequence . However no expression of LDH(X) could be detected in B . psychrosaccharolyticus itself under the conditions chosen for oxygen induction of LDH . The function of the additional, non-expressed enzyme is not known.

FEMS Microbiol Lett, 1990 Feb, 55(3), 261 - 5
Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus; Solaiman DK et al.; A type II restriction endonuclease Sth134I was isolated from Streptococcus thermophilus strain 134 . The enzyme is an isoschizomer of HpaII . The restriction endonuclease is most active at Mg(II) greater than or equal to 5 mM; in the pH range of 7.5-8; temperature of 50 degrees-55 degrees C; and (KCl) or (NaCl) below 100 mM . Double digestion and ligation experiments showed that Sth134I apparently recognized and cleaves DNA at the sequence C1CGG to produce two-base, 5'-protruding ends.

Arch Biochem Biophys, 1990 Feb 1, 276(2), 546 - 53
Purification and properties of xylanase from the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce; Anand L et al.; An extracellular xylanase was purified to homogeneity from the culture filtrate of the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce and its properties were studied . A fourfold purification and a yield of 8% were achieved . The molecular weight of the protein was found to be 22,500 based on electrophoretic mobility and 29,000 by gel filtration behavior . The protein is rich in acidic amino acids, glycine and tyrosine, and poor in sulfur-containing amino acids . The kinetic properties of the enzyme are similar to those of other fungal xylanases . The enzyme shows high affinity toward larchwood xylan (Km = 0.91 mg/ml) and hydrolyzes only xylan . The enzyme becomes inactivated when stored for more than 2 months at -20 degrees C in the dry state . Such an inactivation has not been reported so far for any xylanase . Using chromatographic techniques, one species of protein differing from the native protein in charge but enzymatically active was isolated in low yields . However, a large molecular-weight species of the protein devoid of enzyme activity was isolated in substantial quantities and further characterized . Based on ultracentrifugation and gel electrophoretic studies, it was concluded that this species may be an aggregate of the native protein and that such an aggregation might be taking place on storage in the dry state at -20 degrees C, leading to loss in activity.

J Biomol Struct Dyn, 1990 Feb, 7(4), 935 - 42
DNA substrate specificity of reverse gyrase from extremely thermophilic archaebacteria; Slesarev AI et al.; It has been shown earlier that eukaryotic type I DNA topoisomerases act on duplex DNA regions, while eubacterial type I topoisomerases require single-stranded regions . The present paper demonstrates that the type I topoisomerase from extremely thermophilic archaebacteria, reverse gyrase, winds DNA by binding to single-stranded DNA regions . Thus, type I topoisomerases, both relaxing one in eubacteria and reverse gyrase in extremely thermophilic archaebacteria share a substrate specificity to melted DNA regions . The important consequence of this specificity is that the cellular DNA superhelical stress actively controlled by bacterial topoisomerases is confined to a narrow range characterized by a low stability of the double helix . Hence we suppose that bacterial topoisomerase systems control duplex stability near its minimum, for which purpose they create an appropriate negative superhelicity at moderate temperatures or a positive one at extremely high temperatures, the feedback being ensured by the aforesaid specificity of type I bacterial topoisomerases.

J Bacteriol, 1990 Feb, 172(2), 1157 - 9
Methanogenesis involving a novel carrier of C1 compounds in Methanogenium tationis; Raemakers-Franken PC et al.; The pathway of CO2 reduction to methane in Methanogenium tationis and Methanogenium thermophilicum is similar to that observed in other methanogens . In M . tationis a novel pterin, tatiopterin, is present . This pterin appears to be a structural and functional analog of methanopterin and sarcinapterin . Folate could not substitute for tatiopterin.

Biochemistry, 1990 Jan 30, 29(4), 1009 - 15
Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)(+)-dependent dehydrogenases; Kuroda S et al.; The gene encoding alanine dehydrogenase (EC 1.4.1.1) from a mesophile, Bacillus sphaericus, was cloned, and its complete DNA sequence was determined . In addition, the same gene from a moderate thermophile, B . stearothermophilus, was analyzed in a similar manner . Large parts of the two translated amino acid sequences were confirmed by automated Edman degradation of tryptic peptide fragments . Each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residues corresponding to the subunit (Mr = 39,500-40,000) of the hexameric enzyme . The similarity of amino acid sequence between the two alanine dehydrogenases with distinct thermostabilities is very high (greater than 70%) . The nonidentical residues are clustered in a few regions with relatively short length, which may correlate with the difference in thermal stability of the enzymes . Homology search of the primary structures of both alanine dehydrogenases with those of other pyridine nucleotide-dependent oxidoreductases revealed significant sequence similarity in the regions containing the coenzyme binding domain . Interestingly, several catalytically important residues in lactate and malate dehydrogenases are conserved in the primary structure of alanine dehydrogenases at matched positions with similar mutual distances.

Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1237), 535 - 51; discussion 551-3
Protein structure and function at low temperatures; Jaenicke R; Proteins represent the major components in the living cell that provide the whole repertoire of constituents of cellular organization and metabolism . In the process of evolution, adaptation to extreme conditions mainly referred to temperature, pH and low water activity . With respect to life at low temperatures, effects on protein structure, protein stability and protein folding need consideration . The sequences and topologies of proteins from psychrophilic, mesophilic and thermophilic organisms are found to be highly homologous . Commonly, adaptive changes refer to multiple alterations of the amino acid sequence, which presently cannot be correlated with specific changes of structure and stability; so far it has not been possible to attribute specific increments in the free energy of stabilization to well-defined amino-acid exchanges in an unambiguous way . The stability of proteins is limited at high and low temperatures . Their expression and self-organization may be accomplished under conditions strongly deviating from optimum growth conditions . Molecular adaptation to extremes of temperature seems to be accompanied by a flattening of the temperature profile of the free energy of stabilization . In principle, the free energy of stabilization of proteins is small compared to the total molecular energy . As a consequence, molecular adaptation to extremes of physical conditions only requires marginal alterations of the intermolecular interactions and packing density . Careful statistical and structural analyses indicate that altering the number of ion pairs and hydrophobic interactions allows the flexibility of proteins to be adjusted so that full catalytic function is maintained at varying temperatures.

Eur J Biochem, 1990 Jan 26, 187(2), 321 - 8
Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus . Purification and properties; Pisani FM et al.; A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography . The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate . Molecular mass studies demonstrated that the S . solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits . Comparison of the amino acid composition of beta-galactosidase from S . solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme . A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E . coli . The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.

J Biol Chem, 1990 Jan 25, 265(3), 1360 - 8
Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8; Meinhardt SW et al.; Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8 . Three of these clusters (designated as {N-1H}T, {N-2H}T, and {N-3}T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively . These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I) . They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex . Two additional very low potential iron-sulfur clusters (one binuclear, {N-1L}T, and one tetranuclear, {N-2L}T) were observed in membrane particles . These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively . No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster {N-2}B was found in this bacterial system . In membrane particles isolated from T . thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials {Em9(Q/QH2) = 40, -100, -160, -300 mV} and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide . Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase . Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T . thermophilus HB-8.

Biochemistry, 1990 Jan 23, 29(3), 732 - 7
Telomere G-strand structure and function analyzed by chemical protection, base analogue substitution, and utilization by telomerase in vitro; Henderson ER et al.; Eukaryotic telomeres have a 12-16 nucleotide long deoxyguanosine (dG) rich single-stranded overhang at their molecular termini . Some of the unique features of telomeres are probably attributable to a specialized structure formed by this overhang . In the ciliated protozoan Tetrahymena thermophila, the dG-rich overhang is comprised of approximately two repeats of the sequence d(TTGGGG) . Previous work has shown that the synthetic oligonucleotide d(TTGGGG)4 can form an unusual non-Watson-Crick base-paired structure (the "G-strand structure") containing G-G base pairs and syn-guanines . We have tested the susceptibility of various dGs in this structure to methylation by DMS . At 0-10 degrees C one dG residue is hypersensitive to methylation while others are particularly resistant . By systematically substituting deoxyinosine (dI) for dG in d(TTGGGG)4 we identify N2 groups of guanine essential for formation of the G-strand structure . We show that dI-substituted molecules that cannot form the G-strand structure nonetheless function as substrates for telomere repeat addition in vitro by the telomere lengthening enzyme, telomerase . The implications of these data are discussed.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 131 - 4
Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus; Sen S et al.; Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATCC27634 . Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4 X 10(-7) per donor and 1.2 X 10(-7) per recipient . Transposon transfer from Thermus to Bacillus subtilis was also demonstrated in similar broth matings . Transfer characteristics were consistent with the conjugation mechanism described for Tn916 in mesophiles.

Yi Chuan Xue Bao, 1990, 17(1), 46 - 52
{Expression of xylE gene in Bacillus stearothermophilus}; He XS et al.; Using the gene expression vector pFDC11 for Bacillus stearothermophilus CU21, a recombinant plasmid pFDX1 was constructed, which carries the Pseudomonas-derived xylE gene coding for Catechol 2,3-dioxygenase (CatO2ase) . CatO2ase activity can be detected from CU21 (pFDX1) cells grown at 48 degrees C . This result indicates that the promoterless xylE gene can be expressed in a thermophilic host under the direction of a promoter from a thermophilic bacterium . By selecting Kmr mutants at elevated growth temperature, a segregant CU21-161 was obtained, the xylE gene expression of which at 55 degrees C and 60 degrees C was much higher than that of the parental strain . A method for CatO2ase assay with suspension of intact cells was also reported in this paper.

Biochem Cell Biol, 1990 Jan, 68(1), 274 - 83
Structures of polar lipids from the thermophilic, deep-sea archaeobacterium Methanococcus jannaschii; Ferrante G et al.; Cells of Methanococcus jannaschii, grown at 65 degrees C in a defined medium, contained 7% of lipid composed of 87% polar and 13% neutral components . Within the polar fraction 16 lipids were resolved by thin-layer chromatography, 4 of which were present in trace amounts . Staining reactions demonstrated that the more abundant lipids were glycolipids, aminophospholipids, and an aminophosphoglycolipid . Most of the polar fraction (82%) consisted of five diether lipids, which were purified and their structures were resolved largely through nuclear magnetic resonance, mass spectrometry, and optical rotation methods . Macrocyclic diethers had the head groups phosphoethanolamine-(1----6)-beta-D-glucopyranose, beta-D-glucopyranose, and beta-D-glucopyranosyl-(1----6)-beta-D-glucopyranose . Phosphoethanolamine was identified as a head group for both the noncyclized and macrocylic diether core lipids . The neutral lipids were mainly acyclic C30 isoprenoids, predominantly dihydro-, hexahydro, and octahydro-squalenes.

J Basic Microbiol, 1990, 30(2), 81 - 94
Studies on the mycoflora of Aswan High Dam Lake, Egypt: monthly variations; el-Hissy FT et al.; Fifty-one species and one variety appertaining to twenty one genera of mesophilic fungi were recovered from the monthly samples of marginal water (44 species, 1 variety and 18 genera) and submerged mud (78 species, 1 variety and 30 genera) of Aswan High Dam Lake during the period from July 1985 to December 1986 . The most common species were Aspergillus fumigatus, A . flavus, A . terreus, A . niger and Penicillium funiculosum . The highest fungal populations were almost detected either in October, in December 1985 or in February 1986 . Of the 12 thermophilic and thermotolerant fungal species, A . fumigatus and A . nidulans were the most common . Paecilomyces variotii, Rhizomucor pusillus, Thermomyces lanuginosus, Thermoascus thermophilus and Sporotrichum thermophilum were fairly common in one locality or more . The physico-chemical characteristics of water and mud samples were also followed.

Appl Environ Microbiol, 1990 Jan, 56(1), 87 - 92
Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste; Mackie RI et al.; The rate of {15N}ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste . The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day) . The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor . Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production . In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates . Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h . Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates . The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production . Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.

Mol Cell Biol, 1990 Jan, 10(1), 377 - 81
Termination region in rRNA genes from a eucaryotic thermophile, Thermomyces lanuginosus; Walker K et al.; S1 mapping of the termination region in the ribosomal DNA from a thermophilic fungus, Thermomyces lanuginosus, revealed three distinct termini corresponding to the mature 25S rRNA, a precursor that is 19 nucleotides longer and corresponds to the 37S precursor in yeast cells, and a putative termination site at +96 that bears a limited sequence homology with the SalI box of mammalian cells . An estimate of the secondary structure suggested that the three termini are in close proximity, a feature that may be essential to precursor termination and maturation . The results raise questions regarding recently reported relationships between ribosomal DNA termination and spacer enhancer elements in fungi.

Adv Biochem Eng Biotechnol, 1990, 42, 187 - 209
Biochemistry and biotechnology of amino acid dehydrogenases; Ohshima T et al.; Over the last decade, amino acid dehydrogenases such as alanine dehydrogenase (Ala DH), leucine dehydrogenase (Leu DH), and phenylalanine dehydrogenase (Phe DH) have been applied to the enantiomer-specific synthesis and analysis of various amino acids . In perticular, amino acid dehydrogenases from thermophiles have received much attention because of their high stability . Their productivity was enhanced and the purification facilitated by the gene cloning . The advances in biotechnological applications of these enzymes are based on fundamental studies concerning characteristics of the enzymes and reaction mechanism as described in this chapter . Further elucidation of the structure and function of these enzymes based on genetic engineering and protein engineering may enable their properties to be improved for their future uses in biotechnology.

J Basic Microbiol, 1990, 30(7), 467 - 79
Fungal flora associated with combine harvester wheat and sorghum dusts from Egypt; Abdel-Hafez SI et al.; 107 species and 8 species varieties belonging to 44 genera were collected from combine harvester wheat and sorghum dusts (35 genera and 91 species + 4 varieties) and from the atmosphere of their hay sites (26 genera and 69 species + 4 varieties) on glucose- and cellulose-Czapek's Dox agar at 28 degrees C and 45 degrees C . The mycoflora of wheat and sorghum dusts were basically similar on the two types of media and the most common fungi were: Alternaria alternata, Aspergillus flavus, A . fumigatus, A . niger, A . ochraceus, A . sydowii, A . terreus, Cochliobolus spicifer, Emericella nidulans, Fusarium moniliforme, Penicillium chrysogenum, P . duclauxii, P . funiculosum and P . oxalicum . Truly thermophilic species were frequently encountered from the two substrates: Chaetomium thermophilum, Humicola grisea var . thermoidae, H . insolens, Malbranchea pulchella var., sulphurea, Rhizomucor pusillus, Sporotrichum thermophilum, Talaromyces thermophilus, Thermoascus thermophilus and Thermomyces lanuginosus . The airborne fungi in the two atmospheres were basically similar and the most prevalent species were members of Alternaria (1 species), Aspergillus (18 species + 2 varieties), Chaetomium (2 species), Cochliobolus(3 species), Emericella (3 species + 2 varieties), Fusarium (3 species), Mucor (1 species), Penicillium (14 species) and Stachybotrys (1 species).

Proteins, 1990, 8(2), 173 - 8
Distinct character in hydrophobicity of amino acid compositions of mitochondrial proteins; Nakashima H et al.; A compact mitochondrial gene contains all essential information about the synthesis of mitochondrial proteins which play their roles in a small compartment of the mitochondrium . Almost no noncoding regions have been found through the gene, but a necessary set of tRNAs for the 20 amino acids is provided for biosynthesis, some of them coding different amino acids from those in a usual cell . Since the gene is so compact that the produced proteins would have some characteristic aspects for the mitochondrium, amino acid compositions of mitochondrial proteins (mt-proteins) were examined in the 20-dimensional composition space . The results show that compositions of proteins translated from the mitochondrial genes have a distinct character having more hydrophobic content than others, which is illustrated by a clustered distribution in the multidimensional composition space . The cluster is located at the tail edge of the global distribution pattern of a Gaussian shape for other various kinds of proteins in the space . The mt-proteins are rich in hydrophobic amino acids as is a membrane protein, but are different from other membrane proteins in a lesser content of Val . A good correlation found between the base and amino acid compositions for the mitochondria was examined in comparison to those of organisms such as thermophilic bacterium having an extreme G-C-rich base composition.

Prog Clin Biol Res, 1990, 344, 139 - 58
Molecular structures and evolution of mouse isozyme genes functioning in the malate-aspartate shuttle; Shimada K et al.; To examine molecular mechanisms of transcription of mammalian isozyme genes functioning in the malate-aspartate shuttle and to observe structural and evolutionary relationships, we investigated gene organizations of cAspAT and mAspAT, and cMDH and mMDH, and isolated and characterized cDNAs and genomic DNAs for these isozymes in mice . The deduced amino acid sequences of mouse cAspAT and mAspAT showed about 47%, and those of mouse cMDH and mMDH, about 23% overall homology . Surprisingly, the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and E . coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice . The first duplication of a common ancestral MDH gene should thus have occurred long before the emergence of the eukaryotic cells, and subsequently, the mammalian mMDH and E . coli MDH genes have evolved from one of the duplicates . The mammalian cMDH and Thermus flavus MDH genes have no doubt evolved from one of the other duplicates . Moreover, structural organizations of the two-pairs of isozyme genes indicated that introns antedate the divergence of these mitochondrial and cytosolic isozyme genes . The 5' ends of all four isozyme genes lacked the TATA and CAAT boxes characteristic of eukaryotic promoters but did contain G + C-rich sequences and multiple transcription-initiation sites . We found several highly conserved regions in the 5' flanking sequences between mAspAT and cAspAT, between mMDH and mAspAT, and between cMDH and cAspAT genes.

J Basic Microbiol, 1990, 30(3), 165 - 80
Some physiological studies on fungi isolated from poultry feedstuffs; Megalla SE et al.; A total of 506 isolates of mesophilic, thermophilic and thermotolerant fungi isolated from the poultry feed ingredients included soybean meals, ground maize, cottonseed cake, wheat bran and fish meal, on glucose-Czapek's agar, Littman oxgall agar at 28 degrees C and yeast starch agar (YPSs) at 45 degrees C, were screened for their ability to produce hydrolytic protease enzyme on solid media . Most of the fungal isolates were able to produce such enzymes but with variable capabilities . The highest proteolytic activity was exhibited by some isolates of Penicillium chrysogenum, Aspergillus flavus, Thermoascus thermophilus and Rhizopus chizopodifarmis . Of all fungal isolates screened for proteolytic activity, Penicillium chrysogenum and Thermoascus thermophilus produced the highest amounts of proteases . These two isolates were used to study the effect of some environmental and nutritional factors on their proteolytic activity . It was found that the highest yield of protease by P . chrysogenum (12.5 units) was achieved 3 days after incubation at 30 degrees C . Marked reduction in protease activity was observed at 37 degrees C . The thermophilic fungus T . thermophillus exhibited maximum (18 units) proteolytic activity 6 days after incubation at 45 degrees C . The enzyme yield was reduced to 13 units at 50 degrees C . Among the seven carbon sources tested, sucrose was the most appropriate for maximum protease production by both P . chrysogenum and T . thermophilus (13.2 and 12.8 units, respectively) . Of the sixteen nitrogen sources investigated, NaNO3 was the best inorganic additive nitrogenous salt which induced the highest proteolytic activity by P . chrysogenum and T . thermophilus, whereas DL-tryptophan was the most preferable organic nitrogen compound for maximum protease production by the two fungi tested.

Biochem Int, 1990, 20(5), 1001 - 9
Comparative study of the phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophlus by the tritium topography method; Bobkova EV et al.; A comparative study of thermostability and aminoacid composition of the phenylalanyl-tRNA synthetases from E . coli and Thermus thermophilus HB8 has been carried out . Compared with the mesophilic enzyme, a considerable increase of Pro, Leu, Phe, Arg and decrease of Asx, Ile, Ser, Thr and Lys content have been revealed in the thermophilic protein . Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of both the enzymes . In the E . coli enzyme, Thr residues were also easy to access while on the surface of the thermophilic enzyme there were more Arg residues . The quantitative assay of the surface compositions revealed the increased exposure of the (Leu + Ile) residues on the thermophilic protein as well as of the charged Asx and Arg residues . A possible correlation of the observed effects with thermostability is discussed.

Mol Gen Mikrobiol Virusol, 1990 Jan, (1), 18 - 22
{Testing of G----A mutation in position 110 of a minor intron of beta-globin genes in patients with thalassemia in Azerbaijan}; Fedorov AN et al.; A point mutation G-A in the 110 position of the beta-globin gene small intron has been revealed by cloning and sequencing from the material of a homozygote beta-thalassemia patient in Azerbaijan . In the present study two allele-specific oligonucleotide probes for testing the mutation have been synthesized . Assessment frequency of the mutation among the beta-thalassemia patients in Azerbaijan has been performed with the use of the amplified beta-globin gene fragments obtained by using the thermostable DNA-polymerase from T . thermophilus with the subsequent dot-hybridization in gel of the amplified material with the oligonucleotide probes . The possibility to test the mutation by hybridization of the oligonucleotide probes with the donors and beta-thalassemia patients restricted genomic DNA has been analyzed . Only one of 50 thalassemia alleles of beta-globin genes under study has been shown to possess the mutation mentioned.

Antonie Van Leeuwenhoek, 1990 Jan, 57(1), 1 - 7
Elemental metals as electron sources for biological methane formation from CO2; Belay N et al.; Several elemental metals were examined as potential electron donors for methanogenic bacteria, using both a single tube system where the metal was in direct contact with the cells, and a two-flask system, where metal and cells were not in direct contact, but had contact via the gas phase . With all organisms examined in the direct contact system, Fe degree, Al degree and Zn degree served as electron donors for methanogenesis; some organisms used Ni degree or Sn degree as low-level electron donors . Of the metals tested, methanogenesis from H2 + CO2 was inhibited by direct contact with Zn degree or Cu degree, but not by Fe degree or Al degree . Ni degree and Co degree were inhibitory to some methanogens, with Ni degree being particularly inhibitory to the thermophilic strains tested . With all organisms examined in the two-flask system, Fe degree and Zn degree served as good electron sources for both methanogenesis and growth; Co degree generated a very low level of methane and Cu degree did not work at all . In either system V degree, Ti degree or Cd degree did not serve as electron donors . The results suggest that some elemental metals (notably Fe degree, Al degree and Zn degree) produce gaseous H2 by cathodic depolarization which is then consumed by the methanogen, thus accelerating oxidation of the metal by its metabolic activity . All of these reactions are thermodynamically favorable; however, some other metals that are clearly favorable for such a reaction on thermodynamic grounds (Ti degree and V degree) are very stable and do not serve as electron donors.

Anal Biochem, 1990 Jan, 184(1), 63 - 6
A paper membrane filter assay for ciliate chemoattraction; Leick V et al.; A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms . A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (approximately 10(5) cells/ml) is placed in one compartment and a solution containing attractant in the other . The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting . The assay described is convenient with a signal-to-noise ratio of approximately 10 . It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.

J Biol Chem, 1989 Dec 25, 264(36), 21837 - 41
The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase; Yokoyama K et al.; Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions . Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y . (1977) J . Biol . Chem . 252, 3480-3485) . Here, the properties of purified subunit complexes were compared in detail with those of native TF1 . The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1 . In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different . ATPase activity of the alpha 3 beta 3 delta complex is cold labile . The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex . Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM . Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively . ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed . The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex . The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.

Eur J Biochem, 1989 Dec 22, 186(3), 695 - 700
13C-NMR study of acetate assimilation in Thermoproteus neutrophilus; Schafer S et al.; Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns . Acetate was assimilated via reductive carboxylation of acetyl-CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate . 2-Oxoglutarate was mainly formed via citrate . However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2-oxoglutarate and 5% pyruvate through the reductive citric acid cycle . A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed . The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2-aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.

Biochemistry, 1989 Dec 12, 28(25), 9574 - 8
Thermostability of cytochrome c-552 from the thermophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus; Sanbongi Y et al.; The denaturation of the c-type cytochrome of the thermophilic bacterium Hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra . The melting temperature (T1/2) of cytochrome c-552 in the presence of 1.5 M guanidine hydrochloride was 34 degrees C higher than that of the c-type cytochrome of Pseudomonas aeruginosa cytochrome c-551 . Hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrome c-551 of the mesophilic bacterium P . aeruginosa, even though their amino acid sequences are 56% identical and they have numerous other similarities . However, notwithstanding these similarities between the sequences of the cytochromes c-552 and c-551 that were compared, it is very likely that these differences in stability could be due to some heretofore undefined differences in their spatial structures . It has been suggested that alpha-helix structure and electrostatic interaction could be the source of the stable spatial structure of cytochrome c-552.

Biochim Biophys Acta, 1989 Dec 8, 993(2-3), 266 - 74
Purification and characterization of two cellobiohydrolases from Chaetomium thermophile var . coprophile; Ganju RK et al.; Cellobiohydrolases I and II were purified to homogeneity from culture filtrates of a thermophilic fungus, Chaetomium thermophile var . coprophile, by using a combination of ion-exchange and gel filtration chromatographic procedures . The molecular weights of cellobiohydrolase I and II were estimated to be 60,000 and 40,000 and the enzymes were found to be glycoproteins containing 17 and 22.8% carbohydrate, respectively . The two forms differed in their amino-acid composition mainly with respect to threonine, alanine, methionine and arginine . Antibodies produced against either form of cellobiohydrolases failed to cross-react with the other . The tryptic maps of the two enzymes were found to be different . The temperature optima for cellobiohydrolase I and II were 75 and 70 degrees C, and they were optimally active at pH 5.8 and 6.4, respectively . Both enzymes were stable at higher temperatures and were able to degrade crystalline cellulosic materials.

Biochim Biophys Acta, 1989 Dec 7, 977(3), 329 - 34
Cytochrome c-551 from the thermophilic bacterium PS3 grown under air-limited conditions; Sone N et al.; A small-sized c-type cytochrome, designated cytochrome c-551, was prepared from membrane fraction of the thermophilic bacterium PS3 grown under air-limited conditions by extraction with cholate, precipitation with polyethylene glycol, and successive chromatographies with DEAE-cellulose and Sephacryl S-200 in the presence of a detergent . The purified sample contained approximately 1 mol of heme c per 10,000 g protein; it showed absorption bands at 551, 522 and 416 nm upon reduction, and a Soret peak at 409 nm upon oxidation . This cytochrome showed a single band of 10 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulfate . The isoelectric point of this cytochrome c-551 was pH 4.0 . Cytochrome c-551 was suggested to play an important role in the respiratory chain with a terminal oxidase cytochrome o, which is produced under air-limited conditions, since cytochrome c-551 could mediate electron transfer between cytochrome bc1(b6f) complex and cytochrome o, showing quinol oxidase activity.

J Biochem (Tokyo), 1989 Dec, 106(6), 958 - 60
Hexagonal structure of two-dimensional crystals of the alpha 3 beta 3 complex thermophilic ATP synthase; Yoshimura H et al.; The highly dissociable alpha 3 beta 3 subunit complex (Mr = 319,582) of thermophilic ATP synthase was crystallized on a mercury surface under oxygen . The two-dimensional crystal was compared with that of TF1 (Mr = 385,351, alpha 3 beta 3 gamma delta epsilon subunit complex) by means of computer image processing . The crystals showed the same hexagonal lattice (a = b = 10 nm), despite the difference in their molecular weights . The color images of the two protein molecules were also hexagonal . However, there was an open hole in the image of the alpha 3 beta 3 complex, where small subunits (gamma, delta, and epsilon) of TF1 may have been located . The structure of this heterohexamer is consistent with that deduced from other physical parameters.

Int J Parasitol, 1989 Dec, 19(8), 823 - 34
A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis; Adams M et al.; The present study employs allozyme electrophoresis to characterize and inter-relate 61 isolates of Naegleria . Diploidy was confirmed, with heterozygotes observed at 29 of the 33 loci established and in all but two isolates . With a single exception, isolates clustered at two levels of similarity, either below 21% or above 52% . It is argued that such a major discontinuity provides a sound biological basis for a species concept in Naegleria . On this basis the present species-level taxonomy does not reflect the genetic diversity of the genus . The study recognized 18 genetic groups of species rank . The subspecies N . australiensis italica deserves specific rank; additional thermophilic species not closely related to N . fowleri and N . lovaniensis are recognized; and N . gruberi as currently conceived is a complex of 10 species, at least five of which are represented in the formal culture collections . Most species are genetically too different for relationships to be elucidated by allozyme electrophoresis, supporting the view that some of the times of divergence within the genus are extremely ancient.

Environ Res, 1989 Dec, 50(2), 289 - 95
Pathogenic amoebae in natural thermal waters of three resorts of Hidalgo, Mexico; Rivera F et al.; In a search for free-living amoebae, seven water samples from three thermal water bathing resorts in Tecozautla, Hidalgo, were analyzed during December 1984 . The samples were concentrated by filtration and centrifugation, and inoculated later on monoxenic and axenic media . The identification of the isolates was performed by morphology and isoelectric focusing of isoenzymes and total proteins . Thirty-three strains of free-living amoebae belonging to the genera Naegleria, Acanthamoeba, and Willaertia were isolated . Twenty of these strains belonged to the Naegleria genus, 16 of them were classified as Naegleria spp., and 2 were classified as Naegleria lovaniensis . Noteworthy was the finding of two pathogenic strains of the species Naegleria australiensis . N . australiensis and N . lovaniensis may be considered good indicator organisms, since they live in the same environmental conditions as N . fowleri, the agent of primary amoebic encephalitis (PAM) . On the other hand, amoebae other than Naegleria were isolated and identified as Acathamoeba castellanii (two strains), and Acanthamoeba lugdunensis (one strain), which proved to be pathogenic when tested in mice . Nine more pathogenic strains of the genus Acanthamoeba spp . were isolated together with one strain of Willaertia magna, a thermophilic nonpathogenic amoeba . The chlorination and periodical surveillance of water resorts like the one studied is recommended, in order to prevent the appearance of more cases of PAM or other human diseases associated with pathogenic Acanthamoeba spp.

J Bacteriol, 1989 Dec, 171(12), 6720 - 5
Chromosome map of the thermophilic archaebacterium Thermococcus celer; Noll KM; A physical map for the chromosome of the thermophilic archaebacterium Thermococcus celer Vu13 has been constructed . Thirty-four restriction endonucleases were tested for their ability to generate large restriction fragments from the chromosome of T . celer . Of these, the enzymes NheI, SpeI, and XbaI yielded the fewest fragments when analyzed by pulsed-field electrophoresis . NheI and SpeI each gave 5 fragments, while XbaI gave 12 . The size of the T . celer chromosome was determined from the sum of the apparent sizes of restriction fragments derived from single and double digests by using these enzymes and was found to be 1,890 +/- 27 kilobase pairs . Partial and complete digests allowed the order of all but three small (less than 15 kilobase pairs) fragments to be deduced . These three fragments were assigned positions by using hybridization probes derived from these restriction fragments . The positions of the other fragments were confirmed by using hybridization probes derived in the same manner . The positions of the 5S, 16S, and 23S rRNA genes as well as the 7S RNA gene were located on this map by using cloned portions of these genes as hybridization probes . The 5S rRNA gene was localized 48 to 196 kilobases from the 5' end of the 16S gene . The 7S RNA gene was localized 190 to 504 kilobases from the 3' end of the 23S gene . These analyses demonstrated that the chromosome of T . celer is a single, circular DNA molecule . This is the first such demonstration of the structure of an archaebacterial chromosome.

J Bacteriol, 1989 Dec, 171(12), 6710 - 9
Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains; Grogan DW; Though amenable to routine manipulation and a popular subject of molecular genetic and biochemical studies on archaebacteria, the genus Sulfolobus has remained poorly described in phenotypic terms . To delineate their physiological capabilities and diversity, five laboratory strains, including type strains of the described species Sulfolobus acidocaldarius and S . solfataricus, were compared with respect to a variety of growth and biochemical parameters, including component profile of the surface-layer cell wall, inhibitors of growth, growth rate as a function of temperature and pH, and compounds used as sole sources of carbon or nitrogen . Motility and photoregulated production of an orange pigment were detected in all five strains tested . The results provide new criteria for distinguishing Sulfolobus strains as well as potential tools for the physiological and genetic manipulation of these extreme thermophiles.

Appl Environ Microbiol, 1989 Dec, 55(12), 3208 - 13
Expression of the insecticidal protein gene from Bacillus thuringiensis subsp . aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile; Nakamura K et al.; Expression of the insecticidal protein gene from Bacillus thuringiensis subsp . aizawai IPL7 in B . subtilis MI113 and B . stearothermophilus SIC1 was examined . Production of the protein (130 kilodaltons {KDa}) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B . thuringiensis . When the original gene containing its own promoter was subcloned in B . subtilis, only a small amount of the protein was produced . Therefore, both the promoter for the B . stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy . B . subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C . In contrast, B . stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.

Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9218 - 22
Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme; Murphy FL et al.; A shortened form of the intervening sequence of the self-splicing RNA from Tetrahymena thermophila catalyzes sequence-specific cleavage of RNA . Cleavage site selection involves a base-pairing interaction between the substrate RNA and a binding site within the intervening sequence . Single-base changes in this binding site were previously shown to alter substrate specificity in a predictable manner . To examine the generality with which substrate specificity can be altered, six variant catalytic RNAs (ribozymes) have been produced with two- or three-base changes in the active site . Each ribozyme cleaves its predicted substrate . The conditions required for good reactivity and for discrimination against cleavage at mismatched sites vary and were independently determined for each ribozyme.

FEBS Lett, 1989 Nov 6, 257(2), 219 - 22
Identification of the N-tosyl-L-phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu; Peter ME et al.; EF-Tu from Thermus thermophilus was first labelled with N-{14C}tosyl-L-phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin . The resulting peptides were separated by reversed-phase HPLC . Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76-88 in T . thermophilus EF-Tu . The TPCK reactive site is at Cys-82 . Kinetic measurements of the incorporation of TPCK into native EF-Tu and EF-Tu nicked at position Arg-59 were performed . The results provide evidence that the cleavage of the peptide bond between Arg-59 and Gly-60 does not lead to a dramatic conformational change of EF-Tu at the aa-tRNA binding site.

J Biol Chem, 1989 Nov 5, 264(31), 18392 - 6
Activation of acetate by Methanosarcina thermophila . Purification and characterization of phosphotransacetylase; Lundie LL Jr et al.; Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate . This rate was 10-fold greater than the rate of acetyl phosphate synthesis . The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane . Activity was optimum at pH 7.0, and 35-45 degrees C . The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures . Phosphate and sulfate partially protected against heat inactivation . Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M . thermophila approximated 175 mM . Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity . Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8487 - 91
Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector; Yu GL et al.; A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T . thermophila by microinjection . Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously . However, in one transformant, the circular plasmid initially outreplicated the endogenous rDNA and was the major rDNA form for up to 65 generations . Stable circular replicons have not been reported previously in Tetrahymena . A single point mutation (+G) was identified in the repeated promoter of the plasmid maintained in this transformant . After recovery from the Tetrahymena transformant and recloning in Escherichia coli, the mutated circular plasmid again transformed Tetrahymena with stable maintenance of the circular rDNA plasmid . Transformants containing circular replicons were also obtained by using a similar plasmid from which the repeated promoter, but not the repeated replication origin, had been removed by BAL-31 deletion . We therefore propose that repeated rRNA promoters are deleterious in vivo in Tetrahymena, which normally lacks them . Transformants were obtained in 2-5 days compared with the 7-14 days required for transformation with unmutated rDNA plasmids by recombination . Similar results were obtained when a 550-base-pair segment containing the telomerase RNA gene of T . thermophila was inserted in the polylinker of the plasmid . We suggest that this plasmid is a useful vector system for transformation of Tetrahymena.

Arch Biochem Biophys, 1989 Nov 1, 274(2), 511 - 7
Purification and characterization of an alpha-D-glucuronidase from a thermophilic fungus, Thermoascus aurantiacus; Khandke KM et al.; An alpha-D-glucuronidase was purified from the culture filtrates of Thermoascus aurantiacus . A simple colorimetric method for its assay is reported . The enzyme is a single polypeptide chain with a molecular weight of 118,000 . It acts optimally at pH 4.5 . It shows maximum activity at 65 degrees C . The t 1/2 at 70 degrees C was 40 min . It specifically cleaved the alpha-(1----2) linkage between 4-O-methyl-alpha-D-glucuronic acid and the xylose residue in xylan and several glucurono-xylooligosaccharides.

FEMS Microbiol Lett, 1989 Nov, 53(1-2), 183 - 6
Antigenic conservation of the ureases of spiral- and helical-shaped bacteria colonising the stomachs of man and animals; Newell DG et al.; A monoclonal antibody, CP11, has been produced which is directed against the ureas of Campylobacter pylori . This antibody has been used to look for antigenic cross-reactivity, in other ureolytic and non-ureolytic campylobacters, by immunohistological techniques . It has also been used to investigate the helical-shaped organisms found in the stomach of the human, monkey and cat (CS1) and the ileum of the rat (ST1) . Interestingly the antibody cross-reacted with the gastric helical organisms from the human, monkey and cat but not with the rat helical organism . No cross-reactivity was observed with C . mustelae or the other ureolytic campylobacters, C . nitrofigilis and the urease positive thermophilic campylobacters . These results are discussed in relation to the phylotaxonomy of these organisms.

J Protozool, 1989 Nov-Dec, 36(6), 562 - 7
A thermostable acid alpha-glucosidase from Tetrahymena thermophila: purification and characterization; Banno Y et al.; An acid alpha-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399 . Its general molecular, catalytic and immunological properties were compared to those of the T . pyriformis W enzyme . The enzyme from T . thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T . pyriformis enzyme . The purified enzyme was most active at 56 degrees C and showed resistance to thermal inactivation . The acid alpha-glucosidase appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity . The Km values determined with p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively . The enzyme was antigenically distinct from T . pyriformis acid alpha-glucosidase.

J Bacteriol, 1989 Nov, 171(11), 6307 - 15
S-layer protein gene of Acetogenium kivui: cloning and expression in Escherichia coli and determination of the nucleotide sequence; Peters J et al.; Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure . The outer surface is covered with a hexagonally packed surface (S) layer . The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector . It was expressed under control of a cloned Acetogenium promoter or the lacZ gene . We determined the complete sequence of the structural gene . The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids . It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues . An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A . kivui polypeptide.

J Protozool, 1989 Nov-Dec, 36(6), 582 - 96
Oral assembly in left-handed Tetrahymena thermophila; Nelsen EM et al.; We have investigated oral development in a non-genetically derived left-handed (LH) form of Tetrahymena thermophila, in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal . Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells . In most of these OAs, membranelles are assembled from the cells' anterior to posterior . Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs . Many of these membranelles re-orient to a normal orientation near the end of oral development . Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules . In some cases, the entire OA develops and remains as a 180 degrees rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama . We present a model for these complex developmental outcomes . These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells . The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.

J Protozool, 1989 Nov-Dec, 36(6), 577 - 82
Properties of purified L-ornithine decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila; Eichler W; Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing . The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO2.h-1.mg-1 . The purified enzyme was rather stable at 37 degrees C (14% loss of activity within 1 h) . The molecular and catalytic properties of this enzyme were investigated . The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions . The pH optimum was between 6.0 and 7.0, the Km for the substrate L-ornithine was 0.11 mM, and the Km for the cofactor pyridoxal 5-phosphate was 0.12 microM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated Ki of about 10 mM . The enzyme was inhibited competitively by D-ornithine with a Ki of 1.6 mM and by alpha-difluoromethylornithine with a Ki of 0.15 mM . The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T . thermophila at high concentrations with a half life time of 14 min . Other basic amino acids, e.g . L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989 Nov, 22(4), 249 - 60
Effects of sonication and growth temperature on the cytochrome oxidase activity of Bacillus species; Liu JK et al.; The cytochrome oxidase activity of Bacillus strains was analyzed quantitatively by colorimetric N, N, N', N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase assay . Twenty six type strains were readily grouped, the oxidase positive, the oxidase negative, and the oxidase indeterminate groups . TMPD-dependent oxidase activities for whole cells and sonicated cells were compared . Spectral absorbance analyses of membrane fractions showed that all 26 Bacillus type strains contained the o-type cytochrome oxidase; 19 strains contained cytochrome a + a3 and among which 5 strains contained cytochromed . All of the 10 thermophilic Bacillus strains, which were predominantly oxidase positive, exhibited maximum oxidase activity when grown at optimum temperature (65 degrees C) . The other three "caldo-active" thermophiles exhibited maximum oxidase activities when grown at their optimum temperatures, 70 degrees C to 80 degrees C . Mesophilic B . cereus and B . subtilis exhibited maximum oxidase activity in cells grown at 42 degrees C and 55 degrees C, respectively . In no case did growth temperature induce oxidase negative strains to exhibit an oxidase positive reaction, and vice versa.

Biochimie, 1989 Nov-Dec, 71(11-12), 1185 - 91
Structural analysis of prokaryotic and eukaryotic 5S rRNAs by RNase H; Lorenz S et al.; The availabilities of single-stranded 5S rRNA regions c, d and d' for base pairing interactions were analyzed by using synthetic DNA oligomers . Hybrid formation was detected by the endonucleolytical mode of the RNA-DNA specific action of RNase H . Provided that the hybrid interaction involved 6 successive base pairs, 5S rRNA loop c nucleotides 42-47 displayed accessibility in Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus 5S rRNAs as well as in eukaryotic 5S rRNAs from Saccharomyces carlsbergensis, Rattus rattus and Equisetum arvense . Investigating eubacterial 5S rRNA regions d and d' (nucleotides 71-76 and 99-105, respectively), susceptibility was observed in E . coli 5S rRNA which, however, decreases in B . stearothermophilus and even more so in T . thermophilus 5S rRNA . For additional evaluation of the data obtained by RNase H cleavage, association constants of the hexanucleotides were determined by equilibrium dialysis at 4 degrees C for B . stearothermophilus 5S rRNA . The results obtained reveal that nucleotides 36-41 of B . stearothermophilus 5S rRNA are inaccessible for Watson-Crick interaction, which suggests that this part of loop c is in a structurally constrained configuration, or buried in the tertiary structure or involved in tertiary interactions.

J Cell Biol, 1989 Nov, 109(5), 1983 - 92
Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei; White EM et al.; Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila . Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating . The way in which histone H4 localized to either the macronucleus or the micronucleus suggested that it accumulates in whichever nucleus is replicating . The inability of the micronucleus to accumulate Tetrahymena H1 or heterologous nuclear proteins, even at a period in the cell cycle when it is accumulating H4, suggests that it has a specialized transport system . These studies demonstrate that although the mechanism for localizing proteins to nuclei is highly conserved among eukaryotes, it can differ between two porecontaining nuclei lying in the same cytoplasm.

Arch Biochem Biophys, 1989 Nov 1, 274(2), 501 - 10
Degradation of larchwood xylan by enzymes of a thermophilic fungus, Thermoascus aurantiacus; Khandke KM et al.; Proteins from the culture filtrates of Thermoascus aurantiacus grown on paper were found to hydrolyze larchwood xylan completely to form xylose and 4-O-methyl-alpha-D-glucuronic acid . Partial hydrolysis of xylan by a xylanase purified from the culture filtrates resulted in the formation of neutral xylooligosaccharides of dp from 2 to 6 and acidic xylooligosaccharides of dp from 5 to 8 . Each of these acidic sugars contained a single molecule of 4-O-methyl-alpha-D-glucuronic acid as a branch . Extensive hydrolysis of these oligosaccharides or xylan by xylanase led to the isolation of xylose, xylobiose, and an aldotetrauronic acid as terminal products . The structure of the aldotetrauronic acid was established by NMR as (2(2)-O-alpha-D,4-O-methyl-alpha-D-glucurono)-xylotriose . A beta-glucosidase, also purified from the culture filtrates, hydrolyzed xylan and the neutral or the acidic xylooligosaccharides from the nonreducing end to release only xylose . Neither xylanase nor beta-glucosidase hydrolyzed the beta-(1----4) linkage between the xylose carrying the branch and the adjacent xylose residue on each side.

Arch Biochem Biophys, 1989 Nov 1, 274(2), 491 - 500
Purification of xylanase, beta-glucosidase, endocellulase, and exocellulase from a thermophilic fungus, Thermoascus aurantiacus; Khandke KM et al.; A strain of thermophilic fungus, Thermoascus aurantiacus, was isolated from local soil . From the culture filtrates of the organism grown on blotting paper, a xylanase, beta-glucosidase, exocellulase, and endocellulase were obtained in large amounts in highly purified form by employing ion-exchange and gel-permeation chromatography . The xylanase was crystallized . The xylanase and endocellulase were stable at 70 degrees C for 8 h, whereas the beta-glucosidase and exocellulase were less stable at 70 degrees C.

J Biochem (Tokyo), 1989 Nov, 106(5), 798 - 802
Effects of modification of 4-thiouridine in E . coli tRNA(fMet) on its methyl acceptor activity by thermostable Gm-methylases; Hori H et al.; tRNA(guanosine-2'-)-methyltransferases (Gm-methylases) isolated from extreme thermophiles, Thermus thermophilus strains HB 27 and HB 8, methylate the 2'-OH of the G18 ribose of the GG sequence in the D loop of tRNA, by recognizing the D "loop-stem" structure as a minimal requirement . To examine the role of the consensus uridine residue at position 8 (U8) adjacent to the D "loop-stem" region in the recognition of Gm-methylase, 4-thiouridine at this position (s4U8) in Escherichia coli tRNAfMet was modified reversibly with S-benzylthioisothiourea (sBTIU) or irreversibly by UV light . The initial velocities of the methylation reaction for the sBTIU-modified and the UV-induced cross-linked tRNAs were decreased to 40 and 30%, respectively, of that of the intact tRNA, but the sBTIU-modified tRNA regained almost full activity on reduction with beta-mercaptoethanol . Although both of the modified tRNAfMetS showed larger Km (although to different extents) and slightly smaller Vmax than the intact tRNAfMet, they retained full activities of methylation with tRNA(adenine-1-)-methyltransferase (m1A-methylase) and of aminoacylation with aminoacyl-tRNA synthetase (ARS) fraction as well, both of which were prepared from T . thermophilus strain HB 27 . The 5'-half fragments derived from the sBTIU-modified and cross-linked tRNAfMetS showed methylation efficiency (Vmax/Km) not appreciably different from that of the unmodified 5'-half fragment . These results suggest that the conformation of S4U8 residue of tRNA is deeply involved in the recognition of tRNA by Gm-methylase.

J Biol Chem, 1989 Oct 25, 264(30), 17834 - 7
A unique tRNA intron in the variable loop of the extreme thermophile Thermofilum pendens and its possible evolutionary implications; Kjems J et al.; We describe, for the first time, an intron that is located in the variable loop of a tRNA . It is 18 nucleotides long and occurs within a precursor tRNAGly from the extreme thermophile Thermofilum pendens . The variable loop is less critical for tRNA function than the anticodon arm where other class III introns occur . This raises the possibility that tRNAs with a large variable loop (type II) arose from those with a small variable loop (type I) by retaining a splicing-deficient intron.

J Mol Biol, 1989 Oct 20, 209(4), 635 - 44
DNA polymerase from Sulfolobus acidocaldarius . Replication at high temperature of long stretches of single-stranded DNA; Salhi S et al.; The activity of a homogeneous DNA polymerase from the thermophilic archaebacterium, Sulfolobus acidocaldarius, on a singly primed, single-stranded recombinant phage M13 DNA has been examined . At the optimal temperature (70 to 75 degrees C) this template is efficiently replicated in ten minutes using a ratio of enzyme molecule to primed-template of 0.8 . Analysis of DNA products during the course of polymerization shows that species of quite homogeneous size are observed and that the number of primers extended by the enzyme is constant, whatever the enzyme molecule to primed template ratio is in the range 1/50 to 2, indicating that the 100 x 10(3) Mr DNA polymerase from S . acidocaldarius is randomly recycled on the template molecules . At non-optimal temperature (60 degrees C and 80 degrees C) the distribution of products observed indicated the presence of arrest sequences; some have been shown to be reversible . One of these pausing signals detected at 80 degrees C has been further analysed, and has been found to be DNA sequence-dependent.

Nucleic Acids Res, 1989 Oct 11, 17(19), 7879 - 89
Catalytic activity is retained in the Tetrahymena group I intron despite removal of the large extension of element P5; Joyce GF et al.; We have made sizeable internal deletions within the self-splicing group I intron of Tetrahymena thermophila . Deletions were made in a piecewise manner in order to remove secondary structural elements thought to be extraneous to the catalytic center of the molecule . The resulting deletion mutants retain self-splicing activity, albeit under modified reaction conditions that enhance duplex stability . Considering those portions of the molecule that can be deleted without a loss of catalytic activity, one is left with a catalytic center of approximately 130 nucleotides that is solely responsible for the molecule's activity.

J Cell Sci, 1989 Oct, 94 ( Pt 2), 343 - 54
Cellular localization of the SerH surface antigen in Tetrahymena thermophila; Bolivar I et al.; We have purified the SerH surface antigen of T . thermophila by a novel and simple procedure and produced specific antisera against it . By the use of various immunochemical techniques, we have investigated the intracellular distribution of the antigen and shown that SerH is not only abundant in the cortex but also in the digestive apparatus of the ciliate . In each of these two localizations, SerH occupies a variety of compartments: in the cortex it can be found in the surface coat, the exocytotic mucocysts, the endocytotic parasomal sacs and as an integral protein of the membrane; in the digestive apparatus, SerH is found around ingested bacteria, in the cytoplasm surrounding the cytopharynx and the forming food vacuoles, and, seemingly, as a membrane-associated protein in young food vacuoles . Pulse-chase experiments have shown that feeding dramatically increases the global turnover of SerH . Besides these localizations, SerH is principally found in clumps around dense intracytoplasmic spheres, which could be mucocyst precursors . Indirect evidence is presented that SerH is routed to the peripheral or extracellular compartments via the mucocysts . The antigen is absent from alveolar, nuclear or mitochondrial membranes . We propose that SerH covers any membrane in contact or future contact with the extracellular medium.

Nihon Kyobu Shikkan Gakkai Zasshi, 1989 Oct, 27(10), 1237 - 42
{A case of pulmonary sarcoidosis with calcification in the lung}; Asami H et al.; A 35 year-old male farmer presented with complaints of productive cough and sputum . The chest X-ray films showed reticulonodular shadows bilaterally in the upper and middle lung fields, segmental infiltration in the right lung, and no BHL . Tuberculin reaction was negative . Serum angiotensin converting enzyme level was 26.5 IU/ml . Precipitating antibodies for Thermophilic actinomycetes and M.f . were negative . BAL showed moderate lymphocytosis (24.3%), and CD 4/8 was 1.67 . A biopsy specimen of right scalene lymph node showed epithelioid cell granulomas and TBLB epithelioid cell granulomas and spot-like calcification . Pulmonary sarcoidosis with calcification in the lung is very rare.

Biol Chem Hoppe Seyler, 1989 Oct, 370(10), 1127 - 31
Inhibition of L-arginine iminohydrolase (EC 3.5.3.6) from Tetrahymena thermophila by putrescine and spermidine: feedback control of polyamine biosynthesis; Eichler W; L-Arginine iminohydrolase (arginine deiminase, ADI) from Tetrahymena thermophila was purified approx . 75-fold by means of gel permeation chromatography . The Km of the purified enzyme for L-arginine was 412 +/- 25 microM and L-ornithine inhibited the reaction competitively with a Ki of 985 +/- 105 microM . D-Ornithine was a weak inhibitor with a Ki of greater than 10mM . The polyamines putrescine and spermidine inhibited ADI incompetitively with a Kii of 2.8mM for putrescine and 4.3mM for spermidine . Since the concentrations required for inhibition were within the range of the normal intracellular polyamine concentrations in Tetrahymena (maximally 14mM putrescine and 4mM spermidine), it is suggested that the polyamine effects on ADI are of regulatory nature . Thus, polyamine biosynthesis in Tetrahymena thermophila is regulated not only on the level of ornithine decarboxylase activity, but also on an earlier step, the supply of ODC with substrates.

Biol Chem Hoppe Seyler, 1989 Oct, 370(10), 1113 - 26
Stimulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila . Regulation by L-arginine; Eichler W; Tetrahymena thermophila cells grown in a synthetic nutrient medium for 9 h removed 97% of the free L-arginine but less than 50% of any of the other essential amino acids . The major portion of the arginine was degraded rapidly (76-92%) whereas 5-15% was conserved as intact and only 2.5-10% were incorporated into protein . However, if bovine serum albumin (BSA) was present in the medium as a macromolecular arginine source the incorporation of free arginine into protein was reduced to less than 1% but the degraded fraction was increased . Apparently, the uptake mode of arginine determines its fate: arginine taken up by phagocytosis is bound for protein biosynthesis, arginine taken up by membrane receptors is chanelled to degradation . Media without arginine did not support growth of Tetrahymena . Citrulline and ornithine, the precursors of arginine biosynthesis in yeast and vertebrates, were not able to substitute for arginine . Pronounced morphological changes, e.g . greatly reduced ribosome content, were observed in Tetrahymena cells after 24 h of arginine starvation in otherwise complete medium, but not in cells starved in water, salt solution, or buffer . Thus, arginine is an essential nutrient component for Tetrahymena and the rapid degradation of this compound involving the enzymes arginine deiminase (ADI) and citrulline hydrolase (CH) might be of regulatory importance for the unicellular, as it is the case with acetylcholine and catecholamines in mammalian organisms . Since the product of these enzymes, L-ornithine, is the substrate for the regulatory key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), the effects of the presence of absence of arginine on the activities of each particular enzyme of the pathway were studied, including ODC and the enzyme ornithine-oxo-acid aminotransferase (O delta T), which is a competitor of ODC for the common substrate . The arginine-degradative pathway was stimulated by extracellular free but not by peptide-bound arginine and was modulated by extracellular protein which induced phagocytosis; O delta T was stimulated with a time lag . The stimulation of ODC was in a reciprocal relation to the arginine concentration and enhanced by phagocytosis and previous arginine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)

Appl Environ Microbiol, 1989 Oct, 55(10), 2669 - 74
Improved medium for recovery and enumeration of the farmer's lung organism, Saccharomonospora viridis; Amner W et al.; A new medium, which we propose to call R8, was developed for the isolation and enumeration of the thermophilic actinomycete, Saccharomonospora viridis . This organism has been implicated in a range of hypersensitivity pneumonitides, including farmer's lung, but is generally isolated in small numbers from contaminated environments . Recovery of S . viridis from moldy hay and mushroom compost on R8 medium was compared with recovery on conventional media . S . viridis was isolated from both substrates but in highest numbers and most consistently on the R8 medium . The selectivity of this medium was best observed when the sedimentation chamber method was used for hay samples . Here S . viridis accounted for up to 80% of the total number of actinomycetes recovered on R8 and could not be recovered on rifampin selective medium under the same conditions . R8 was also found to be an efficient recovery medium for a range of thermophilic actinomycetes from mushroom compost and for another allergenic species, Faenia rectivirgula, from moldy hay . Contamination of isolation plates by thermophilic bacilli was reduced on R8 compared with the activity on half-strength tryptone soy agar, supplemented with 0.2% casein hydrolysate, and this, together with specific improvements in S . viridis growth, accounts for the selective effect . It is possible that the occurrence of S . viridis and its role as a causative agent of hypersensitivity pnuemonitis have been underestimated by the use of suboptimal recovery protocols . It is hoped that use of R8 in conjunction with dilution plate techniques will generate information on the ecology of S . viridis and contribute to health risk assessment studies.

J Dairy Sci, 1989 Oct, 72(10), 2444 - 51
Growth kinetics of Streptococcus thermophilus at subbacteriostatic penicillin G concentrations; Yondem F et al.; Streptococcus thermophilus may be subjected to the effects of penicillin G in contaminated milk used for yogurt production . Sensitivity of this microorganism to penicillin G has been conventionally determined by the help of penicillin G-impregnated disks placed on solid media . It was observed that the bacteriostatic penicillin G concentration was much greater in liquid media than in solid media . The conventional disk method may not be appropriate for antibiotic sensitivity determinations if the microorganisms will be used in liquid culture . A simple mathematical model simulated the growth of S . thermophilus in liquid culture . Numerical values of this model's parameters were regarded as the measure of the antibiotic effect on the culture . In penicillin G containing fresh medium, small concentrations of antibiotic decreased the specific growth rate considerably . Increasing the antibiotic concentration caused only slight additional decline . Antibiotic shock, i.e., rapidly introducing penicillin G into an actively growing antibiotic-free culture, stopped growth of the penicillin G-resistant microorganisms, and no death was observed, but a fraction of the microorganisms were killed in the wild culture . Both the wild and the resistant cultures recovered from the shock in a few hours . Addition of penicillin G-resistant microorganisms together with the antibiotic dosage into the wild culture prevented death.

Antonie Van Leeuwenhoek, 1989 Oct, 56(3), 201 - 9
Distribution of clinically important thermophilic actinomycetes in vegetable substrates and soil in north-western India; Gangwar M et al.; Medically important thermophilic actinomycetes were isolated from 218 (64%) of 341 samples of vegetable substrates and soil examined from sites in north-western India . Thermoactinomyces vulgaris (T . candidus) was the commonest species, occurring in 56% of samples, followed by Saccharomonospora viridis in 29%, Thermoactinomyces thalpophilus in 27%, Faenia rectivirgula (Micropolyspora faeni) in 21% and Thermoactinomyces sacchari in 14% . T . vulgaris and T . thalpophilus were isolated from all types of substrate examined, with T . vulgaris always more common than T . thalpophilus . Of the other thermophilic actinomycetes, F . rectivirgula was isolated predominantly from hay (44%) and S . viridis (56%) and T . sacchari (44%) from sugar-cane bagasse . The largest populations of T . vulgaris and T . thalpophilus were found in paddy straw, followed by T . sacchari, S . viridis and F . rectivirgula in sugar-cane bagasse . The widespread occurrence of these clinically important thermophilic actinomycetes suggests that exposure of humans and animals to them may be frequent in north-western India . Studies are required to determine the prevalence of extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by thermophilic actinomycetes in the local population.

EMBO J, 1989 Oct, 8(10), 3135 - 9
Reverse gyrase binding to DNA alters the double helix structure and produces single-strand cleavage in the absence of ATP; Jaxel C et al.; Stoichiometric amounts of pure reverse gyrase, a type I topoisomerase from the archaebacterium Sulfolobus acidocaldarius were incubated at 75 degrees C with circular DNA containing a single-chain scission . After covalent closure by a thermophilic ligase and removal of bound protein molecules, negatively supercoiled DNA was produced . This finding, obtained in the absence of ATP, contrasts with the ATP-dependent positive supercoiling catalyzed by reverse gyrase and is interpreted as the result of enzyme binding to DNA at high temperature . Another consequence of reverse gyrase stoichiometric binding to DNA is the formation of a cleavable complex which results in the production of single-strand breaks in the presence of detergent . Like eubacterial type I topoisomerase (protein omega), reverse gyrase is tightly attached to the 5' termini of the cleaved DNA . In the light of these results, a comparison is tentatively made between reverse gyrase and the eubacterial type I (omega) and type II (gyrase) topoisomerases.

J Biochem Biophys Methods, 1989 Oct, 19(4), 281 - 6
M(III)-facilitated recovery and concentration of enzymes from mesophilic and thermophilic organisms; Collingwood TN et al.; Six enzymes isolated from organisms of widely differing thermal growth optima were flocculated from solution at constant pH by addition of Fe(III) solution . In all cases the enzyme concentration was 1 g.l-1 or less . Flocculation profiles were generated for each enzyme over a range of Fe(III) levels . The concentrated enzymes were recovered from the Fe(III)/protein complex by solubilisation with citrate and dithionite followed by precipitation with ammonium sulphate . In all cases approximately 70-80% enzyme recovery was achieved . Enzyme thermal stability did not appear to be important and protein concentration had no effect on the efficiency of enzyme recovery over the range of 0.01-1 g.l-1 . Approximately 30 mmol Fe(III)/l of enzyme solution facilitated optimal enzyme recovery for all solutions studied . For protein concentrations up to 1 g.l-1 a 100-fold enzyme concentration factor can be expected.

J Biochem (Tokyo), 1989 Oct, 106(4), 612 - 5
A protein that accumulates during starvation in Tetrahymena nuclei; Suda M et al.; Tetrahymena pyriformis was starved in 50 mM Tris-HCl, pH 7.5, at 28 degrees C . The number of cells did not change appreciably under the starvation conditions . Nuclear proteins of unstarved cells and cells starved for 1, 2, 4, and 7 d were analyzed by SDS-polyacrylamide gel electrophoresis . Most of the large amount of nonhistone proteins present in the unstarved cell nucleus disappeared with the starvation time . However, the relative amounts of the high mobility group protein and histones did not change appreciably . On the other hand, a protein with a molecular weight of ca . 16,000 gradually accumulated in the nucleus on starvation . This protein was extracted with 0.25 M HCl, but was not soluble in 0.5 M perchloric acid . The amino acid composition and molecular weight of this protein were similar to those of HMG protein LG-2 of T . thermophila . Some lysyl endopeptidase peptides of this protein were found to have amino acid sequences present in LG-2, thus we tentatively named it an LG-2-like protein.

Nucleic Acids Res, 1989 Sep 25, 17(18), 7263 - 72
Sequence microheterogeneity is generated at junctions of programmed DNA deletions in Tetrahymena thermophila; Austerberry CF et al.; Regulated DNA deletions are known to occur to thousands of specific DNA segments in Tetrahymena during macronuclear development . In this study we determined the precision of this event by examining the junction sequences produced by three different deletions in many independent caryonidal lines . 0.9 kb deletions in region M produce at least 3 types of junction sequences, of which two have been determined and found to be different by 4 bp . The alternative 0.6 kb deletions in this region are much less variable . 1.1 kb deletions in region R, known from a previous study to be slightly variable, produce two types of junction sequences which are different from each other by 3 bp . Thus, developmentally regulated deletions in Tetrahymena can produce sequence microheterogeneity at their junctions . This process contributes significantly to the diversification of Tetrahymena's somatic genome.

Nucleic Acids Res, 1989 Sep 25, 17(18), 7283 - 94
Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158; van der Lelie D et al.; Plasmids pMV158 and pTB913, originating from Streptococcus agalactiae and a thermophilic Bacillus respectively, were sequenced to completion . Both contained a BA3-type minus origin of replication and an RSA-site, believed to constitute a site-specific recombination site . These two regions were more than 99% homologous to the corresponding regions of the Staphylococcus aureus plasmid pUB110 . Deleting the BA3-type minus origin resulted in the accumulation of a considerable amount of single-stranded DNA, both in L . lactis subsp . lactis and B . subtilis, indicating that this minus origin was functional in both bacterial species . Like pUB110, both plasmids contained an open reading frame encoding a putative plasmid recombination enzyme (Pre protein), which was located downstream of the RSA-site . On the basis of sequence comparisons between pUB110, pMV158, pTB913, pT181, pE194, pNE131 and pT48 two distinct families of RSA-sites and Pre proteins could be distinguished.

J Mol Biol, 1989 Sep 20, 209(2), 327 - 8
Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus; Trakhanov S et al.; Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam . The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A . The diffraction patterns extend to better than 20 A.

Biochim Biophys Acta, 1989 Sep 15, 992(3), 320 - 6
Beta-aminoglutaric acid is a major soluble component of Methanococcus thermolithotrophicus; Robertson DE et al.; 13C- and 15N-NMR spectroscopy have been used to identify beta-aminoglutaric acid (beta-glutamic) as a major soluble component of the thermophilic, autotrophic marine methanogen Methanococcus thermolithotrophicus . This rare, non-protein amino acid has been recognized as a major dissolved free amino acid in marine sediments, but the microorganism responsible for its production has not previously been identified . The concentration of beta-aminoglutarate (beta-glutamate) is about one half that of free alpha-glutamate and increases (relative to the alpha-isomer) as cells enter the stationary phase . Analysis of the 13C label distribution in a 13CO2-pulse/12CO2-chase experiment shows that label enters the beta-aminoglutarate pool after it has decayed from other small soluble molecules . This implies that beta-aminoglutarate is a catabolic product of the cells . Preliminary biosynthesis studies with labeled precursors indicate that only a single acetate moiety is incorporated in this unusual compound . This information is used to suggest possible biosynthetic pathways.

Eur J Biochem, 1989 Sep 15, 184(2), 313 - 9
Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum; Heda GD et al.; The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum . The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa . No evidence for a second form of the enzyme lacking small subunits was obtained . C . tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature . Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid . The Km for ribulose bisphosphate for the carboxylase activity of the C . tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs . Amino acid composition and immunological analyses of C . tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability . It is hypothesized that thermal stability of C . tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.

Zentralbl Bakteriol, 1989 Sep, 271(3), 263 - 71
Electron microscopic, biochemical and physiological studies of Bifidobacterium pseudolongum SS-24 and Bifidobacterium thermophilum SS-19; Kudo H et al.; Comparative studies of physiology, biochemical characteristics, and morphology by electron microscopy were conducted on Bifidobacterium pseudolongum SS-24 isolated from dogs and Bifidobacterium thermophilum SS-19 isolated from swine . Both B . pseudolongum and B . thermophilum grow unusually rapidly in the rumen fluid medium of Scott and Dehority, and reached a maximum of optical density after only 6 to 7 h of incubation . B . pseudolongum and B . thermophilum showed similar patterns of results for 21 biochemical characteristics tested, with a difference found only for N-acetyl-glucosaminidase . Scanning electron micrographs revealed that B . pseudolongum produced extensive amounts of extracellular material . The cell walls of B . pseudolongum and B . thermophilum were totally different . Transmission electron micrographs of ruthenium red-stained preparations of B . pseudolongum showed a very thick (ca . 0.2 microns) Gram-positive cell wall, whereas B . thermophilum was found to have a thin (ca . 0.05 microns) Gram-positive cell wall.

J Antibiot (Tokyo), 1989 Sep, 42(9), 1362 - 9
Isolation and characterization of new thiol protease inhibitors estatins A and B; Yaginuma S et al.; New thiol protease inhibitors, estatins A and B, were isolated from the culture filtrate of Myceliophthora thermophila M4323 . The basic, water-soluble inhibitors were characterized as having an agmatine, trans-epoxysuccinic acid and L-phenylalanine or L-tyrosine moieties in the structure . The molecular formulas C18H25N5O5 and C18H25N5O6 for A and B were indicated by elemental analysis and fast atom bombardment MS . Estatins were specific inhibitors against thiol proteases such as papain, ficin and bromelain . They suppressed IgE antibody production in mice, but not IgG.

Mol Cell Biol, 1989 Sep, 9(9), 3657 - 66
The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing; Barfod ET et al.; Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G) . The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron . Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site) . Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site . Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice . When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7 . Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount . In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay . Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation . Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.

J Cell Biol, 1989 Sep, 109(3), 1007 - 14
Histone acetylation in conjugating Tetrahymena thermophila; Pfeffer U et al.; We have monitored histone acetylation during conjugation of the ciliated protozoan Tetrahymena thermophila using antibodies against the tetraacetylated form of H4 histone (Pfeffer, U., N . Ferrari, and G . Vidali . 1986 . J . Biol . Chem . 261:2496-2498) . During meiosis, the three prezygotic divisions, fertilization, and the first postzygotic division, micronuclei, do not contain highly acetylated forms of H4 histone . However, after the second postzygotic division, when anteriorly located micronuclei begin to develop into new macronuclei, they are strongly stained by the anti-tetraacetylated H4 histone antibody . In the old macronucleus, histones are actively deacetylated when it has ceased to transcribe but before it is eliminated . Histone acetylation processes analyzed here appear to be correlated to the commitment to transcription rather than to the transcription process itself . This is in good correlation with evidence we have obtained in chick erythrocyte nuclei during reactivation upon fusion with mammalian cells (Pfeffer, U., N . Ferrari, F . Tosetti, and G . Vidali . 1988 . Exp . Cell Res . 178:25-30) . Furthermore, it becomes clear from our data that histone acetylation occurs in close correlation to the position of nuclei within the cytoplasm of T . thermophila . Mechanisms that control differential histone acetylation and deacetylation are discussed.

Indian J Pediatr, 1989 Sep-Oct, 56(5), 657 - 60
Campylobacter diarrhea in children in Trivandrum; Devi KI et al.; One hundred and fifty two stool samples from patients belonging to the pediatric age group clinically diagnosed as acute diarrhea/dysentery were processed for thermophilic campylobacters . C . jejuni was isolated from 9 samples (5.9%) . Five of the C . jejuni isolates were from children who presented with bloody diarrhea and 4 were from those who had watery diarrhoea . Though the pathogenic role of C . jejuni in these cases is not proved, this study indicates the prevalence of the organism in Trivandrum district.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6484 - 7
The alpha 3 beta 3 complex, the catalytic core of F1-ATPase; Miwa K et al.; The alpha 3 beta 3 complex was reconstituted from alpha and beta subunits of the thermophilic bacterium PS3 F1-ATPase (TF1) and then isolated . It is less stable at high and low temperatures than TF1, and the complex dissociates into subunits during native polyacrylamide gel electrophoresis . The alpha 3 beta 3 complex has about 20% of the ATPase activity of TF1 . Its enzymic properties are similar to those of the native TF1, exhibiting similar cooperative kinetics as a function of ATP concentration, similar substrate specificity for nucleotide triphosphates, and the presence of two peaks in its temperature-activity profile . Differing from TF1, the ATPase activity of the alpha 3 beta 3 complex is insensitive to N3- inhibition, its divalent cation specificity is less stringent, and its optimum pH shifts to the alkaline side . The addition of the gamma subunit to the alpha 3 beta 3 complex leads to the formation of the alpha 3 beta 3 gamma complex, indicating that the alpha 3 beta 3 complex is an intermediate in the process of assembly of the holoenzyme from each subunit . These results definitely show that the essential structure for eliciting the ATPase activity of F1-ATPase is trimeric alpha beta pairs and that the kinetic cooperativity of the F1-ATPase is an inherent property of this trimeric structure but is not due to the presence of single-copy subunits . In this sense, the alpha 3 beta 3 complex is the catalytic core of F1-ATPase.

Eur J Biochem, 1989 Sep 1, 184(1), 151 - 6
13C-NMR study of autotrophic CO2 fixation in Thermoproteus neutrophilus; Schafer S et al.; The pathway of autotrophic CO2 fixation has been investigated in the extremely thermophilic sulfur-respiring anaerobic archaebacterium Thermoproteus neutrophilus . {1,4-13C2}Succinate was used as a tracer since this compound was incorporated in small amounts virtually into all cell compounds without affecting the organism's ability to synthesize all cell constituents from CO2 . Three representative amino acids, glutamate, aspartate and alanine were isolated from cells after growth for several generations in the presence of {1,4-13C2}succinate and their labelling patterns were determined by 13C NMR spectroscopy . The data is consistent with CO2 fixation by a reductive citric acid cycle, as proposed earlier for the green sulfur bacterium Chlorobium limicola, the sulfate-reducing Desulfobacter hydrogenophilus and the microaerophilic Knallgasbacterium Hydrogenobacter thermophilus . The presence of a reductive citric acid cycle in archaebacteria indicates that this CO2 fixation mechanism which is an alternative to the Calvin cycle is present in many anaerobic or facultative anaerobic microorganisms.

Biochemistry, 1989 Aug 22, 28(17), 6888 - 94
Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme; Grosshans CA et al.; A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates . We have now examined the metal ion requirements of this reaction . Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity . Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage . Thus, these ions can be eliminated as cofactors for the reaction . While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM . These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis . Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity . It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry . Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.

Biochemistry, 1989 Aug 22, 28(17), 6949 - 54
F1 ATPase from the thermophilic bacterium PS3 (TF1) shows ATP modulation of oxygen exchange; Kasho VN et al.; The ATPase from the ATP synthase of the thermophilic bacterium PS3 (TF1), unlike F1 ATPase from other sources, does not retain bound ATP, ADP, and Pi at a catalytic site under conditions for single-site catalysis {Yohda, M., & Yoshida, M . (1987) J . Biochem . 102, 875-883} . This raised a question as to whether catalysis by TF1 involved alternating participation of catalytic sites . The possibility remained, however, that there might be transient but catalytically significant retention of bound reactants at catalytic sites when the medium ATP concentration was relatively low . To test for this, the extent of water oxygen incorporation into Pi formed by ATP hydrolysis was measured at various ATP concentrations . During ATP hydrolysis at both 45 and 60 degrees C, the extent of water oxygen incorporation into the Pi formed increased markedly as the ATP concentration was lowered to the micromolar range, with greater modulation observed at 60 degrees C . Most of the product Pi formed arose by a single catalytic pathway, but measurable amounts of Pi were formed by a pathway with high oxygen exchange . This may result from the presence of some poorly active enzyme . The results are consistent with sequential participation of three catalytic sites on the TF1 as predicted by the binding change mechanism.

FEBS Lett, 1989 Aug 14, 253(1-2), 269 - 72
Identification of a mutation in Escherichia coli F1-ATPase beta-subunit conferring resistance to aurovertin; Lee RS et al.; A mutation conferring aurovertin resistance on Escherichia coli F1-ATPase was identified as R398----H in the F1 beta-subunit . Beta-subunit from the mutant does not bind aurovertin; therefore our results suggest the region of sequence around residue beta-398 is involved in aurovertin binding . Since nucleotide and aurovertin binding to isolated beta-subunit are not mutually exclusive, the data further suggest that the beta-subunit catalytic nucleotide-binding domain does not include residue 398 . The mutation prevented aurovertin inhibition of ATPase at pH 6 and 8.5, implying charge on the arginine side-chain is not a major determinant of aurovertin binding or that the pK of R398 is shifted due to a peculiar environment . The equivalent residue is usually arginine in F1 beta-subunits of different species; notably in the aurovertin-insensitive thermophilic bacterium PS3 F1-ATPase, this residue is phenylalanine.

FEBS Lett, 1989 Aug 14, 253(1-2), 257 - 63
Identification of photosystem I components from the cyanobacterium, Synechococcus vulcanus by N-terminal sequencing; Koike H et al.; The photosystem I core complex isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, is composed of eight low-molecular-mass proteins of 18, 14, 12, 9.5, 9, 6.5, 5 and 4.1 kDa in addition to the PS I chlorophyll protein . N-terminal amino acid sequences of all these components were determined and compared with those of higher plants . Clearly, the 9.5 kDa component corresponds to the protein which carries the non-heme iron-sulfur centers A and B . This protein is so poorly visualized by staining that it has probably been overlooked in gel electrophoresis analyses . The 18, 14, 12 and 9 kDa components show appreciable homology with respective subunits of higher plant PS I . In contrast, the 6.5, 5 and 4.1 kDa components do not correspond to any known proteins except that the sequence of the 4.1 kDa component matches an unidentified open reading frame (ORF) 42 (liverwort) or ORF44 (tobacco) of chloroplast DNA.

FEBS Lett, 1989 Aug 14, 253(1-2), 178 - 82
N-terminal sequencing of low-molecular-mass components in cyanobacterial photosystem II core complex . Two components correspond to unidentified open reading frames of plant chloroplast DNA; Ikeuchi M et al.; We recently reported the presence of several low-molecular-mass protein components in the PS II O2-evolving core complex from the thermophilic cyanobacterium, Synechococcus vulcanus {(1989) FEBS Lett . 244, 391-396} . Here we have characterized the three components (4.1, 4.7, 5 kDa) of the same cyanobacterial core complex by N-terminal sequencing . There were two components in the 4.7 kDa region, both having a blocked N-terminus . One has a sequence highly homologous to open reading frame 34 of plant chloroplast DNA (tentatively designated psbM), while the other has a sequence partially homologous to open reading frame 43 of chloroplast DNA (designated psbN), although neither of the two gene products has yet been confirmed in chloroplasts . The cyanobacterial 4.1 kDa protein partially corresponds to the 4.1 kDa nuclear-encoded core component of higher plant PS II . The cyanobacterial 5 kDa component, however, shows a sequence that is unrelated to any other known proteins.

Biochim Biophys Acta, 1989 Aug 14, 1008(3), 309 - 14
Primary structure of the chromosomal protein MC1 from the archaebacterium Methanosarcina sp . CHTI 55; Chartier F et al.; The DNA of the thermophilic archaebacterium Methanosarcina sp . CHTI 55 has been shown to be associated with two proteins called MC1 and MC2, of molecular mass 11 kDa and 17 kDa (Chartier et al . (1988) Biochim . Biophys . Acta 951, 149-156) . The most abundant of these proteins, protein MC1, can protect DNA against thermal denaturation . In the present paper we report the covalent structure of protein MC1 and its effect on transcription of DNA in vitro . The covalent structure was determined from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid and arginine residues . The amino-acid sequence of protein MC1 from Methanosarcina sp . CHTI 55 is closely related to that of the protein MC1 (previously called HMb) isolated from Methanosarcina barkeri strain MS: among the nine substitutions observed between the two proteins seven are conservative . Transcription of DNA in vitro is stimulated by protein MC1 at low protein-to-DNA ratio but is inhibited at a ratio higher than 0.1 (w/w), which is the one determined in the bacterial deoxyribonucleoprotein complex.

Biochemistry, 1989 Aug 8, 28(16), 6549 - 55
Amino acid sequence of alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii; Peretz M et al.; The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented . The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence . These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I) . The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37,652 . The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today . Of these, only 18 residues (5%) are strictly conserved: 11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.

J Biol Chem, 1989 Aug 5, 264(22), 13005 - 11
The aminoacylation of structurally variant phenylalanine tRNAs from mitochondria and various nonmitochondrial sources by bovine mitochondrial phenylalanyl-tRNA synthetase; Kumazawa Y et al.; Bovine mitochondrial (mt) phenylalanine tRNA (tRNAPhe) was purified on a large scale using a new hybridization assay method developed by the authors . Although its melting profile suggested a loose higher order structure, presumably influenced by the apparent loss of D loop-T loop interaction necessary for forming a rigid L-shaped tertiary structure, its aminoacylation capacity catalyzed by mt phenylalanyl-tRNA synthetase (PheRS) was nearly equal to that of Escherichia coli tRNAPhe . Misaminoacylation was not observed for the mt tRNAPhe-mt PheRS system . Comparing the aminoacylation efficiencies of several combinations of tRNAPheS and PheRSs from various sources, including bovine mitochondria, bovine and yeast cytosols, E . coli, Thermus thermophilus, and Sulfolobus acidocaldarius, it was clarified that mt PheRS was able to aminoacylate all the above mentioned tRNAPhe species, albeit with varying degrees of efficiency . This broad charging spectrum suggests that mt PheRS possesses a relatively simple recognition mechanism toward its substrate, tRNAPhe.

Eur J Cell Biol, 1989 Aug, 49(2), 225 - 35
Characterization of pre-rRNA components in ribosomal precursor particles from macronuclei of Tetrahymena thermophila; Muller B et al.; Ribosomal precursor particles were extracted from purified macronuclei of Tetrahymena thermophila and separated in sucrose gradients . The RNA components of major particle fractions were isolated and analyzed by Northern blot hybridization using cloned rDNA fragments . A tentative scheme of preribosome maturation was established based on the RNA constituents and corresponding processing steps occurring in the different particle classes . The primary transcript of ribosomal genes, the unspliced precursor rRNA, was found in some experiments in the upper region (less than 40S) of sucrose gradients run for short times . This is in accordance with earlier results by others indicating that slowly sedimenting ribonucleoprotein (RNP) structures may exist as a transitory stage of preribosome formation . Usually, however, unspliced pre-rRNA was only found in 80S preribosomes, where splicing occurred as indicated by the presence of splice intermediates and products only in this fraction . In addition, the further processing of spliced pre-rRNA at three major sites in variable temporal order took place in the 80S preribosomes, i.e., (i) the cleavage at or near the 5'end of the 17S rRNA sequence, (ii) the central cleavage in the internal transcribed spacer (ITS2) between the 5.8S and 26S rRNA sequence, and (iii) the cleavage in the ITS1 at or near the 3' end of the 17S rRNA sequence . Only the latter event was found to result more or less immediately in the division of the 80S preribosomes into separate precursors (p40S and 60S) of the small and large ribosomal subunits . If the alternative pre-rRNA cleavage site in the ITS2 was used first the 80S preribosomes retained their integrity . The conversion of the p40S precursors into nuclear 40S subribosomal particles was correlated with the processing of pre-17S rRNA into 17S rRNA . In the 60S ribosomal precursor particles the processing of pre-26S rRNA, including the formation of precursors (ITS and 7S RNA) to 5.8S rRNA, occurred . A substantial proportion of 26S rRNA molecules isolated from these particles already contained the central hidden break as indicated by the presence of 26S rRNA alpha- and beta-subfragments . The major pre-rRNA processing by-products, IVS and ETS RNA, were partly associated with preribosomes and partly present as free RNAs in the supernatant of sucrose gradients . This indicates that they are liberated and degraded mainly outside the particles in which they are formed . In contrast, the initiation fragment (IF), a small promoter-proximal transcript, was exclusively associated with large particles.(ABSTRACT TRUNCATED AT 400 WORDS)

J Cell Sci, 1989 Aug, 93 ( Pt 4), 691 - 703
Rearrangement of the cytoskeleton and nuclear transfer in Tetrahymena thermophila cells fused by electric field; Gaertig J et al.; This paper reports on electrofusion of Tetrahymena thermophila and on the reorganization of the cytoskeleton in fused cells . Important factors influencing the fusion yield are the number of electric pulses, the strength of alternate current field and cell density . The process of cell fusion consists of a mutual intermingling of cell membranes following their deformation at the contact zone, followed by the formation of cytoplasmic bridges and simultaneous disruption of portions of the submembranous cytoskeleton (epiplasm) and alveolar sacs . The course of further changes in cell organization depends on the polarity of fused cells . Homopolar fusion partners integrate by gradual translocation of portions of cortical cytoskeletal elements . In contrast, cortical integration of heteropolar fused cells is limited . Cytoskeletal integration is particularly promoted if the cells are incubated in non-growing conditions . Cortical integration leads to a high frequency of micronuclear transfer when a micronucleate strain is used as a donor and an amicronucleate strain is used as a recipient in the fusion experiments.

Biochemistry, 1989 Jul 25, 28(15), 6237 - 44
Strand specificity of the topoisomerase II mediated double-stranded DNA cleavage reaction; Andersen AH et al.; The strand specificity of topoisomerase II mediated DNA cleavage was analyzed at the nucleotide level by characterizing the enzyme's interaction with a strong DNA recognition site . This site was isolated from the promoter region of the extrachromosomal rRNA genes of Tetrahymena thermophila and was recognized by type II topoisomerases from a variety of phylogenetically diverse eukaryotic organisms, including Drosophila, Tetrahymena, and calf thymus . When incubated with this site, topoisomerase II was found to introduce single-stranded breaks (i.e., nicks) in addition to double-stranded breaks in the nucleic acid backbone . Although the nucleotide position of cleavage on both the noncoding and coding strands of the rDNA remained unchanged, the relative ratios of single- and double-stranded DNA breaks could be varied by altering reaction conditions . Under all conditions which promoted topoisomerase II mediated DNA nicking, the enzyme displayed a 3-10-fold specificity for cleavage at the noncoding strand of its recognition site . To determine whether this specificity of topoisomerase II was due to a faster forward rate of cleavage of the noncoding strand or a slower rate of its religation, a DNA religation assay was performed . Results indicated that both the noncoding and coding strands were religated by the enzyme at approximately the same rate . Therefore, the DNA strand preference of topoisomerase II appears to be embodied in the enzyme's forward cleavage reaction.

FEMS Microbiol Lett, 1989 Jul 15, 51(1), 131 - 6
Transcription initiation and a RNA polymerase binding site upstream of the purE gene of the archaebacterium Methanobacterium thermoautotrophicum strain delta H; Brown JW et al.; DNA-dependent RNA-polymerase (RNAP) purified from the thermophilic archaebacterium Methanobacterium thermoautotrophicum strain delta H has been shown to bind specifically to DNA in the intergenic region upstream of the purE gene cloned from this species . The RNAP binding site has been limited to a 41 bp region of DNA which contains the Box A archaebacterial promoter sequence, 5'ATTAAATA . Transcription of the purE gene appears to initiate in vivo at three locations 20-22 bp downstream of the Box A sequence and 27-29 bp upstream of the ATG translation initiation codon of the purE gene.

Biochim Biophys Acta, 1989 Jul 13, 975(2), 293 - 8
EXAFS studies of the isolated bovine heart Rieske {2Fe-2S}1+(1+,2+) cluster; Powers L et al.; Recently the involvement of one or, more likely, two nitrogen-ligands in the Rieske-type {2Fe-2S} cluster has been reported based on the chemical assay and various spectroscopic analyses, such as EPR, Mossbauer, ENDOR, and resonance Raman, of isolated Thermus thermophilus HB-8 protein by Fee and his collaborators . Similarly, the presence of at least one nitrogen ligand was shown in the mitochondrial Rieske {2Fe-2S} cluster . We have conducted EXAFS studies of the Rieske {2Fe-2S} protein isolated from the cytochrome bc1 complex of bovine heart mitochondria . Standard analysis could not distinguish one or two nitrogen ligands per cluster . However, one nitrogen and three cysteine ligands per cluster was found to be, possibly, a better solution in more comprehensive analysis procedures.

Microbiologica, 1989 Jul, 12(3), 181 - 8
Antibodies to Campylobacter pylori in patients with idiopathic dyspepsia; Landini MP et al.; We used Western Blotting analysis to determine the immune profile to Campylobacter pylori polypeptides in: A) sera from patients with idiopathic dyspepsia and bacteriological evidence of C . pylori gastric colonization, B) sera from patients with the same symptoms but no bacteriological evidence of C . pylori infection and C) healthy subjects . To avoid interference of aspecific reactions due to antigenic cross reactivity with other thermophilic Campylobacter species, antisera were raised in rabbits against C . pylori as well as against C . coli and C . jejuni . Some bands (with an approximate molecular weight of 118, 85, 40, 34, 28, 18 and 12 Kd) which can be considered specific for C . pylori were identified and the IgG reaction to some of them (40, 34, 28 Kd) was shown to be significantly higher in patients with bacteriological evidence of C . pylori infection than in the other two groups . IgM reactivity to two bacterial proteins of molecular weight 118 and 40 Kd was particularly evident in the second group of patients suggesting a possible diagnostic tool to identify C . pylori infection at a very early stage.

Allergy, 1989 Jul, 44(5), 314 - 21
Characterization of Micropolyspora faeni antigens by human antibodies and immunoblot analysis; Iranitalab M et al.; IgG, IgM and IgA antibody responses against Micropolyspora faeni (Mf) antigens were studied by means of immunoblotting experiments using 70 sera derived from three groups of farmers, namely patients with extrinsic allergic alveolitis (EAA) due to thermophilic actinomycetes (n = 25), patients without EAA but with hay exposure (n = 14), and patients suspected to have EAA (n = 31), and 27 sera from two groups of control persons (healthy laboratory workers, n = 13; healthy farmers, n = 14) . Patients with EAA showed IgG, IgM and IgA antibody responses mainly against the antigens with molecular weights (MW) of 11, 12, 25, 35 and 60 kD ("major antigens"), and in addition, but less often, against six antigens with MW in the range of 15 to 62.5 kD ("minor antigens") . The other two groups of patients and also the exposed control persons showed very similar results; however, the antibody response in healthy farmers was substantially weaker in comparison to the three groups of patients and was almost limited to the major antigens with MW 11, 25 and 60 kD . Although patients with proven EAA had higher amounts of antibodies, there was no correlation between this antibody response and the onset of disease . The results indicate the necessity of including at least the major antigens with MW of 11, 25 and 60 kD in all extracts used for in vitro diagnosis of Mf-induced EAA.

Ir J Med Sci, 1989 Jul, 158(7), 173 - 4
Farmers lung: a three year survey and comparison of ELISA and CIEP techniques in antibody detection; Melinn M et al.; The incidence of detection of antibodies against antigens derived from two thermophilic actinomycetes in patients clinically suspected of having Farmer's Lung was assessed . Approximately 25% of samples submitted over a three year period were found to be positive in either the ELISA test or the CIEP test for antigens derived from M . faeni and T . vulgaris . The ELISA test proved to be more sensitive than the CIEP test in the detection of antibodies directed against both organisms.

J Bacteriol, 1989 Jul, 171(7), 3788 - 95
Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus; Speelmans G et al.; Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus . Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+) . The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons . Lithium ions, but no other cations tested, could replace sodium ions in serine transport . The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system . To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C . fervidus . Serine transport could be driven by a membrane potential generated by the proton pump . Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake . The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM) . The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake . A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.

Chin Med J (Engl), 1989 Jul, 102(7), 563 - 7
Pathogenesis of extrinsic allergic alveolitis and pulmonary fibrosis induced by streptomyces thermohygroscopicus; Che DY et al.; There were 48 strains of thermophilic actinomyces isolated from the specimens of mouldy hay and sputum of the patients suffering from farmer's lung (FL) . Streptomyces thermohygroscopicus (STHs), one strain of them, was used for this investigation . The microorganisms were injected into the lungs of rabbits and rats by thyrocrico- or tracheocentesis . The results showed that the pathological changes in the lungs including macrophage alveolitis, granuloma formation and diffuse interstitial were similar to that induced by other thermophilic actinomyces . IgG and C3 deposition in the lesions were also observed by immunofluorescence examination . Specific immunocomplexes in the sera of some animals were detected by ELISA with the STH-antisera . The results suggested that STHs was possibly one of the pathogens responsible for FL in China's countryside.

Biochimie, 1989 Jul, 71(7), 805 - 11
Methylated amino acids in the proteins of the cytoplasmic ribosome of Tetrahymena thermophila; Guerin MF et al.; Two-dimensional electrophoretic analysis of proteins of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila labeled in vivo with L-{14C1}methionine and L-{3H-methyl}methionine identifies one heavily methylated protein in each ribosomal subunit . These proteins, S31 and L21, each contain N epsilon-trimethyl-lysine.

J Appl Bacteriol, 1989 Jun, 66(6), 477 - 90
A two-year study of the distribution of 'thermophilic' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype; Fricker CR et al.; The incidence of 'thermophilic' campylobacters in foods and environmental samples has been studied over a two-year period . Of 781 environmental samples, 529 (67%) were found to contain campylobacters, and campylobacters were isolated from 835 (39%) of 2116 food samples . Sewage was almost always contaminated with campylobacters (96.6% of samples) and of the food samples both poultry (55.5%) and offal (47.0%) were commonly contaminated . Determination of the heat-stable serotypes of all strains isolated from these sources and of 921 strains isolated from human faeces showed that there was a wide distribution of serotypes in most types of sample . Serotype Pen 2 was the commonest type found in human faeces (18.9%) and it was also commonest in offal (21.3%), beef (40.0%), sewage (17.7%) and was the third commonest type in poultry . A comparison of culture media and conditions for optimal production of both cytotoxic and cytotonic enterotoxins showed that Brucella Broth incubated under microaerobic conditions for 24 h at 42 degrees C was suitable for both toxins . Detection of cytotoxic activity was most sensitive using HeLa cells . The sensitivities of two ELISA systems and a Chinese Hamster Ovary tissue culture assay for detection of cytotonic enterotoxin were comparable . Not all strains isolated from cases of enteritis in human beings produced toxin; 23.1% produced cytotonic enterotoxin and 17.5% produced cytotoxin . There was no correlation between serotype and toxin production . The wide distribution of campylobacters, indistinguishable from those isolated from cases of enteritis in human beings, leads us to conclude that simplistic statements suggesting that one particular type of food is primarily responsible for cases of human disease should not be made.

J Med Genet, 1989 Jun, 26(6), 363 - 7
Prenatal diagnosis of beta thalassaemia based on restriction endonuclease analysis of amplified fetal DNA; Pirastu M et al.; In the Mediterranean area, 50% of the beta thalassaemia mutations abolish or create a restriction endonuclease site in the beta globin gene . This study describes a new procedure for prenatal detection of these beta thalassaemia defects based on the direct visualisation, on an ethidium bromide stained polyacrylamide gel, of the discrete DNA fragments produced by restriction endonuclease digestion of fetal DNA, enzymatically amplified using the DNA polymerase from the thermophilus bacterium Thermus aquaticus . We applied this procedure to the Sardinian population to detect the nonsense mutation at codon 39 and the frameshift at codon 6 of the beta globin gene; these are the most frequent beta thalassaemia mutations in this population, accounting for 95% and 2.2% of the beta thalassaemia chromosomes . The main advantages of this procedure are simplicity (no radioactivity), sensitivity (0.2 microgram of DNA), and rapidity (12 hours) . The very small amount of fetal material required makes amniotic fluid cell culture unnecessary and may decrease the fetal loss rate associated with trophoblast sampling . By circumventing the use of radioactive and non-radioactive probes, the spread of this technology to the high risk areas will be facilitated.

J Bacteriol, 1989 Jun, 171(6), 2933 - 41
Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit; Hartmann RK et al.; A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping . The gene represents an isolated transcription unit encoding solely 16S rRNA . Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters . The promoter-leader region was very similar to the E . coli rrn P2 promoter-leader segment that is responsible for antitermination . The T . thermophilus leader region was approximately 85 nucleotides shorter than its E . coli P2 counterpart . Potential processing intermediates were correlated with a proposed secondary structure of T . thermophilus pre-16S rRNA.

Mol Cell Biol, 1989 Jun, 9(6), 2598 - 605
Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA; Capowski EE et al.; We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila . Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites . Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena . Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA . In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth . This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation . We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.

Biotechnol Appl Biochem, 1989 Jun, 11(3), 307 - 11
Overproduction of thermostable leucine dehydrogenase of Bacillus stearothermophilus and its one-step purification from recombinant cells of Escherichia coli; Oka M et al.; We have cloned the leucine dehydrogenase (EC 1.4.1.9) gene from a thermophile, Bacillus stearothermophilus, into Escherichia coli MV1184 with a vector plasmid, pUC119 . The cloned cells produced a large amount of the thermostable enzyme, which corresponds to about 60% of the total soluble protein . The enzyme was purified to more than 95% homogeneity by only one step, heat treatment of the cell-extracts, with an average yield of 75 mg/g of wet cells (obtained from 100 ml of the culture).

FEBS Lett, 1989 May 22, 249(1), 67 - 9
Alpha 3 beta 3 complex of thermophilic ATP synthase . Catalysis without the gamma-subunit; Kagawa Y et al.; A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex . The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa) . Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit . A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes . The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.

Nature, 1989 May 11, 339(6220), 145 - 7
Phylogenetic analysis based on rRNA sequences supports the archaebacterial rather than the eocyte tree; Gouy M et al.; How many primary lineages of life exist and what are their evolutionary relationships? These are fundamental but highly controversial issues . Woese and co-workers propose that archaebacteria, eubacteria and eukaryotes are the three primary lines of descent and their relationships can be represented by Fig . 1a (the 'archaebacterial tree') if one neglects the root of the tree . In contrast, Lake claims that archaebacteria are paraphyletic, and he groups eocytes (extremely thermophilic, sulphur-dependent bacteria) with eukaryotes, and halobacteria with eubacteria (the 'eocyte tree', Fig . 1b) . Lake's view has gained considerable support as a result of an analysis of small subunit ribosomal RNA sequence data by a new approach, the evolutionary parsimony method . Here we report that analysis of small subunit data by the neighbour-joining and maximum parasimony methods favours the archaebacterial tree and that computer simulations using either the archaebacterial or the eocyte tree as a model tree show that the probability of recovering the model tree is very high (greater than 90 per cent) for both the neighbour-joining and maximum parsimony methods but is relatively low for the evolutionary parsimony method . Moreover, analysis of large subunit rRNA sequences by all three methods strongly favours the archaebacterial tree.

Appl Environ Microbiol, 1989 May, 55(5), 1093 - 9
Levels of bacteria, fungi, and endotoxin in bulk and aerosolized corn silage; Dutkiewicz J et al.; Three samples of silage taken from the surface of a silo and from depths of 20 and 45 cm in the silo were studied for identification of the potential agents causing symptoms of organic dust toxic syndrome . The samples were examined by dilution plating before and after aerosolization in an acoustical dust generator . Aerosol samples were collected by liquid impinger and filter cassettes . The samples were examined for total aerobic bacteria, anaerobic bacteria, gram-negative bacteria, lactobacilli, listeriae, thermophilic actinomycetes, fungi, and endotoxin . Very high levels of total aerobic bacteria and fungi were found in the surface sample (up to 10(9) CFU/g in the bulk sample and up to 10(9) CFU/m3 after aerosolization), whereas the corresponding values from the deepest site were 100 to 50,000 times lower . Aspergillus fumigatus predominated among the fungi, whereas Bacillus and gram-negative organisms (Pseudomonas, Alcaligenes, Citrobacter, and Klebsiella species) prevailed among bacteria . Thermophilic actinomycetes occurred in numbers up to 10(7) CFU/g in the bulk samples, whereas anaerobic bacteria, lactobacilli, and listeriae were only few or absent . The concentration of endotoxin was high in the surface sample (up to 211.4 Endotoxin Units/mg) and about 200-fold lower in the sample from the deepest site . The results show that contact with dust from the surface of silage carries the risk of exposure to high concentrations of microorganisms, of which A . fumigatus and endotoxin-producing bacteria are the most probable disease agents.

Appl Environ Microbiol, 1989 May, 55(5), 1078 - 81
Nickel transport by the thermophilic acetogen Acetogenium kivui; Yang HC et al.; Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiCl2 . The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the mid- to late exponential phases of growth . Nickel transport was linear during the transport assay period and displayed saturation kinetics . The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 microM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively . The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel . Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose- and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.

J Clin Microbiol, 1989 May, 27(5), 938 - 43
Cellular fatty acid composition of Campylobacter pylori from primates and ferrets compared with those of other campylobacters; Goodwin CS et al.; The cellular fatty acid profiles of newly described campylobacters were determined on a polar, capillary column . Six isolates of the gastric spiral organism, Campylobacter pylori subsp . mustelae, from ferrets from Australia, England, and the United States were all found to have a similar fatty acid profile which was different from that of C . pylori from humans; C . pylori subsp . mustelae did not have 3-hydroxyoctadecanoic acid (3-OH C18:0) and had much less tetradecanoic acid (C14:0) and much more hexadecanoic acid (C16:0) . Inasmuch as Lambert et al . (M.A . Lambert, C.M . Patton, T.J . Barrett, and C.W . Moss, J . Clin . Microbiol . 25:706-713, 1987) have proposed that campylobacters can be grouped by cellular fatty acid composition, we propose this organism should be in a new gas-liquid chromatography (GLC) group, group J . Seven isolates of gastric spiral organisms from macaque monkeys and baboons, including three from Macaca nemestrina, and one isolate from a pig were found to have fatty acid profiles very similar to that of C . pylori; but a second type of organism (type B) from M . nemestrina had a unique profile without 19-carbon cyclopropane fatty acid (C19:0 cyc) but with 3-hydroxy tetradecanoic acid (OH C14:0), which is not present in other gastric spiral bacteria . We propose that this organism (nemestrina type B) should be in a new GLC group, group K . The cellular fatty acid profile of seven isolates of C . jejuni subsp . doylei was found to be similar to that for C . jejuni, but with possibly significant differences in that the former did not have 3-OH C14:0 but did have 3-hydroxyhexadecanoic acid (3-OH C16:0) and had more C14:0 than did C . jejuni . Two strains of urease-positive thermophilic campylobacters were found to have a profile similar to that of "C . cinaedi" and thus should be included with them in GLC group D . We confirm that C . sputorum has a unique cellular fatty acid composition and suggest that it should be in a new group, group H.

FEMS Microbiol Lett, 1989 May, 50(1-2), 65 - 9
Genome comparison of Lactococcus strains by pulsed-field gel electrophoresis; Le Bourgeois P et al.; The two restriction enzymes SmaI and ApaI were found to produce distributions of DNA fragments useful for genome analysis of some lactic acid bacteria (Lactococcus lactis and Streptococcus salivarius subsp . thermophilus) by pulsed-field gel electrophoresis . The genome size was estimated to be 1750 to 2500 kb depending on the species . Each strain displayed unique restriction patterns; nevertheless, the percentage of the comigrating fragments of two isogenic or closely related strains was about 80% and fell to 20-40% when the patterns of two non-related strains of the same species or two strains belonging to different species were compared.

Zentralbl Bakteriol, 1989 May, 271(1), 127 - 34
Distribution of serotypes and biotypes of thermophilic campylobacters in the Federal Republic of Germany: a comparison with other countries; Bar W et al.; Thermophilic Campylobacter isolates from infected humans from the southwest of the FRG were bio- and serotyped according to Lior's scheme . Serotyping was performed with a commercially available test kit (Sopar, Brussels) . C . jejuni represented about 87% of the isolates with Biotype I representing about 50% of the isolates . C . coli was found in 12% of the isolates and C . laridis in less than 1% . The most frequently identified serotypes were LIO 8, LIO 7 and LIO 4 . The results are compared with investigations from seven other countries and with one investigation from northern Germany.

Z Naturforsch {C}, 1989 May-Jun, 44(5-6), 345 - 52
Design and applications of sensitive enzyme immunoassays specific for clostridial enoate reductases; Krause G et al.; Rabbit antisera raised against purified enoate reductase from Clostridium tyrobutyricum (DSM 1460) and horse-radish peroxidase-conjugated staphylococcal protein A or anti-rabbit immunoglobulin G, respectively, were used to develop enzyme immunoassays . Sensitivity limits of the assay are about 250 pg antigen if the enzyme immunoassays are performed on membrane filters and examined visually, and 20 pg for tests in aqueous solution with spectrophotometrical evaluation . The procedures were applied for dot and Western blots as well as colony lifts . Immunological distances between enoate reductases from different clostridia were determined and the amounts of antigen present in bacterial crude extracts were estimated . In crude extracts of C . thermoaceticum a protein of approximately the size of the enoate reductase and its subunits from C . tyrobutyricum was immunologically detected . Gel filtration chromatography of identically pretreated crude extracts from C . thermoaceticum and C . tyrobutyricum produced immunological signals at similar molecular weights and revealed a lesser tendency of the presumed thermophilic enoate reductase from C . thermoaceticum to disintegrate into its subunits and fragments as compared to its mesophilic counterpart.

J Gen Microbiol, 1989 May, 135 ( Pt 5), 1315 - 24
Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA; Wu LJ et al.; An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr . The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA . The transformation frequency (transformants per regenerant) was 0.5 to 1.0 . Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 {2.5 x 10(5) transformants (microgram DNA)-1} and pTHT15 {1.8 x 10(5) transformants (micrograms DNA)-1}, respectively . Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli . Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C . In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures . The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively . The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.

J Bacteriol, 1989 May, 171(5), 2384 - 90
Isolation, characterization, and biological activity of the Methanococcus thermolithotrophicus ferredoxin; Hatchikian EC et al.; A ferredoxin has been isolated from the thermophilic methanogen Methanococcus thermolithotrophicus . The native protein was a monomer exhibiting a molecular weight of 7,262, calculated from the amino acid composition . Its absorption spectrum had two maxima at 390 and 283 nm, with an absorbance ratio A390/A283 of 0.79 . The absorption at 390 nm (E = 29 mM-1 cm-1) and the content of iron of the protein are in agreement with the presence of two 4Fe-4S clusters in M . thermolithotrophicus ferredoxin . Its amino acid composition showed the presence of eight cysteine residues, which is the required number of cysteines for the binding of two 4Fe-4S clusters . The protein was characterized by the lack of histidine, arginine, and leucine and a high content of valine . It was unusually stable to high temperatures but not to oxygen . The ESR spectrum of the protein in the oxidized state showed a minor signal at g = 2.01, corresponding to an oxidized 3Fe-4S cluster . The protein, which was difficult to reduce with dithionite or reduced mediators, exhibited in its reduced state a spectrum typical of two interacting reduced 4Fe-4S clusters . M . thermolithotrophicus ferredoxin functioned as an electron acceptor for the CO dehydrogenase complex with an extract free of ferredoxin . No reaction was detected with F420 or hydrogenase.

Mol Gen Genet, 1989 May, 217(1), 70 - 6
Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases; Grabnitz F et al.; The nucleotide sequence of the bglB gene, coding for the thermostable beta-glucosidase B of Clostridium thermocellum was determined . The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of beta-glucosidase B purified from Escherichia coli . The derived amino acid sequence corresponding to a polypeptide of Mr 84,100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide . The protein bears no resemblance to other bacterial beta-glucosidase sequences . However, extensive regions of homology were identified between the C . thermocellum enzyme and fungal beta-glucosidases . The N-terminal homologous region contains an amino acid sequence very similar to the active site of beta-glucosidase A3 from Aspergillus wentii . The striking sequence similarities between C . thermocellum beta-glucosidase B and Kluyveromyces fragilis beta-glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.

Mol Gen Genet, 1989 May, 217(1), 105 - 10
Identification and characterization of the genes encoding three structural proteins of the Thermoproteus tenax virus TTV1; Neumann H et al.; Three structural proteins, TP1, TP2 and TP3, of the virus TTV1 of the thermophilic archaebacterium Thermoproteus tenax strain Kra1 were mapped within the viral genome by locating the amino-terminal amino acid sequences of these proteins in the TTV1 DNA sequence . The derived amino acid sequences comprise 113, 139 and 160 amino acid residues, respectively . All three proteins are hydrophobic . The three genes are not linked, but transcribed in the same direction . No Shine-Dalgarno sequences are found in the vicinity of the initiation codons of these three genes . By Northern analysis, four mRNAs of 0.5 kb, 0.8 kb, 1.1 kb and 1.8 kb in size were found to be encoded in the region and in the vicinity of the genes, the shortest one (t1) encoding TP3 and the longest one (t4) encoding TP2 . No transcript from the region encoding TP1 has been found so far . A transcriptional start site was mapped for the transcript t1 . Its upstream sequence was similar to the putative consensus sequence for archaebacterial promoters.

Biochimie, 1989 May, 71(5), 655 - 65
Ribosomal proteins of Tetrahymena thermophila . Correlation of one- and two-dimensional electrophoretic migration patterns and characterization of additional small and large subunit proteins; Petridou B et al.; Further analysis of the protein complement of the cytoplasmic ribosome of the protozoon Tetrahymena thermophila has led to the identification and characterization of seven additional proteins, three in the small and four in the large subunit of this ribosome . Several of these proteins are poorly soluble or insoluble in the absence of high concentrations of urea and are not seen in the electrophoretic distribution patterns of ribosomal proteins in two-dimensional polyacrylamide gels unless 6 M urea is added to electrode buffers in contact with protein samples (first dimension) and first-dimension gels (second dimension) . The migration patterns of the 40S and 60S subunits of the T . thermophila ribosome in one-dimensional polyacrylamide SDS gels and in two-dimensional gels prepared by means of the basic-acidic system of Kaltschmidt and Wittmann**, and the basic-SDS system of Zinker and Warner*** have been correlated.

J Dairy Sci, 1989 May, 72(5), 1142 - 8
Effect of threonine and glycine concentrations on threonine aldolase activity of yogurt microorganisms during growth in a modified milk prepared by ultrafiltration; Marranzini RM et al.; To evaluate the combined effects of threonine and glycine concentrations during growth on threonine aldolase activity (EC 2.1.2.1) of yogurt microorganisms, Streptococcus thermophilus and Lactobacillus bulgaricus, a modified milk growth medium was prepared using UF to deplete the free amino acid level . Threonine and glycine were added according to a 2x2x2 factorial design at 5 or 195 microg.ml(-1) along with a standard amino acid mixture . Acetaldehyde production and threonine aldolase activity were evaluated utilizing headspace gas chromatography . Results showed that threonine and glycine concentrations did not affect growth or titratable acidity . The high concentration of threonine in combination with low glycine in the growth medium resulted in increased acetaldehyde synthesis by both microorganisms . Conversely, high glycine with low threonine decreased acetaldehyde synthesis . High threonine and low glycine increased threonine aldolase activity of cell-free extracts from S . thermophilus and L . bulgaricus, whereas high glycine and low threonine reduced threonine aldolase activity of both microorganisms.

Biochimie, 1989 May, 71(5), 667 - 79
Protein-RNA crosslinking in the subunits of the cytoplasmic ribosome of Tetrahymena thermophila; Petridou B et al.; Use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to introduce RNA-protein crosslinks in the 40S and 60S subunits of the cytoplasmic ribosome of Tetrahymena thermophila is described, and proteins linked covalently to 17S and 26S ribosomal RNAs are identified . RNA-protein crosslinking is accompanied by extensive dimerization and aggregation of ribosomal subunits probably due to formation of interparticle protein-protein crosslinks.

Biol Chem Hoppe Seyler, 1989 May, 370(5), 451 - 66
Polyamine effects on DNA-directed RNA polymerases in the ciliate Tetrahymena thermophila . In vivo- and in vitro-experiments suggesting highly specific regulative interactions; Eichler W et al.; Stimulation of growth in resting cultures of the ciliated protozoan Tetrahymena thermophila stimulated putrescine formation in a manner coordinated to transcription and partly to DNA-polymerisation . The stimulation of polyamine biosynthesis involved increased formation of the precursor L-ornithine from L-arginine as well as stimulation of the regulative key enzyme ornithine decarboxylase . In order to characterize the interrelationships between polyamines and transcription, which were suggested by these in vivo-assays, in vitro-assays were performed employing isolated pure macronuclei from Tetrahymena thermophila . The results obtained were: i) The diamine putrescine and the polyamines spermidine and spermine stimulated the incorporation of {4-14C}UTP into RNA time- and concentration dependently . This stimulation was not due to changes in the ionic strength nor to substitution for divalent cations (e.g . Mg2+ or Mn2+) . The di- and polyamines did not alter the Km of RNA polymerases for the substrates, e.g . UTP . ii) Purified yeast-RNA, when added to the in vitro transcription system at concentrations capable of stimulating purified ornithine decarboxylase from Tetrahymena inhibited the RNA polymerases . The inhibitory effect of RNA on the polymerases was partly antagonized by spermidine and spermine but not by putrescine whereas the residual polymerase activity was stimulated by all three bases . iii) The stimulating effects of the di- and polyamines were synergistic but not absolutely additive, suggesting different targets for their actions . iv) Stimulation of RNA polymerases by putrescine, spermidine, or spermine after inhibition of particular enzymes by alpha-amanitin allowed to distinguish the effects on the three polymerases (I, II, and III): putrescine was not specific for any of the polymerases; spermidine was most active stimulating polymerase I and spermine was most active stimulating polymerase II, less active stimulating polymerase I but strongly inhibitory to polymerase III . v) The results obtained in the previous experiments were confirmed by electrophoretic analysis of the products formed in the in vitro transcription assays which furthermore showed that the differences between the experiments with or without polyamines were most pronounced if partly denatured calf thymus DNA was present in the assay mixture . This finding and the inhibition by RNA suggest that the factor influenced most by the polyamines is the binding of the RNA polymerases to the DNA target.

J Protozool, 1989 May-Jun, 36(3), 304 - 7
Identification of a cDNA coding for the SerH3 surface protein of Tetrahymena thermophila; Doerder FP et al.; We have identified a Tetrahymena thermophila cDNA-containing plasmid (pC6) which hybridizes to a 1.47-kB RNA whose changes in cellular concentration parallel the changes in synthetic rate of a major cell surface protein . From a molecular and genetic analysis of strains expressing the gene (SerH3) encoding this protein, and of strains expressing immunologically distinct alleles of this gene, we conclude that pC6 encodes a portion of the SerH3 allele.

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